KR20200078200A - 크리스퍼 연관 단백질 및 엑소뉴클레아제를 포함하는 변형된 크리스퍼 연관 단백질 - Google Patents

크리스퍼 연관 단백질 및 엑소뉴클레아제를 포함하는 변형된 크리스퍼 연관 단백질 Download PDF

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KR20200078200A
KR20200078200A KR1020180167870A KR20180167870A KR20200078200A KR 20200078200 A KR20200078200 A KR 20200078200A KR 1020180167870 A KR1020180167870 A KR 1020180167870A KR 20180167870 A KR20180167870 A KR 20180167870A KR 20200078200 A KR20200078200 A KR 20200078200A
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박종진
윤지영
최선미
박미진
박슬기
박영훈
이정혁
임정학
김동욱
최성화
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Abstract

본원 발명은 크리스퍼 연관 단백질 및 엑소뉴클레아제인 RecJ를 포함하는 유전자 편집 기능이 향상된 크리스퍼 플러스 단백질에 관한 것이다. 본원 발명의 일 구체예로 RecJ가 크리스퍼 연관 단백질에 결합된 크리스퍼 플러스 단백질은 종래의 크리스 연관 단백질에 비해 녹아웃 및 녹인 효율이 증가하였다. 따라서, 본 발명의 크리스퍼 플러스는 유전자 편집 효율이 향상된 유전자 가위를 제공하며, 이를 이용할 경우 보다 안정적이고 효율적으로 유전자를 편집할 수 있다. 따라서, 상기 크리스퍼 플러스 단백질은 종래의 크리스퍼 연관 단백질을 대체할 수 있는 효율적인 유전자 편집 기능을 보유한 단백질로서 사업성이 우수할 것으로 예상된다.

Description

크리스퍼 연관 단백질 및 엑소뉴클레아제를 포함하는 변형된 크리스퍼 연관 단백질{MODIFIED CRISPR ASSOCIATED PROTEIN COMPRISING CRISPR ASSOCIATED PROTEIN AND EXONUCLEASE AND USE THEREOF}
본 발명은 엑소뉴클레아제가 크리스퍼 연관 단백질에 결합된 변형된 크리스퍼 연관 단백질 및 이의 용도에 관한 것이다.
유전체 교정(Genome editing)이란 생명체의 유전정보를 자유롭게 교정하는 기술이다. 생명과학분야의 진보와 유전체 서열 분석 기술의 발전을 통해 우리는 다양한 유전정보에 대해 폭넓게 이해할 수 있게 되었다. 예를 들어 동식물의 번식, 질병과 성장, 다양한 인간 유전질병을 일으키는 유전자 변이, 바이오연료의 생산 등을 위한 유전자에 대한 이해는 이미 확보된 상황이지만 이를 직접적으로 활용하여 생명체를 개선하고, 인간 질병을 치료하는 수준에까지 이르기 위해서는 그 이상의 기술 진보가 필수적이다.
유전체 교정 기술은 인간을 포함하여 동물, 식물, 미생물의 유전정보를 변화시켜 그 활용범위를 획기적으로 확장시킬 수 있다. 유전자가위는 원하는 유전정보를 정확히 자를 수 있도록 설계되어 만들어지는 분자 도구로 유전체 교정 기술에서 핵심역할을 하고 있다. 유전자 서열분석 분야를 한 차원 발전시켰던 차세대시퀀싱(Next generation sequencing) 기술과 같이, 유전자가위는 유전정보 활용의 속도와 그 범위를 확장시키고 새로운 산업분야를 창출해 내는 핵심 기술이 되고 있다.
지금까지 개발된 유전자 가위는 그 순서에 따라 3세대로 나눌 수 있다. 1세대 유전자 가위는 ZFN(Zinc Finger Nuclease), 2세대 유전자 가위는 TALEN(Transcription Activator-Like Effector Nuclease), 가장 최근에 연구된 CRISPR(Clustered regularly interspaced short palindromic repeat)-Cas(CRISPR-associated) 9은 3세대 유전자 가위다.
CRISPRs는 유전자 서열이 밝혀진 박테리아의 대략 40% 및 유전자 서열이 밝혀진 고세균의 90%의 유전체에서 발견되는 여러 짧은 직접 반복을 포함하는 좌위이다. Cas 단백질은 CRISPR RNA(crRNA) 및 trans-activating crRNA(tracrRNA)로 명명된 두 개의 RNA와 복합체를 형성했을 때, 활성 엔도뉴클레아제(endonuclease)를 형성하고, 그렇게 함으로써 파지 또는 플라스미드의 침입에서 외부 유전적 요소를 분해하여 숙주 세포를 보호한다. crRNA는 전달에 외부 침입자로부터 점유되었던 숙주 유전체의 CRISPR 요소로부터 전사된다.
이러한 CRISPR-Cas 시스템 유래의 RNA-가이드 뉴클레아제(RNA-guided nuclease)는 유전체를 교정할 수 있는 수단을 제공해준다. 특히, 단일 가이드 RNA(sgRNA)와 Cas 단백질을 이용하여 세포 및 기관의 유전체를 편집할 수 있는 기술과 관련된 연구들이 활발히 진행되어왔다. 최근, Cpf1 단백질(Prevotella 및 Francisella 1 유래)이 CRISPR-Cas 시스템의 또 다른 뉴클레아제 단백질로서 보고되었고(B. Zetsche, et al., 2015), 이에 따라 유전체 교정에 있어서 선택의 폭이 넓어졌다. Cpf1은 5' 돌출부 형태로 절단하는 점, 가이드 RNA의 길이가 더 짧은 점, 시드(seed) 서열 및 절단 위치 사이가 더 길다는 점에서 여러 장점을 가진다. 그럼에도 여전히 보다 우수한 유전자 가위를 개발하기 위하여 많은 연구가 이루어지 있는 실정이다.
B. Zetsche, et al., Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system, Cell, 2015, 163, 759-771.
본 발명자들은 유전체 교정에 있어서 기존에 알려진 크리스퍼 연관 단백질보다 더 효과적인 단백질을 개발하기 위하여 지속적으로 연구한 결과, 표적 핵산 서열을 인식하여 절단하는 효율이 향상될 뿐 아니라, 녹인 효율이 증가된, 엑소뉴클레아제를 포함하는 변형된 크리스퍼 연관 단백질을 개발하여 본 발명을 완성하였다. 따라서, 본 발명의 목적은 표적 핵산 서열을 인식하여 절단하는 효율이 향상된 변형된 크리스퍼 연관 단백질을 제공하는 것이다.
상기 목적을 달성하기 위하여, 크리스퍼 연관 단백질에 엑소뉴클레아제 중 RecJ가 결합된 변형된 크리스퍼 연관 단백질을 제공한다.
RecJ가 크리스퍼 연관 단백질에 결합된 크리스퍼 플러스 단백질은 크리스 연관 단백질에 비해 녹아웃(Knockout) 및 녹인(Knockin) 효율이 증가하였다. 따라서, 본원의 변형된 크리스퍼 연관 단백질은 유전자 편집 효율이 향상된 유전자 가위를 제공하며, 이를 이용할 경우 보다 안정적이고 효율적으로 유전자를 편집할 수 있다.
도 1은 변형된 Cas9 단백질을 체외에서 절단하여 분석한 것이다. 각 RNP의 농도는 25 nM이다. 별표(*)는 약 600 bp에서 나타나는 절단된 템플릿(template)을 의미한다. 각 겔 이미지의 하단 부분에 절단 효율을 표시하였다.
도 2는 SpCas9 및 SpCas9-RecJ에 의한 원형 dsDNA 분해를 확인한 것이다. 선형 dsDNA는 DNA 전기 영동에서 뚜렷하고 두꺼운 dsDNA 크기를 보였다. SpCas9/gRNA C apoproteins를 배양한 후, SpCas9은 시간에 상관없이 단일의 선명하고 두꺼운 dsDNA를 나타내었다. 그러나, SpCas9-RecJ/gRNA C는 60-180분 배양 후에 흐릿하며, 약하고, 얇은, 꼬리가 끌리는 dsDNA형태를 나타내었는데, 이는 DNA 분해를 의미한다.
도 3은 반응 시간에 따른 유전자 편집 효율을 나타낸 것이다. 이는 T7E1 엔도뉴클레아제 활성의 산물이며, 삽입(insertion)-결실률(deletion)(% indel)과 비교하여 측정하였다.
도 4는 CCR5 및 DHCR7에 대한 SpyCas9, SpyCas9-RecJ 및 SpyCas9-T5 간의 HDR 효율을 비교하여 나타낸 것이다. CCR5 및 DHCR7 유전자자리(locus)에서 SpyCas9, SpyCas9-RecJ 및 SpyCas9-T5의 HDR 효율을 나타낸 것이다.
도 5 및 도 6은 유전자 편집 효율을 확인하기 위하여, HEK293 세포에서 SpCas9 및 SpCas9-RecJ의 In vitro 절단 에세이를 수행한 결과를 나타낸다.
도 7은 Cas9 및 Cas9-RecJ이 인간 PD-1 및 CCR5의 유전자에 미치는 온-타겟 효과를 나타낸 것이다.
도 8은 인간 CCR5에 대한 Cas9 및 Cas9-RecJ의 오프 타겟 효과를 확인한 것이다.
도 9는 5개의 오프 타겟팅된 유전자의 In vitro 절단 에세이를 나타낸 것이다.
도 10은 선형 dsDNA를 이용한 In vitro 절단 에세이를 통하여, FnCpf1, FnCpf1-RecJ 및 SpCas9이 가지고 있는 엑소뉴클레아제 기능을 확인한 것이다.
도 11은 선형 dsDNA를 이용하여 FnCpf1 또는 FnCpf1-RecJ 엑소뉴클레아제에 의한 In vitro 절단 에세이 결과를 나타낸 것이다.
도 12는 RecJ를 포함하는 변형된 크리스퍼 연관 단백질의 구조를 나타낸 것이다. A는 SpyCas9 또는 FnCpf1의 C 말단에 RecJ가 융합된 형태이다. B는 SpyCas9 또는 FnCpf1의 N 말단에 RecJ가 결합된 형태이다. C는 SpyCas9 또는 FnCpf1의 C 말단에는 RecJ가 N 말단에는 DBP(DNA Binding Protein)이 결합된 형태이다. 이때, His tag은 6개의 His로 구성되며, BPNLS는 핵 위치화 시그널(nuclear localization signal)을 의미한다.
도 13은 CCR5 및 DHCR7에 대한 SpyCas9의 sgRNA 결합 사이트를 나타낸 것이다. 인간 CCR5(A) 및 인간 DHCR7(B)에 대한 SpyCas9의 sgRNA 결합 영역을 각각 노란색과 핑크색으로 나타낸 것이다. PAM 서열은 녹색으로 표시하였다. 화살표는 뉴클레아제 절단 위치를 나타낸다.
도 14는 CCR5 및 DHCR7에 대한 FnCpf1의 crRNA 결합 사이트를 나타낸 것이다. 인간 CCR5(A) 및 인간 DMNT1(B)에 대한 FnCpf1의 crRNA 결합 영역을 각각 붉은색과 오렌지색으로 나타낸 것이다. PAM 서열은 파란색으로 표시하였다. 화살표는 뉴클레아제 절단 위치를 나타낸다.
도 15 및 도 16은 SpyCas9과 SpyCas9의 C 말단에 다양한 단백질이 결합된 변형된 SpyCas9 사이의 편집 효율을 비교한 것이다. SpyCas9의 C 말단에 GFP, hTdT, 및 엑소뉴클레아제로 알려진 RecJ, RecE, lambda, mungbean, and T5가 결합된 형태이다. 도면에서 나타낸 바와 같이 Knock-out 및 Knock-in 효율이 서로 상이하다.
도 17은 SpyCas9과 SpyCas9-RecJ의 Knock-out 편집 효율을 비교한 것이다. A는 SpyCas9-RecJ의 구조를 나타낸 것이다. B는 대조군, SpyCas9 및 SpyCas9-RecJ 간의 유전자 편집 효율을 나타낸 것이다. C는 CCR5 및 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 SpyCas9-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 18은 SpyCas9과 SpyCas9-RecJ의 Knock-in 편집 효율을 비교한 것이다. A는 SpyCas9-RecJ의 구조를 나타낸 것이다. B는 대조군, SpyCas9 및 SpyCas9-RecJ 간의 유전자 편집 효율을 나타낸 것이다. C는 CCR5 및 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 SpyCas9-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 19는 및 20은 SpyCas9과 SpyCas9의 N 말단 또는 N/C 양말단에 다양한 단백질이 결합된 변형된 SpyCas9 사이의 편집 효율을 비교한 것이다. 변형된 SpyCas9은 SpyCas9의 N 말단에 RecJ, RecE, GFP, 단일 가닥 DNA 결합 단백질(single stranded DNA binding protein, SSB) 및 이중 가닥 DNA 결합 단백질(double stranded DNA binding protein, DSB)이 결합되어 있다. 또한, 변형된 SpyCas9은 SpyCas9의 C 말단에 RecJ가 결합되어 있고, N 말단에 SSB 또는 DSB이 결합되어 있다. 도면에서 나타낸 바와 같이 Knock-out 및 Knock-in 효율은 서로 상이하다.
도 21은 SpyCas9과 RecJ-SpyCas9 간의 Knock-out 편집 효율을 비교한 것이다. A는 RecJ-SpyCas9의 구조를 나타낸 것이다. B는 대조군, SpyCas9 및 RecJ-SpyCas9의 유전자 편집 효율을 나타낸 것이다. C는 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 RecJ-SpyCas9의 편집 효율 변화를 백분율로 나타낸 것이다.
도 22는 SpyCas9과 RecJ-SpyCas9의 Knock-in 편집 효율을 비교한 것이다. A는 RecJ-SpyCas9의 구조를 나타낸 것이다. B는 대조군, SpyCas9 및 RecJ-SpyCas9 간의 유전자 편집 효율을 나타낸 것이다. C는 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 RecJ-SpyCas9의 편집 효율 변화를 백분율로 나타낸 것이다.
도 23은 SpyCas9과 SSB-SpyCas9-RecJ의 Knock-out 편집 효율을 비교한 것이다. A는 SSB-SpyCas9-RecJ의 구조를 나타낸 것이다. B는 대조군, SpyCas9 및 SSB-SpyCas9-RecJ의 유전자 편집 효율을 나타낸 것이다. C는 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 SSB-SpyCas9-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 24는 SpyCas9과 DSB-SpyCas9-RecJ 간의 Knock-out 편집 효율을 비교한 것이다. A는 DSB-SpyCas9-RecJ의 구조를 나타낸 것이다. B는 대조군, SpyCas9 및 DSB-SpyCas9-RecJ 간의 유전자 편집 효율을 나타낸 것이다. C는 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 DSB-SpyCas9-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 25 및 도 26은 FnCpf1과 FnCpf1의 C 말단에 다양한 단백질이 결합된 변형된 FnCpf1 사이의 편집 효율을 비교한 것이다. FnCpf1의 C 말단에 GFP, hTdT, 및 엑소뉴클레아제로 알려진 RecJ, RecE, lambda, mungbean, 및 T5가 결합된 형태이다. 도면에서 나타낸 바와 같이 Knock-out 및 Knock-in 효율은 서로 상이하다.
도 27은 FnCpf1 및 FnCpf1-RecJ의 Knock-out 편집 효율을 비교한 것이다. A는 FnCpf1-RecJ의 구조를 나타낸 것이다. B는 대조군, FnCpf1 및 FnCpf1-RecJ의 유전자 편집 효율을 나타낸 것이다. C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 FnCpf1-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 28은 FnCpf1 및 FnCpf1-RecJ의 Knock-in 편집 효율을 비교한 것이다. A는 FnCpf1-RecJ의 구조를 나타낸 것이다. B는 대조군, FnCpf1 및 FnCpf1-RecJ 간의 유전자 편집 효율을 나타낸 것이다. C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 FnCpf1-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 29 및 30은 FnCpf1과 FnCpf1의 N 말단 또는 N/C 양말단에 다양한 단백질이 결합된 변형된 FnCpf1 사이의 편집 효율을 비교한 것이다. 변형된 FnCpf1은 FnCpf1의 N 말단에 RecJ, RecE, GFP, 단일 가닥 DNA 결합 단백질(single stranded DNA binding protein, SSB) 및 이중 가락 DNA 결합 단백질(double stranded DNA binding protein, DSB)이 결합되어 있다. 또한, 변형된 FnCpf1은 FnCpf1의 C 말단에 RecJ가 결합되어 있고, N 말단에 SSB가 결합되어 있다. 도면에서 나타낸 바와 같이 Knock-out 및 Knock-in 효율은 서로 상이하다.
도 31은 FnCpf1 및 RecJ-FnCpf1의 Knock-out 편집 효율을 비교한 것이다. A는 RecJ-FnCpf1의 구조를 나타낸 것이다. B는 대조군, RecJ-FnCpf1 간의 유전자 편집 효율을 나타낸 것이다. C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 RecJ-FnCpf1의 편집 효율 변화를 백분율로 나타낸 것이다.
도 32는 FnCpf1 및 RecJ-FnCpf1 간의 Knock-in 편집 효율을 비교한 것이다. A는 RecJ-FnCpf1의 구조를 나타낸 것이다. B는 대조군, RecJ-FnCpf1의 유전자 편집 효율을 나타낸 것이다. C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 RecJ-FnCpf1의 편집 효율 변화를 백분율로 나타낸 것이다.
도 33은 FnCpf1과 SSB-FnCpf1-RecJ의 Knock-out 편집 효율을 비교한 것이다. A는 SSB-FnCpf1-RecJ의 구조를 나타낸 것이다. B는 대조군, FnCpf1 및 SSB-FnCpf1-RecJ의 유전자 편집 효율을 나타낸 것이다. C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 SSB-FnCpf1-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 34는 FnCpf1과 SSB-FnCpf1-RecJ의 Knock-in 편집 효율을 비교한 것이다. A는 SSB-FnCpf1-RecJ의 구조를 나타낸 것이다. B는 대조군, FnCpf1 및 SSB-FnCpf1-RecJ의 유전자 편집 효율을 나타낸 것이다. C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 SSB-FnCpf1-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 35a, 도 35b 및 도 35c는 SpyCas9 및 SpyCas9의 C 말단에 단백질이 결합된 변형된 SpyCas9에 유도되는 비상동적인 말단 연결에 있어서, ssODN 및 노코다졸(nocodazole)이 미치는 영향을 확인한 것이다. 도 35a는 ssODN 및 노코다졸이 처리되지 않았을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 35b는 ssODN 및 노코다졸이 처리되었을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 35c는 ssODN 및 노코다졸의 존재 유무에 따른 효율을 비교한 것이다. ssODN 및 노코다졸은 Knock-out의 경우, SpyCas9 및 변형된 SpyCas9의 효율에는 큰 영향이 없음을 확인하였다.
도 36a, 도 36b 및 도 36c는 SpyCas9 및 SpyCas9의 N 말단 또는 N/C 양말단에 단백질이 결합된 변형된 SpyCas9에 유도되는 비상동적인 말단 연결에 있어서, ssODN 및 노코다졸이 미치는 영향을 확인한 것이다. 도 36a는 ssODN 및 노코다졸이 처리되지 않았을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 36b는 ssODN 및 노코다졸이 처리되었을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 36c는 ssODN 및 노코다졸의 존재 유무에 따른 효율을 비교한 것이다. ssODN 및 노코다졸은 Knock-out의 경우, SpyCas9 및 변형된 SpyCas9의 효율에는 큰 영향이 없음을 확인하였다.
도 37a, 도 37b 및 도 37c는 FnCpf1 및 FnCpf1의 C 말단에 단백질이 결합된 변형된 FnCpf1에 유도되는 비상동적인 말단 연결에 있어서, ssODN 및 노코다졸(nocodazole)이 미치는 영향을 확인한 것이다. 도 37a는 ssODN 및 노코다졸이 처리되지 않았을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 37b는 ssODN 및 노코다졸이 처리되었을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 37c는 ssODN 및 노코다졸의 존재 유무에 따른 효율을 비교한 것이다. ssODN 및 노코다졸은 Knock-out의 경우, FnCpf1 및 변형된 FnCpf1의 효율을 향상 시킨다는 점을 확인하였다.
도 38a, 도 38b 및 도 38c는 FnCpf1 및 FnCpf1의 N 말단 또는 N/C 양말단에 단백질이 결합된 변형된 FnCpf1에 유도되는 비상동적인 말단 연결에 있어서, ssODN 및 노코다졸이 미치는 영향을 확인한 것이다. 도 38a는 ssODN 및 노코다졸이 처리되지 않았을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 38b는 ssODN 및 노코다졸이 처리되었을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 38c는 ssODN 및 노코다졸의 존재 유무에 따른 효율을 비교한 것이다. ssODN 및 노코다졸은 Knock-out의 경우, FnCpf1 및 변형된 FnCpf1의 효율에는 큰 영향이 없음을 확인하였다.
도 39a 및 도 39b는 각각의 실험에 대한 타당성을 나타낸 것이다. 최상단에는 on-target 서열을 나타낸 것이며, 미스매치된 핵산은 다른 색으로 표시하였다. GFLAS01은 음성대조군이며; GFLAS02 및 GFLAS05는 SpyCas9이며; GFLAS03, GFLAS04, GFLAS06 및 GFLAS07은 Cas9-RecJ를 이용하여 실험한 결과를 나타낸다.
도 40은 Cas9 및 Cas9-RecJ간의 오프-타겟팅 효율을 비교한 것이다. 5개의 인간 CCR5로 오프-타겟을 테스트하였다. A는 CCR5에 대한 오프-타겟 후보 서열을 나타낸 것이다. B는 딥 시퀀싱(deep sequencing)을 하기 위한 증폭물을 나타낸 것이다.
도 41은 Cas9 및 Cas9-RecJ의 증폭물의 딥 시퀀싱 결과를 나타낸 것이다. Cas9-RecJ은 KCNJ6, CNTPNA2, 및 Ch.5에서는 Cas9에 비해 오프-타겟이 나타나는 비율이 낮다는 것을 확인하였다.
도 42는 SpyCas9의 단백질 서열 및 핵산 서열을 도식화한 것이다.
도 43은 SpyCas9-RecJ의 단백질 서열 및 핵산 서열을 도식화한 것이다.
도 44는 RecJ-SpyCas9의 단백질 서열 및 핵산 서열을 도식화한 것이다.
도 45는 SSB-SpyCas9-RecJ의 단백질 서열 및 핵산 서열을 도식화한 것이다.
도 46은 DSB-SpyCas9-RecJ의 단백질 서열 및 핵산 서열을 도식화한 것이다.
도 47은 FnCpf1의 단백질 서열 및 핵산 서열을 도식화한 것이다.
도 48은 FnCpf1-RecJ의 단백질 서열 및 핵산 서열을 도식화한 것이다.
도 49는 RecJ-FnCpf1의 단백질 서열 및 핵산 서열을 도식화한 것이다.
도 50은 SSB-FnCpf1-RecJ의 단백질 서열 및 핵산 서열을 도식화한 것이다.
도 51은 변형된 크리스퍼 연관 단백질 및 sgRNA를 이용하여 식물체의 유전자를 편집하는 실험 방법의 개요를 나타낸 것이다.
도 52는 유전자 편집을 하기 위해 준비된 식물체를 나타낸 것이다.
도 53은 식물체의 타겟 위치를 도식화한 것이다.
도 54는 차세대 염기서열 분석법(Next-Generation Sequencing, NGS)을 통해 유전자 편집 후의 식물체의 핵산 서열을 분석한 것이다.
도 55는 변형된 크리스퍼 연관 단백질의 삽입-결실 효율을 나타낸 것이다.
도 56은 변형된 크리스퍼 연관 단백질의 삽입-결실 효율을 나타낸 것이다.
이하 본원 발명을 상세히 설명한다.
본 발명은 일 측면으로 크리스퍼 연관 단백질 및 엑소뉴클레아제인 RecJ를 포함하는 변형된 크리스퍼 연관 단백질을 제공한다. 이때, 상기 변형된 크리스퍼 연관 단백질은 이하에서 크리스퍼 플러스 단백질이라고 지칭할 수 있다.
본 명세서에서 사용된 용어, "크리스퍼 플러스 단백질"이란, 크리스퍼 연관 단백질의 유전자 편집 효율이 개선된 크리스퍼 연관 단백질을 지칭한다. 이때, 크리스퍼 연관 단백질의 편집 효율의 개선은 크리스퍼 연관 단백질 이외의 단백질을 결합시킴으로써 달성할 수 있다.
상기 크리스퍼 플러스 단백질은 유전자 편집 기능이 향상되었다. 이때, 유전자 편집 기능이란 녹아웃(Knockout) 및 녹인(Knockin) 기능을 의미한다. 녹아웃은 크리스퍼 연관 단백질이 타겟 핵산 서열과 결합하였을 시에, 타겟 핵산의 일부를 결실시키거나, 타겟 핵산의 일부에 뉴클레오티드를 삽입한 경우를 의미한다. 또한, 녹인이란 외래 유전자가 타겟 부위에 삽입되는 것을 의미한다.
상기 RecJ는 크리스퍼 연관 단백질의 N 말단 또는 C 말단에 결합될 수 있다. 또한, 크리스퍼 연관 단백질과 RecJ는 직접 결합될 수도 있다. 또한, 상기 크리스퍼 연관 단백질 및 RecJ는 링커 1을 통해 연결되는 것일 수 있다.
구체적으로, 상기 크리스퍼 플러스 단백질은 다음 구조 중 어느 하나일 수 있다:
<구조식 1>
크리스퍼 연관 단백질-RecJ
<구조식 2>
크리스퍼 연관 단백질-링커 1-RecJ
<구조식 3>
RecJ-크리스퍼 연관 단백질
<구조식 4>
RecJ-링커 1-크리스퍼 연관 단백질
상기 구조식 1 내지 4의 융합 단백질은 N 말단에서 C 말단으로 결합된 것을 나타낸다. 이때, 상기 구조식 1 내지 4에 표시된 링커 1은 1 내지 20개의 아미노산으로 구성된 것일 수 있다. 또한, 상기 링커 1은 5 내지 10개의 아미노산일 수 있으며, 6개, 7개, 8개, 9개, 10개의 아미노산으로 구성될 수 있다. 일 구체예의 링커 1은 서열번호 17, 18, 19, 20, 21, 22, 23 또는 24로 표시되는 아미노산을 가질 수 있다.
본 명세서에서 사용된 용어, "크리스퍼 연관 단백질"은 Cas 효소, CRISPR 효소, CRISPR 단백질, Cas 단백질 및 CRISPR Cas는 일반적으로 상호교환적으로 사용될 수 있다.
또한, 상기 크리스퍼 연관 단백질은 스트렙토코커스(Streptococcus), 캄필로박터(Campylobacter), 니트라티프락토르(Nitratifractor), 스타필로코커스(Staphylococcus), 파르비바쿨룸(Parvibaculum), 로세부리아(Roseburia), 네이세리아(Neisseria), 글루콘아세토박터(Gluconacetobacter), 아조스피릴룸(Azospirillum), 스파에로카에타(Sphaerochaeta), 락토바실러스(Lactobacillus), 유박테리움(Eubacterium), 코리네박터(Corynebacter), 카르노박테리움(Carnobacterium), 로도박터(Rhodobacter), 리스테리아(Listeria), 팔루디박터(Paludibacter), 클로스트리디움(Clostridium), 라크노스피라세아에(Lachnospiraceae), 클로스트리디아리디움(Clostridiaridium), 렙토트리키아(Leptotrichia), 프란시셀라(Francisella), 레지오넬라(Legionella), 알리사이클로바실러스(Alicyclobacillus), 메타노메티오필러스(Methanomethyophilus), 포르피로모나스(Porphyromonas), 프레보텔라(Prevotella), 박테로이데테스(Bacteroidetes), 헬코코커스(Helcococcus), 레토스피라(Letospira), 데설포비브리오(Desulfovibrio), 데설포나트로눔(Desulfonatronum), 오피투타세아에(Opitutaceae), 투베리바실러스(Tuberibacillus), 바실러스(Bacillus), 브레비바실러스(Brevibacilus), 메틸로박테리움(Methylobacterium) 또는 아시다미노코커스(Acidaminococcus)를 포함하는 속에서 유래된 것일 수 있다.
또한, 상기 크리스퍼 연관 단백질은 Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 및 Csf4로 구성된 군으로부터 선택되는 어느 하나 일 수 있다. 일 구체예로 상기 크리스퍼 연관 단백질은 Cas9 또는 Cas12a 일 수 있다. 일 실시예로 Cas9은 SpyCas9일 수 있다. 또한, Cpf1은 FnCpf1, AsCpf1 또는 LbCpf1 일 수 있으며, 일 실시예로 Cpf1은 FnCpf1 일 수 있다. 상기 크리스퍼 연관 단백질은 종래에 알려진 서열과 95%, 96%, 97%, 98%, 99%의 상동성을 가질 수 있다.
이때, 상기 RecJ는 엑소뉴클레아제로 알려진 단백질이다. 이때, 상기 RecJ의 일 구체예로는 에스케리치아(Escherichia) 속 유래의 RecJ(서열번호 1), 스트렙토코코스(Streptococcus) 속 유래의 RecJ(서열번호 3), 락토코코스(Lactococcus) 속 유래의 RecJ(서열번호 5), 바실러스(Bacillus) 속 유래의 RecJ(서열번호 7), 에르리치아(Ehrlichia) 속 유래의 RecJ(서열번호 9), 클라미디아(Chlamydia) 속 유래의 RecJ(서열번호 11), 피네골디아(Finegoldia) 속 유래의 RecJ(서열번호 13), 또는 스트렙토코코스(Streptococcus) 속 유래의 RecJ(서열번호 15)일 수 있다.
본 발명은 또 다른 측면으로, 크리스퍼 연관 단백질, RecJ 및 DNA 결합 단백질(DNA binding protein, DBP)를 포함하는 크리스퍼 플러스 단백질 일 수다.
상기 RecJ 및 DBP는 크리스퍼 연관 단백질의 N 말단 또는 C 말단에 결합될 수 있다. 또한, 이때, 상기 크리스퍼 연관 단백질, RecJ 및 DBP는 링커을 통해 연결되는 것일 수 있다. 이때, DBP는 SSB(single strand binding protein) 또는 DSB(double strand binding protein) 일 수 있다. 상기 크리스퍼 플러스 단백질의 일시예는 다음 구조 중 어느 하나일 수 있다:
<구조식 5>
DBP-크리스퍼 연관 단백질-RecJ
<구조식 6>
DBP-링커 2-크리스퍼 연관 단백질-RecJ
<구조식 7>
DBP-링커 2-크리스퍼 연관 단백질-링커 1-RecJ
상기 구조식 5 내지 7의 융합 단백질은 N 말단에서 C 말단으로 결합된 것이다. 크리스퍼 연관 단백질, RecJ 및 DBP는 직접 결합될 수도 있으며, 링커를 통해 연결될 수 있다. 이때, 링커 1은 상술한 바와 같다. 또한, 상기 구조식 5 내지 7에 표시된 링커 2는 1 내지 20개의 아미노산으로 구성된 것일 수 있다. 또한, 상기 링커 2는 5 내지 10개의 아미노산일 수 있으며, 6개, 7개, 8개, 9개, 10개의 아미노산으로 구성될 수 있다. 일 구체예의 링커 2는 서열번호 17, 18, 19, 20, 21, 22, 23 또는 24로 표시되는 아미노산 서열을 가질 수 있다.
본 발명은 또 다른 측면으로, 크리스퍼 연관 단백질 및 RecJ를 포함하는 크리스퍼 플러스 단백질 및 크리스퍼 연관 단백질, RecJ 및 DBP를 포함하는 크리스퍼 플러스 단백질에 추가적으로 추가적으로 핵 위치화 시그널(nuclear localization signal, NLS)을 적어도 하나를 더 포함하는 크리스퍼 플러스 단백질 일 수 있다. 이때, 핵 위치화 시그널은 bpNLS(biparticle nuclear localization sequence)일 수 있다. 적어도 하나 이상의 NLS가 C-말단 또는 N-말단 부착될 수 있다. 이때, 일 구체예에서는 상기 핵 위치화 시그널은 융합 단백질의 C 말단에 위치할 수 있다. 상기 핵 위치화 시그널은 크리스퍼 플러스 단백질의 활성에는 영향을 미치지 않으나, 크리스퍼 플러스 단백질이 세포의 핵내로 이동할 수 있도록 도와주는 펩타이드를 의미한다.
또한, 본 발명의 다른 측면은, 상기 크리스퍼 플러스 단백질에 추가적으로 추가적으로 His tag를 더 포함할 수 있다. 이때, 상기 His tag는 크리스퍼 플러스 단백질의 C 말단에 위치할 수 있다. 상기 His tag는 크리스퍼 플러스 단백질의 활성에는 영향을 미치지 않으나, 크리스퍼 플러스 단백질의 분리 정제에 사용될 수 있다.
또한, 상기 크리스퍼 플러스 단백질은 서열번호 31, 41, 49, 53, 57, 71 또는 77으로 표시되는 아미노산 서열을 가지는 것 일 수 있다.
본 명세서에서 사용된 용어, "PAM"은 프로토스페이서 인접 모티프(protospacer adjacent motif) 또는 PAM-유사 모티프는 크리스퍼 연관 단백질 복합체가 타겟 유전자좌에 결합하는 것을 지시한다.
본 발명의 일 구체예에서, 크리스퍼 연관 단백질은 FnCpf1과 관련이 있는 PAM은 5' TTN이며, 여기서 N은 A/C/G 또는 T이다. PAM은 타겟 유전자의 5' 말단의 상류에 위치할 수 있다. Cpf1 패밀리의 T-풍부 PAM은 AT-풍부 게놈의 표적화 및 편집을 가능하게 한다. 반면, Cas9 단백질의 PAM은 타겟 유전자의 3' 말단 하류에 위치할 수 있다.
본 발명은 또 다른 측면으로, 상술한 크리스퍼 플러스 단백질을 코딩하는 폴리뉴클레오티드를 제공한다.
상기 뉴클레오티드는 신호서열(signal sequence) 또는 리더 서열(leader sequence)을 추가적으로 포함할 수 있다. 여기에서 사용된 용어 "신호서열"은 목적 단백질의 분비를 지시하는 신호펩타이드를 코딩하는 핵산을 의미한다. 상기 신호펩타이드는 숙주 세포에서 번역된 후에 절단된다. 구체적으로, 본원 발명의 신호서열은 ER(endoplasmic reticulum) 막을 관통하는 단백질의 이동을 개시하는 아미노산 서열을 코딩하는 뉴클레오티드 일 수 있다.
신호서열은 당업계에 그 특징이 잘 알려져 있으며, 통상 16 내지 30개의 아미노산 잔기를 포함하나, 그보다 더 많거나 적은 아미노산 잔기를 포함할 수 있다. 통상적인 신호 펩타이드는 기본 N-말단 영역, 중심 소수성 영역, 및 보다 극성인(polar) C-말단 영역의 세 영역으로 구성된다. 중심 소수성 영역은 미성숙 폴리펩티드가 이동하는 동안 막지질 이중층을 통하여 신호서열을 고정시키는 4 내지 12개의 소수성 잔기를 포함할 수 있다.
이때, 상기 크리스퍼 플러스 단백질을 코딩하는 뉴클레오티드의 일 실시예는 서열번호 32, 42, 50, 54, 58, 72 또는 72로 표시되는 서열을 가질 수 있다.
본 발명의 또 다른 측면으로, 상기 폴리뉴클레오티드가 적재된 벡터를 제공한다.
본원 발명에서 사용된 용어 "벡터"는 숙주 세포에 도입되어 숙주 세포 유전체 내로 재조합 및 삽입될 수 있다. 또는 상기 벡터는 에피좀으로서 자발적으로 복제될 수 있는 뉴클레오티드 서열을 포함하는 핵산 수단으로 이해된다. 상기 벡터는 선형 핵산, 플라스미드, 파지미드, 코스미드, RNA 벡터, 바이러스 벡터 및 이의 유사체들을 포함한다. 바이러스 벡터의 예로는 레트로바이러스, 아데노바이러스, 및 아데노-관련 바이러스를 포함하나 이에 제한되지 않는다. 또한, 상기 플라스미드는 항생제 내성 유전자와 같은 선별 마커를 포함할 수 있고, 플라스미드를 유지하는 숙주 세포는 선택적인 조건하에서 배양될 수 있다.
본 발명의 또 다른 측면으로, 상기 벡터가 도입된 숙주 세포를 제공한다.
상기 숙주 세포는 벡터를 도입하여 본원의 크리스퍼 플러스 단백질을 생산할 수 있다면 종류에 한정되는 것은 아니다. 일 구체예로 상기 숙주세포는 대장균일 수 있다.
본 발명의 또 다른 측면으로, 상기 크리스퍼 플러스 단백질 및 crRNA가 결합된 RNP 복합체를 제공한다.
일 구체예에서, 상기 복합체의 핵산 성분는 직접 반복 서열에 연결된 가이드 서열을 포함할 수 있다. 상기 직접 반복 서열은 하나 이상의 줄기 루프(stem loop) 또는 최적화된 2차 구조를 포함할 수 있다. 바람직한 구현예에서, 직접 반복은 최소한의 길이가 16 nt이고 단일 줄기 루프를 가질 수 있다. 추가 구현예에서, 직접 반복은 길이가 16 nt보다 더 길며, 바람직하게는 17 nt 초과이고, 하나 초과의 줄기 루프 또는 최적화된 2차 구조를 가질 수 있다. 바람직한 구현예에서, 직접 반복은 변형되어 하나 이상의 단백질-결합 RNA 압타머를 포함할 수 있다.
이때, 상기 crRNA는 타겟 유전자에 상보적으로 결합할 수 있는 핵산 서열을 갖는다. 이때, 타겟 유전자는 세포 내 DNA 분자에 포함될 수 있다. 세포는 원핵 세포 또는 진핵 세포일 수 있다. 세포는 포유동물 세포일 수 있다. 포유동물 세포는 비인간 영장류, 소, 돼지, 설치류 또는 마우스 세포일 수 있다. 세포는 비포유동물 진핵 세포, 예컨대 가금류, 어류 또는 새우일 수 있다. 세포는 또한 식물 세포일 수 있다. 식물 세포는 카사바, 옥수수, 수수, 밀, 담배, 애기장대, 채소 또는 쌀 일 수 있다.
본 발명에 의해 세포에 도입된 변형은, 세포 및 세포 의 자손이 생물학적 생성물, 예컨대 항체, 전분, 알코올 또는 기타 다른 원하는 세포 산출물의 개선된 생성을 위하여 변경되도록 하는 것일 수 있다. 본 발명에 의해 세포에 도입된 변형은 세포 및 세포의 자손이 생성된 생물학적 생성물을 변화시키는 변경을 포함하도록 하는 것일 수 있다.
또한, 본 발명의 또 다른 측면으로, 상기 RNP 복합체를 유효성분으로 포함하는 유전자 편집용 조성물 또는 유전자 편집용 키트를 제공한다.
이하, 본원 발명을 하기 실시예에 의하여 더욱 상세하게 설명한다. 단, 하기 실시예는 본원 발명을 예시하기 위한 것일 뿐, 본원 발명의 범위가 이들만으로 한정되는 것은 아니다.
I. 크리스퍼 플러스 단백질의 제조 및 활성 확인
제조예 1. Cpf1 및 RecJ외의 다른 변형된 단백질이 결합된 변형된 크리스퍼 연관 단백질 제조
이콜라이 단백질 발현 벡터 pET28a (addgene, Watertown, MA, USA) 를 NotI 제한 효소를 사용하여서 FnCpf1 (3,902 bp)의 정방향 프라이머 (CGTCGACAAGCTTGCGGCCGCATGTCAATTTATCAAGAATT) 와 역방향 프라이머 (TGGTGGTGCTCGAGTGCGGCCGCTTgGTTATTCCTATTCTGCAC) 를 이용하여 pY002 (addgene, Watertown, MA, USA)를 중합효소 연쇄반응으로 얻은 산물을 깁슨 어셈블리 킷트 (NEB, Ipswich, MA, USA) 방법으로 50도에 25분 인큐베이션으로 통하여 클로닝하였다. 결과적으로 얻어진 pET28a-FnCpf1의 말단에 PspXI 제한효소를 사용하여서 RecJ (1,731 bp), RecE (2,598 bp), SSB (348 bp)는 이콜라이 지놈에서 증폭하였고, mungbean (1,065 bp)은 숙주 나물에서 mRNA 산물을 얻어서 cDNA로 전환한 다음 코딩 영역을 얻을 수 있었다. T5 (828 bp), DSB (267 bp)는 GenScript (Piscataway, NJ, USA)에서 합성하여서 사용하였다. GFP (714 bp) 서열은 생명공학연구원에서 개발한 pCTV-GFP 벡터에서 증폭하여서 사용하였고, hTdT (1,527 bp)는 GenScript에서 DNTT(NM_004088) ORF Clone을 주문하여서 증폭하였다. 증폭된 중합효소반응 산물은 깁슨 어셈블리 방법을 통해서 Cpf1의 말단에 삽입되었다.
name sequence size function
RecJF1 GCGGCCGCACTCGACctgcagGTGAAACAACAGATACAACTTCGT 45 RecJ full-length in back
RecJR1 ccggcctttttcgtggccgccggccttttctgcagAATTGGCCAGATATTGTCGATGATA 60 RecJ full-length in back
RecJR2 TCAGTGGTGGTGGTGGTGGTGctttttcttttttgcctggccggcctttttcgtggccgc 60 RecJ full-length in back
MungbeanF1 CGGCCGCACTCGACctgcagATGCAAACGTTACAGATGAGT 41 Mungbean Nuclease in back
MungbeanR1 cgtggccgccggccttttctgcagACCATTGTAAGAGATAGGTC 44 Mungbean Nuclease in back
T5exonuF1 CGGCCGCACTCGACctgcagATGGCTTCCCGTCGTAATCTAATG 44 T5 exonuclease in back
T5exonuR5 gtggccgccggccttttctgcagTTGTTCTGCAATCTCCAAAATA 45 T5 exonuclease in back
SSBF1 GCGGCaGCACTCGACctgcagATGGCCAGCAGAGGCGTAAACAAGG 46 SSB isolation in back
SSBR1 tcgtggccgccggccttttctgcagACGACCACCCAGCATCTGCATGGT 49 SSB isolation in back
RecEF1 GCGGGCGCACTCGACctgcagATGAGCACAAAACCACTCTTCC 43 RecE in back
RecER1 ttcgtggccgccggccttttctgcagGTCATTTGCATATTCCTTAGCCCA 50 RecE in back
DSBF1 AGCGGCCGCACTCGACctcgagATGGCTAAAAAAGAAATGGTTGAATT 21 with DSBR1 in back
DSBR1 ctttttcgtggccgccggccttttctcgagTTTAGAGAAAACTGTGTCA 37 with DSBF1 in back
T5exoF5 GGTGGACAGCAAATGGGTCGCGGATCCATGGCTTCCCGTCGTAATCTAA 49 T5 full-length in back
T5exoR5 GCTTGTCGACGGAGCTCGAATTCGGATCCTTGTTCTGCAATCTCCAAA 48 T5 full-length in back
pAGROF2 gtctcagctgggaggcgacgaaATGAGTAAAGGAGAAGAACTTTT 45 GFP in back
pAGROR2 gcggaggggttggatcaaagtgaaAATAACCTCTCCTTCTTTTTC 45 GFP in back
HsTdTF2 GCGGCCGCACTCGACctcgagATGGATCCACCACGAGCGTCCCAC 45 HsTdT full length in back
HsTdTR2 ttcgtggccgccggccttttctcgagGGCATTTCTTTCCCACGGTTCAATAT 52 HsTdT full length in back
제조예 2. 타겟 sgRNA 또는 crRNA의 제작
제조예 2.1. CCR5 및 DHCR7을 타겟팅하는 sgRNA의 제작
CCR5 및 DHCR7을 타겟팅하며, SpyCas9와 함께 이용될 수 있는 sgRNA를 제작하였다. sgRNA 결합사이트는 도면 13에 나타낸 바와 같다. 인간 CCR5(A) 및 인간 DHCR7(B)에 대한 SpyCas9의 sgRNA 결합 영역을 각각 노란색과 핑크색으로 나타낸 것이다. PAM 서열은 녹색으로 표시하였다. 화살표는 뉴클레아제 절단 위치를 나타낸다.
제조예 2.2. CCR5 및 DHCR7을 타겟팅하는 crRNA의 제작
CCR5 및 DHCR7을 타겟팅하며, FnCpf1과 함께 이용될 수 있는 crRNA를 제작하였다. crRNA 결합사이트는 도면 14에 나타낸 바와 같다. 인간 CCR5(A) 및 인간 DMNT1(B)에 대한 FnCpf1의 crRNA 결합 영역을 각각 붉은색과 오렌지색으로 나타낸 것이다. PAM 서열은 파란색으로 표시하였다. 화살표는 뉴클레아제 절단 위치를 나타낸다.
실시예 1. Cas9 및 RecJ가 결합된 변형된 크리스퍼 연관 단백질 제조
실시예 1.1. SpCas9 제조
이콜라이 단백질 발현 벡터 pET28a를 NotI 제한 효소를 사용하여서 SpCas9(4,104 bp)의 정방향 프라이머 (CCGTCGACAAGCTTGCGGCCGCATGGACAAGAAGTACAGCATCGGCCTGGA) 와 역방향 프라이머 (TGAGCCAGCTGGGCGGCGACGCGGCCGCACTCGACctcgagaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagCACCACCACCACCACCACTGA) 를 이용하여 pET28a/Cas9-cys (addgene, Watertown, MA, USA)에서 중합효소 연쇄반응으로 얻은 산물을 깁슨 어셈블리 킷트 (NEB, Ipswich, MA, USA) 방법으로 50도에 25분 인큐베이션으로 통하여 클로닝하였다. 서열 정보는 도42-도50에 명시하고 있다.
실시예 1.2. RecJ이 결합된 SpCas9의 제조
결과적으로 얻어진 pET28a-SpCas9의 말단에 PspXI 제한효소를 사용하고 SpCas9 앞쪽에는 BamHI 제한 효소를 사용하여서 RecJ (1,731 bp), RecE (2,598 bp), SSB (348 bp)는 이콜라이 지놈에서 증폭하였고, mungbean (1,065 bp)은 숙주 나물에서 mRNA 산물을 얻어서 cDNA로 전환한 다음 코딩 영역을 얻을 수 있었다. T5 (828 bp), DSB (267 bp)는 GenScript (Piscataway, NJ, USA)에서 합성하여서 사용하였다. GFP (714 bp) 서열은 생명공학연구원에서 개발한 pCTV-GFP 벡터에서 증폭하여서 사용하였고, hTdT (1,527 bp)는 GenScript에서 DNTT(NM_004088) ORF Clone을 주문하여서 증폭하였다. 증폭된 중합효소반응 산물은 깁슨 어셈블리 방법을 통해서 50도에 25분 인큐베이션을 통해서 SpCas9의 말단과 상단에 각각 삽입되었다.
name sequence size function
RecJF2 GGACAGCAAATGGGTCGCGGATCCGTGAAACAACAGATACAACTTCGT 45 RecJ full-length in front
RecJR2 TTGTCGACGGAGCTCGAATTCGGATCCAATTGGCCAGATATTGTCGATGATA 60 RecJ full-length in front
MungbeanF2 GGACAGCAAATGGGTCGCGGATCCATGCAAACGTTACAGATGAGT 41 Mungbean Nuclease in front
MungbeanR2 TTGTCGACGGAGCTCGAATTCGGATCCACCATTGTAAGAGATAGGTC 44 Mungbean Nuclease in front
T5exonuF2 GGACAGCAAATGGGTCGCGGATCCATGGCTTCCCGTCGTAATCTAATG 44 T5 exonuclease in front
T5exonuR6 TTGTCGACGGAGCTCGAATTCGGATCCTTGTTCTGCAATCTCCAAAATA 45 T5 exonuclease in front
SSBF2 GGACAGCAAATGGGTCGCGGATCCATGGCCAGCAGAGGCGTAAACAAGG 46 SSB isolation in front
SSBR2 TTGTCGACGGAGCTCGAATTCGGATCCACGACCACCCAGCATCTGCATGGT 49 SSB isolation in front
RecEF2 GGACAGCAAATGGGTCGCGGATCCATGAGCACAAAACCACTCTTCC 43 RecE in front
RecER2 TTGTCGACGGAGCTCGAATTCGGATCCGTCATTTGCATATTCCTTAGCCCA 50 RecE in front
DSBF2 GGACAGCAAATGGGTCGCGGATCCATGGCTAAAAAAGAAATGGTT 21 with DSBR1 in front
DSBR2 TTGTCGACGGAGCTCGAATTCGGATCCTTTAGAGAAAACTGTGT 37 with DSBF1 in front
pAGROF3 gtctcagctgggaggcgacgaaATGAGTAAAGGAGAAGAACTTTT 45 GFP in front
pAGROR3 TTGTCGACGGAGCTCGAATTCGGATCCAATAACCTCTCCTTCTTTTTC 45 GFP in front
HsTdTF3 GCGGCCGCACTCGACctcgagATGGATCCACCACGAGCGTCCCAC 45 HsTdT full length in front
HsTdTR3 TTGTCGACGGAGCTCGAATTCGGATCCGGCATTTCTTTCCCACGGTTCAATAT 52 HsTdT full length in front
RecJF1 GCGGCCGCACTCGACctgcagGTGAAACAACAGATACAACTTCGT 45 RecJ full-length in back
RecJR1 ccggcctttttcgtggccgccggccttttctgcagAATTGGCCAGATATTGTCGATGATA 60 RecJ full-length in back
RecJR2 TCAGTGGTGGTGGTGGTGGTGctttttcttttttgcctggccggcctttttcgtggccgc 60 RecJ full-length in back
MungbeanF1 CGGCCGCACTCGACctgcagATGCAAACGTTACAGATGAGT 41 Mungbean Nuclease in back
MungbeanR1 cgtggccgccggccttttctgcagACCATTGTAAGAGATAGGTC 44 Mungbean Nuclease in back
T5exonuF1 CGGCCGCACTCGACctgcagATGGCTTCCCGTCGTAATCTAATG 44 T5 exonuclease in back
T5exonuR5 gtggccgccggccttttctgcagTTGTTCTGCAATCTCCAAAATA 45 T5 exonuclease in back
SSBF1 GCGGCaGCACTCGACctgcagATGGCCAGCAGAGGCGTAAACAAGG 46 SSB isolation in back
SSBR1 tcgtggccgccggccttttctgcagACGACCACCCAGCATCTGCATGGT 49 SSB isolation in back
RecEF1 GCGGGCGCACTCGACctgcagATGAGCACAAAACCACTCTTCC 43 RecE in back
RecER1 ttcgtggccgccggccttttctgcagGTCATTTGCATATTCCTTAGCCCA 50 RecE in back
DSBF1 AGCGGCCGCACTCGACctcgagATGGCTAAAAAAGAAATGGTTGAATT 21 with DSBR1 in back
DSBR1 ctttttcgtggccgccggccttttctcgagTTTAGAGAAAACTGTGTCA 37 with DSBF1 in back
T5exoF5 GGTGGACAGCAAATGGGTCGCGGATCCATGGCTTCCCGTCGTAATCTAA 49 T5 full-length in back
T5exoR5 GCTTGTCGACGGAGCTCGAATTCGGATCCTTGTTCTGCAATCTCCAAA 48 T5 full-length in back
pAGROF2 gtctcagctgggaggcgacgaaATGAGTAAAGGAGAAGAACTTTT 45 GFP in back
pAGROR2 gcggaggggttggatcaaagtgaaAATAACCTCTCCTTCTTTTTC 45 GFP in back
HsTdTF2 GCGGCCGCACTCGACctcgagATGGATCCACCACGAGCGTCCCAC 45 HsTdT full length in back
HsTdTR2 ttcgtggccgccggccttttctcgagGGCATTTCTTTCCCACGGTTCAATAT 52 HsTdT full length in back
실시예 2. Cpf1 및 RecJ가 결합된 변형된 크리스퍼 연관 단백질 제조
실시예 2.1. FnCpf1 제조
이콜라이 단백질 발현 벡터 pET28a를 NotI 제한 효소를 사용하여서 FnCpf1 (3,902 bp)의 정방향 프라이머 (CGTCGACAAGCTTGCGGCCGCATGTCAATTTATCAAGAATT) 와 역방향 프라이머 (TGGTGGTGCTCGAGTGCGGCCGCTTgGTTATTCCTATTCTGCAC) 를 이용하여 pY002 (addgene, Watertown, MA, USA) 을 중합효소 연쇄반응으로 얻은 산물을 깁슨 어셈블리 킷트 (NEB, Ipswich, MA, USA) 방법으로 50도에 25분 인큐베이션으로 통하여 클로닝하였다. 서열 정보는 도42-도50에 명시하고 있다.
실시예 2.2. RecJ이 결합된 변형된 FnCpf1의 제조
해당 플라스미드를 지닌 대장균 세포를 카나마이신 항생제가 포함된 LB 배지에 접종하여 37℃에서 밤새 배양한다. 다음 날 배양액을 LB 20 mL에 접종하여 2~3시간 키운 뒤 이를 LB 1L에 넣어 OD600이 0.5~0.7이 될 때까지 37℃에서 배양한다. 1 mM IPTG를 넣고 18℃에서 20시간 배양한다. 다음 날 배양액을 4000 rpm에서 30분간 원심분리하여 상층액은 버리고 펠릿에 용해 완충액(lysis buffer, 5 mM imidazole, 20 mM Tris, 500 mM sodium chloride, 1 mM DTT, 1 mM PMSF, pH 8.0)을 넣어 현탁한다. 현탁액을 초음파-파쇄(sonication)한 후 15000 xG에서 1시간 동안 원심분리하여 상층액만을 분리하고 히스티딘 태깅 컬럼을 사용한 FPLC에 주입하여 단백질을 분리한다. 분리한 단백질 분획을 다시 탈염(desalting) 컬럼을 사용한 FPLC에 주입하여 단백질을 분리 후 해당 분획을 농축한다. 농축된 단백질을 Bradford assay로 정량한 뒤 -80℃에 분주하여 보관한다. 변형된 FnCpf1의 구체예를 도 12에 나타내었다.
실험예 1. 변형된 Cas9 단백질의 절단 효율 확인 : 선형 dsDNA
단백질과 gRNA는 몰농도 비율이 1:1.2가 되도록 준비하였다. 반응 부피 20 μL로 하여 단백질 25 nM에 gRNA 30 nM을 섞은 후 20분 상온에 두고 RNP가 형성되도록 하였다. SpCas9-BPNLS의 분자량을 기준으로 각 단백질의 분자량을 반영하여 넣어줄 단백질의 양을 계산하였다. SpCas9-BPNLS의 분자량은 166.580 kDa이며 25 nM에 해당하는 양은 83.5 g이다. gRNA는 CCR5 유전자를 타겟으로하며 약 33,500 g/mol의 농도를 가지고 있고 30 nM에 해당하는 양은 20 ng이다. 타겟 유전자인 CCR5 주형의 크기는 약 1.4 kb이다.
1.5 kb 크기의 타겟 유전자인 CCR5 주형 DNA 500 ng을 RNP 복합체와 섞은 후 37℃에서 1시간 반응시켰다. 2% agarose gel에 전기영동한 후 겔 이미지를 수득하였다. 이를 이용하여 주형 DNA와 RNP 복합체가 작용하고 남은 주형 DNA의 비율을 계산하여, 단백질의 뉴클레아제 활성 효율을 수득하였다. 절단 후에는 약 600 bp 정도의 밴드가 나타났다(도 1).
실험예 2. 변형된 Cas9 단백질의 절단 효율 확인 : 원형 dsDNA
TAAGG 또는 AAAC 오버행을 가지는 목표서열 20개의 정방향과 역방향 단일 가닥 올리고뉴클레오타이드 2개로 이루어져 있으며, 95℃에서 5분 후, 55℃에서 10분의 인큐베이션하여 올리고뉴클레오타이드가 이차구조를 가지지 않도록 하면서 20개의 정방향과 역방향의 올리고뉴클레오타이드의 단일 가닥을 상보적으로 결합시켜서 20개 베이스페어를 가지는 이중 가닥의 선형으로 정렬시켰다. 이중 가닥의 올리고뉴클레오타이드는 Cas9에 대한 gRNA 지지체를 포함하는 클로닝 벡터의 2개 BsaI 사이트 (A↓TAGGTGAGACCGCAGGTCTCG↓GTTTT)에 클로닝하였다.
결과적으로, sgRNA는 pGEM_447 vector(서열번호 102)를 목표로하는 20개의 목표서열 (목표서열; AAGCCCGGCCGAAATGACCA) 이 T7 프로모터와 sgRNA 지지체 사이에 위치하는 클로닝 벡터에서 T7 프로모터를 주형으로 하는 정방향 프라이머 (정방향 프라이머 서열; ATTCTAATACGACTCACTATAGG)와 sgRNA 지지체의 마지막 20 개의 서열을 주형으로 하는 역방향 프라이머 (역방향 프라이머 서열; GCACCGACTCGGTGCCACTT)를 사용하여서 중합효소연쇄방반응으로 얻어지는 PCR 산물을 MEGAshortscript™ High Yield Transcription Kit(Invitrogen, Grand Island, NY)를 이용하여 RNA 산물인 sgRNA 산물을 얻었다. RNA 산물은 DNase I (NEB, Ipswich, MA)을 이용하여 dsDNA 주형을 제거하기 위해 절단하였고, MEGAclear™ 전사 Clean-Up kit (Invitrogen, Grand Island, NY)를 이용하여 정제하였다.
절단 시험 전의 Cas9-sgRNA 복합체는 NEB2.1 완충액(NEB, Ipswich, MA)을 이용하여 37℃에서 10분간 배양하였다. 절단 시험은 20 μL의 반응 내 0.2-1.6 μg Cas9 단백질, 500 ng sgRNA 및 500 ng dsDNA 기질로 구성되어 있으며, 37℃에서 0 내지 180분 동안 수행되었다. 절단된 dsDNA는 40분 동안 DNA 전기 영동으로 확인하였다(도 2).
실험예 3. 시간에 따른 변형된 Cas9 단백질의 편집 효율 확인
HEK293T 세포를 10% 태아 소혈청인 FBS와 P/S가 함유된 DMEM 배지에서 37℃, 5% CO2 배양기에서 유지시켰다. 트랜스펙션 전날 150,000 세포를 24 well 플레이트로 옮겼다. Cas9 변이 단백질을 세포에 트렌스펙션 시키기 위해 각각 정제된 Cas9 변이체 단백질을 25 ㎕ Opti-MEM 혈청 환원 배지에 첨가한 후 120 ng의 가이드 RNA를 첨가하였다. Cas9 단백질에 대한 가이드 RNA의 몰비는 거의 1.2 : 1의 비율로 유지하였다. 혼합물을 부드럽게 두드린 후 잘 혼합하여 10분 동안 실온에서 배양하였다. 다른 튜브에는 2 ㎕의 lipofectamine 3000을 25 ㎕ Opti-MEM 혈청 환원 배지에 넣어주었고, 희석된 25 ㎕의 시약은 가이드 RNA와 Cas9 단백질 복합체에 넣고 15분 동안 상온에서 배양한 뒤 총 50 ㎕의 혼합물을 세포에 넣어주었다.
게놈 DNA 추출은 제조사의 지시에 따라 PureLink Genomic DNA 키트를 사용하여 진행하였다. 간단하게 세포를 PBS로 1회 세척하고 트립신으로 37℃에서 5분 동안 인큐베이션하였다. 그 후. 세포를 얻기 위해 1.2 ml의 성장 배지를 세포에 첨가 한 후 상온, 250xg에서 5분 동안 스핀다운시켰다. 게놈상 인간 DHCR7의 유전자 증폭을 위해, 두 개의 상이한 PCR 프라이머 쌍이 사용되었다. 전방 프라이머 1: 5'-CAGTAGAGCAGGCATGTTGAGT-3'와 역방향 프라이머 1 : 5'-GTGAAGGTGTATCAAACGCTGA-3’를 사용하였고, 이는 T7E1 분석 후 크기가 각각 427 및 205의 절단된 2개의 DNA 밴드를 나타낸다. 반면, 다음의 다른 프라이머 쌍을 사용할 때는 절단된 단일 DNA 단편만 생성된다 : 정방향 프라이머 2 : 5'-GGGAAACCACTGGCCTTGG-3'; 역방향 프라이머 2 : 5'-GAGCCAGGATCCATGTCCCA-3'.
게놈 DNA를 증폭 후 Wizard® SV 젤 및 PCR 클린업 시스템을 사용하여 정제하였다. 200 ng의 DNA를 PCR 튜브에 넣고 NEB 완충액 2번을 1 ㎕와 증류수 9 ㎕를 넣었다. 이후, 혼합물을 다음 조건을 사용하여 PCR 기계에서 재구성하였다. 98℃에서 5분 동안 변성 후 -2℃/초로 85℃ 온도를 낮추고 -0.1℃/초로 25℃까지 온도를 낮춘다. 이후 이형이중가닥의 변성을 막기 위해 4℃로 유지시킨다. 1 ㎕의 T7E1 엔도뉴클레아제를 혼합물에 첨가하고 37℃에서 60분 동안 배양한다.
유전자 변형의 분석을 위해, 2% 겔을 사용하여 이미지를 얻고, Alphaimager 2200 이미지 분석 소프트웨어를 분석에 사용하였다. 유전자 변형에 대한 비율은 1- [(1-fraction cleaved) 1/2]식을 이용하여 구했다. 그 결과를 도 3에 나타내었다.
실험예 4. 인간 유전자 CCR5 및 DHCR7에 대한 SpyCas9, SpyCas9-RecJ 및 SpyCas9-T5의 HDR(Homology Dependent Repair) 효율 분석
HEK293T 세포를 10% 태아 소혈청인 FBS와 P/S가 함유된 DMEM 배지에서 37℃, 5% CO2 배양기에서 유지시켰다. HEK293T 세포를 3x106 밀도로 10 cm 배양접시에 배양하였다. 세포의 증식 정도는 70% 이하로 유지하였다. 이후 형질전환 16시간 전 200 ng/ml의 노코다졸(Sigma)을 세포에 처리하였고, 동기화된 세포를 유전자 도입 실험에 사용하였다.
외부 유전자인 ssODN(single-stranded oligodeoxynucleotides)을 도입시키기 위해 2x105의 HEK293T 세포를 형질전환 실험에 이용하였다. 각각 33 pmole의 SpCas9, SpCas9-RecJ, SpCas9-T5 단백질과 66 pmole의 CCR5 sgRNA를 이용하였다. 도입 유전자로는 ssODN 20 pmole을 사용하였다. 형질전환을 위해 Lonza사의 4D-Nucleofector 기기를 이용한 전기천공법을 사용하였다. 24시간 후 invitrogen사의 PureLink™ Genomic DNA Mini Kit를 사용하여 genomic DNA를 추출하였다.
편집된 부분을 가지고 있는 gDNA와 그렇지 않은 gDNA 샘플을 이용하여 표적 부위를 증폭하였다. 이후 일루미나의 프로토콜대로 PCR과 정제 과정을 거친 후 MiniSeq 장비를 이용하여 표적 부위에 대한 딥 시퀀싱(deep sequencing) 분석을 진행하였다. HDR 분석 결과는 도 4에 나타내었다. 이때, 분석에 이용된 프라이서 서열은 표 3에 기재된 바와 같다.
Proteins Genes Adapter primer sequences (5’-3’)
SpyCas9 CCR5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAGGGCAACTAAATACATTCT (서열번호 81)
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAACACCAGTGAGTAGAGCGG (서열번호 82)
DHCR7 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGTTTGAGCAACAGTTCTCC (서열번호 83)
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGACGATGTCCACCACAG (서열번호 84)
FnCpf1 CCR5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGTATTTCTGTTCAGATCAC (서열번호 85)
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCCATCAATTATAGAAAGCC (서열번호 86)
DNMT1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTGCACACAGCAGGCCTTTG (서열번호 87)
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCCAATAAGTGGCAGAGTGC (서열번호 88)
실험예 5. SpCas9 및 SpCas9-RecJ에 의한 오프-타겟 효과 확인
SpCas9과 SpCas9-RecJ 단백질을 이용하여 인간 CCR5 유전자를 표적으로 했을 때 표적 부분 외 다른 부분에 대한 영향을 알아보기 위해 스페이서(spacer) 서열 부분이 유사한 다음의 4가지 유전자를 선택하였고 서열은 하기 표 4와 같다.
Target Gene Spacer sequence
On CCR5 TGACATCAATTATTATACAT (서열번호 89)
off#1 ADCY5 TGACATCAATTATTATAgAT (서열번호 90)
off#2 KCNJ6 TGACATCAcTTATTATgCAT (서열번호 91)
off#3 CNTNAP2 TGACATaAATTATTcTACAT (서열번호 92)
off#4 Chr. 5 N/A TGAaATCAATTATcATAgAT (서열번호 93)
Mock, SpCas9, SpCas9-RecJ를 처리한 gDNA로부터 표적 외 부분 ADCY5, KCNJ6, CNTNAP2, Chr.5 N/A에 대한 각각의 프라이머를 이용하여 증폭시켰다. 이후 정제 과정과 제조사의 지시에 따라 시퀀싱 라이브러리를 제조하였고 일루미나 MiniSeq 장비를 이용하여 표적 부위에 대한 딥 시퀀싱 분석을 진행하였다. 그 결과를 도 5 및 도 6에 나타내었다. 이때, 분석에 이용된 프라이서 서열은 표 5에 기재된 바와 같다.
Gene Target specific primer sequences Adapter primer sequences
ADCY5 GTCCCATGACAGGCGTGTAT TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAAGAGTGTAGAGGAGGACAG
GCTCCCACCTTAGTGCTCTG GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAAAAGGAGATGCTTGGCAC
KCNJ6 TGGAGCCATTGGTTTGCATC TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAGACCAAAAAACCTCATGGAT
GCCTGGCCAAGTTTCAGTTA GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTTGAGGAAGACAAGTCATGT
CNTNAP2 TGCAGTGCAGACTCTTTCCA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGCAGACTCTTTCCATACTGT
AAGGACACAGGGCAACTGAA GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAAATCAAATTTCATGTGTCCA
Chr. 5 N/A TGTGGAACGAGTGGTGACAG TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCAGAAGTTCCTGAAATTATCC
GACCAAACCACATTCTTCTCAC GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACATGAGAGATAGTGATATGT
실시예 6. Cas9 및 Cas9-RecJ의 PD-1 및 CCR5에 대한 온-타겟 효과 확인
인간 PD-1 및 CCR5의 동일한 유전자 자리에 대한 Cas9 및 Cas9-RecJ 아포단백질(apoproteins)을 지질 매개 형질감염(lipid mediated transfection) 방법으로 HEK293 세포에 형질감염 시켰다. 게놈 DNA 추출을 위해 0시간 내지 48시간의 다른 시점에서 세포를 수득하였다. 이때, O 시간은 대조군이다. CCR5를 확인하기 위한 544 bp 밴드 및 PD-1을 확인하기 위한 524 bp 밴드를 생성하기 위하여 PCR을 수행하였다. T7E1 엔도뉴클레아제 어세이를 수행할 경우, CCR5에서는 274 bp 및 271 bp의 작은 단편이 나타나고, PD-1에서는 319 bp 및 205 bp의 작은 단편이 나타났다. 이러한 것을 통하여 삽입-결실율(indel)을 측정할 수 있었다. 이에 대한 결과를 도 7에 나타내었다.
실험예 7. Cas9 및 Cas9-RecJ에 의한 오프-타겟 효과 확인
인간 CCR5 sgRNA와 Cas9 및 Cas9-RecJ를 이용시 5개의 오프 타겟팅된 유전자를 나타낸 것이다. ADCY5 641bps; KCNJ6, 566 bps; CNTPNA2, 300 bps; Chr.5, 605bps. 오프-타겟 효과를 도 8에 나타내었다.
또한, 5개의 오프-타겟된 유전자의 In vitro 절단 에세이를 분석하였다. 예상된 절단된 DNA의 크기는 ADCY5에서 377 bp 및 264 bp이고; KCNJ6에서 352 bp 및 214 bp이며; CNTPNA2에서 183 bp 및 117 bp이며; Ch. 5에서 355 bp 및 250 bp이다. 절단은 오프 타겟팅 효과를 확인하기에는 명확하지 않았다. In vitro 에세이를 근거로, SpCas9-RecJ의 오프 타겟팅 정도는 SpCas9과 차이가 없었다. 이에 대한 결과를 도 9에 나타내었다.
실험예 8. 변형된 FnCpf1 단백질의 절단 효율 확인
단백질과 gRNA는 몰농도 비율이 1:1.2가 되도록 준비하였다. 반응 부피 20 μL로 하여 단백질 50 nM에 crRNA 60 nM을 섞은 후 20분 상온에 두고 RNP가 형성되도록 하였다. crRNA는 CCR5 유전자를 타겟으로하며, 타겟 유전자인 CCR5 주형의 크기는 약 1.4 kb이다.
1.5 kb 크기의 타겟 유전자인 CCR5 주형 DNA 500 ng을 RNP 복합체와 섞은 후 37℃에서 1시간 반응시킨다. 2% agarose gel에 전기영동한 후 겔 이미지를 수득하였다. 이를 이용하여 주형 DNA와 RNP 복합체가 작용하고 남은 주형 DNA의 비율을 계산하여, 단백질의 뉴클레아제 활성 효율을 얻었다.
FnCpf1, FnCpf1-RecJ, 또는 SpCas9에 의한 선형 dsDNA가 절단됨을 확인하기 위한 실험을 수행하였다. 선형 dsDNA는 PCR을 통해 증폭되고 정제되었다. Cas9 sgRNA 및 Cpf1 sgRNA 사이트는 서로 근접한 곳에 위치한다. SpCas9 및 FnCpf1 단백질에 사용된 sgRNA는 각각 5’-NGG-3’PAM(protospacer adjacent motif)이 상단(upstream)에는 존재하며, 5’-TTTN-3’PAM이 하단(downstream)에 존재한다. 절단되기 전의 온전한 dsDNA는 1,200 bp 위치에서 단일 밴드로 관찰되었다. 상기 dsDNA는 절단된 후 두개 모두 600 bp 밴드에 위치하였다. 그러나, FnCpf1-RecJ를 이용할 경우, RecJ의 엑소뉴클레아제 활성에 의해 상기 절단된 밴드가 사라짐을 확인하였다(도 10).
FnCpf1 또는 FnCpf1-RecJ 엑소뉴클레아제에 의한 선형 dsDNA In vitro 절단 에세이를 수행하였다. 선형 dsDNA는 FnCpf1 또는 FnCpf1-RecJ에 의해 절단되었다. 선형 dsDNA는 플라스미드로부터 PCR을 이용하여 증폭되고 분리되었다. sgRNA는 FnCpf1 단백질을 타겟 사이트로 위치시키기 위해 사용되었으며, 5’-TTTN-3’PAM이 하단(downstream)에 존재한다. 절단되기 전의 온전한 dsDNA는 1,200 bp 위치에서 단일 밴드로 관찰되었다. 상기 dsDNA는 절단되면 두개 모두 600 bp 밴드에 위치하였다. 그러나, FnCpf1-RecJ를 이용할 경우, RecJ의 엑소뉴클레아제 활성에 의해 상기 절단된 밴드가 사라졌다. 반응 시간을 증가시킴에 따라, FnCpf1 및 FnCpf1-RecJ 반응 혼합물에서 원래의 밴드가 흐려짐을 확인하였다(도 11).
실험예 9. 변형된 Cas9 단백질의 편집 효율 확인
HEK293T 세포를 10% 태아 소혈청인 FBS와 P/S가 함유된 DMEM 배지에서 37℃, 5% CO2 배양기에서 유지시켰다. 녹인 실험의 효율을 높이기 위해 HEK293T 세포를 3x106 밀도로 10 cm 배양접시에 배양한 후 형질전환 16시간 전 200 ng/ml의 노코다졸(Sigma)을 세포에 처리하였고, 동기화된 세포를 유전자 도입 실험에 사용하였다. 사용한 외부 유전자인 ssODN(single-stranded oligodeoxynucleotides)의 서열은 다음과 같다:
CCR5: 5' TGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTAT
ACATATGCATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCC
DHCR7: 5' ATGGGCCCCAGTGTGACTGCCTGCATCCGTCCTCGCAGGGAGGTGGACTGGT
TTTGAATTCCACTGGCGAGCGTCATCTTCCTACTGCTGTTCGCCCCCTTCATCG
외부 유전자인 ssODN(single-stranded oligodeoxynucleotides)을 도입시키기 위해 2x105의 HEK293T 세포를 형질전환 실험에 이용하였다. 각각 33 pmole의 단백질과 66 pmole의 sgRNA를 이용하였다. 도입 유전자로는 ssODN 20 pmole을 사용하였다. 형질전환을 위해 Lonza사의 4D-Nucleofector 기기를 이용한 전기천공법을 사용하였다. 24시간 후 invitrogen사의 PureLink™ Genomic DNA Mini Kit를 사용하여 genomic DNA를 추출하였다.
편집된 부분을 가지고 있는 gDNA와 그렇지 않은 gDNA 샘플을 이용하여 표적 부위를 증폭하였다. 이후 일루미나의 프로토콜대로 PCR과 정제 과정을 거친 후 MiniSeq 장비를 이용하여 표적 부위에 대한 딥 시퀀싱(deep sequencing) 분석을 진행하였다. 시퀀싱을 위해 사용된 어댑터 프라이머 서열은 다음과 같다.
CCR5 (F): 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAGGGCAACTAAATACATTCT
CCR5 (R): 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAACACCAGTGAGTAGAGCGG
DHCR7 (F): 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGTTTGAGCAACAGTTCTCC
DHCR7 (R): 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGACGATGTCCACCACAG
SpyCas9과 SpyCas9의 C 말단에 다양한 단백질이 결합된 변형된 SpyCas9의 CCR5 및 DHCR7에 대한 유전자 편집 효율을 도 15 및 도 16에 나타내었다. SpyCas9의 C 말단에 GFP, hTdT, 및 엑소뉴클레아제로 알려진 RecJ, RecE, lambda, mungbean, and T5가 결합된 형태이다. 도면에서 나타낸 바와 같이 Knock-out 및 Knock-in 효율이 서로 상이하였다.
사용한 변형된 Cas9-RecJ의 구조 및 이의 Knock-out 및 Knock-in 효율을 도 17 및 도 18에 나타내었다. 도 17A는 SpyCas9-RecJ의 구조를 나타낸 것이다. 도 17B는 대조군, SpyCas9 및 SpyCas9-RecJ 간의 유전자 편집 효율을 나타낸 것이다. 도 17C는 CCR5 및 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 SpyCas9-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 18A는 SpyCas9-RecJ의 구조를 나타낸 것이다. 도 18B는 대조군, SpyCas9 및 SpyCas9-RecJ 간의 유전자 편집 효율을 나타낸 것이다. 도 18C는 CCR5 및 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 SpyCas9-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
또한, SpyCas9과 SpyCas9의 N 말단 또는 N/C 양말단에 다양한 단백질이 결합된 변형된 SpyCas9의 DHCR7에 대한 유전자 편집 효율을 도 19는 및 20에 나타내었다. 변형된 SpyCas9은 SpyCas9의 N 말단에 RecJ, RecE, GFP, 단일 가닥 DNA 결합 단백질(SSB) 및 이중 가락 DNA 결합 단백질(DSB)이 결합되어 있다. 또한, 변형된 SpyCas9은 SpyCas9의 C 말단에 RecJ가 결합되어 있고, N 말단에 SSB 또는 DSB이 결합되어 있다. 도면에서 나타낸 바와 같이 Knock-out 및 Knock-in 효율이 서로 상이하였다.
도 21A는 RecJ-SpyCas9의 구조를 나타낸 것이다. 도 21B는 대조군, SpyCas9 및 RecJ-SpyCas9 간의 유전자 편집 효율을 나타낸 것이다. 도 21C는 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 RecJ-SpyCas9의 편집 효율 변화를 백분율로 나타낸 것이다.
도 22A는 RecJ-SpyCas9의 구조를 나타낸 것이다. 도 22B는 대조군, SpyCas9 및 RecJ-SpyCas9 간의 유전자 편집 효율을 나타낸 것이다. 도 22C는 CCR5 및 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 RecJ-SpyCas9의 편집 효율 변화를 백분율로 나타낸 것이다.
도 23A는 SSB-SpyCas9-RecJ의 구조를 나타낸 것이다. 도 23B는 대조군, SpyCas9 및 SSB-SpyCas9-RecJ 간의 유전자 편집 효율을 나타낸 것이다. 도 23C는 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 SSB-SpyCas9-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 24A는 DSB-SpyCas9-RecJ의 구조를 나타낸 것이다. 도 24B는 대조군, SpyCas9 및 DSB-SpyCas9-RecJ 간의 유전자 편집 효율을 나타낸 것이다. 도 24C는 DHCR7 유전자의 편집 정도를 확인하여, SpyCas9 대비 DSB-SpyCas9-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
실험예 10. 변형된 Cpf1 단백질의 편집 효율 확인
HEK293T 세포를 10% 태아 소혈청인 FBS와 P/S가 함유된 DMEM 배지에서 37℃, 5% CO2 배양기에서 유지시켰다. 녹인 실험의 효율을 높이기 위해 HEK293T 세포를 3x106 밀도로 10 cm 배양접시에 배양한 후 형질전환 16시간 전 200 ng/ml의 노코다졸(Sigma)을 세포에 처리하였고, 동기화된 세포를 유전자 도입 실험에 사용하였다. 사용한 외부 유전자인 ssODN(single-stranded oligodeoxynucleotides)의 서열은 다음과 같다.
CCR5: 5' AATTCTCTGAGGCTTTCTTTTAAATATACATAAGGAACTTTCGGAGTGAAGGG
AGAGTTTCATATGGTCAATAACTTGATGCATGTGAAGGGGAGATAAAAAGGTT
DNMT1: 5' TGGCCCTGGGGCCGTTTCCCTCACTCCTGCTCGGTGAATTTGGCTCAGCAGGC
ACCTGCCGAATTCTCAGCTGCTCACTTGAGCCTCTGGGTCTAGAACCCTCTGG
외부 유전자인 ssODN(single-stranded oligodeoxynucleotides)을 도입시키기 위해 2x105의 HEK293T 세포를 형질전환 실험에 이용하였다. 각각 33 pmole의 단백질과 66 pmole의 sgRNA를 이용하였다. 도입 유전자로는 ssODN 20 pmole을 사용하였다. 형질전환을 위해 Lonza사의 4D-Nucleofector 기기를 이용한 전기천공법을 사용하였다. 24시간 후 invitrogen사의 PureLink™ Genomic DNA Mini Kit를 사용하여 genomic DNA를 추출하였다.
편집된 부분을 가지고 있는 gDNA와 그렇지 않은 gDNA 샘플을 이용하여 표적 부위를 증폭하였다. 이후 일루미나의 프로토콜대로 PCR과 정제 과정을 거친 후 MiniSeq 장비를 이용하여 표적 부위에 대한 딥 시퀀싱(deep sequencing) 분석을 진행하였다. 시퀀싱을 위해 사용된 어댑터 프라이머 서열은 다음과 같다.
CCR5 (F): 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGTATTTCTGTTCAGATCAC
CCR5 (R): 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCCATCAATTATAGAAAGCC
DNMT1 (F): 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTGCACACAGCAGGCCTTTG
DNMT1 (R): 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCCAATAAGTGGCAGAGTGC
FnCpf1과 FnCpf1의 C 말단에 다양한 단백질이 결합된 변형된 FnCpf1의 CCR5 및 DNMT1 유전자에 대한 유전자 편집 효율을 도 25 및 도 26에 나타내었다. FnCpf1의 C 말단에 GFP, hTdT, 및 엑소뉴클레아제로 알려진 RecJ, RecE, lambda, mungbean, 및 T5이 결합된 형태이다. 도면에서 나타낸 바와 같이 Knock-out 및 Knock-in 효율이 서로 상이하다.
도 27A는 FnCpf1-RecJ의 구조를 나타낸 것이다. 도 27B는 대조군, FnCpf1 및 FnCpf1-RecJ 간의 유전자 편집 효율을 나타낸 것이다. 도 27C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 FnCpf1-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 28A는 FnCpf1-RecJ의 구조를 나타낸 것이다. 도 28B는 대조군, FnCpf1 및 FnCpf1-RecJ 간의 유전자 편집 효율을 나타낸 것이다. 도 28C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 FnCpf1-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
FnCpf1과 FnCpf1의 N 말단 또는 N/C 양말단에 다양한 단백질이 결합된 변형된 FnCpf1의 유전자 편집 효율을 도 29 및 도 30에 나타내었다. 변형된 FnCpf1은 FnCpf1의 N 말단에 RecJ, RecE, GFP, 단일 가닥 DNA 결합 단백질(single stranded DNA binding protein, SSB) 및 이중 가락 DNA 결합 단백질(double stranded DNA binding protein, DSB)이 결합되어 있다. 또한, 변형된 FnCpf1은 FnCpf1의 C 말단에 RecJ가 결합되어 있고, N 말단에 SSB이 결합되어 있다. 도면에서 나타낸 바와 같이 Knock-out 및 Knock-in 효율이 서로 상이하였다.
도 31A는 RecJ-FnCpf1의 구조를 나타낸 것이다. 도 31B는 대조군, RecJ-FnCpf1 간의 유전자 편집 효율을 나타낸 것이다. 도 31C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 RecJ-FnCpf1의 편집 효율 변화를 백분율로 나타낸 것이다.
도 32A는 RecJ-FnCpf1의 구조를 나타낸 것이다. 도 32B는 대조군, RecJ-FnCpf1 간의 유전자 편집 효율을 나타낸 것이다. 도 32C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 RecJ-FnCpf1의 편집 효율 변화를 백분율로 나타낸 것이다.
도 33A는 SSB-FnCpf1-RecJ의 구조를 나타낸 것이다. 도 33B는 대조군, FnCpf1 및 SSB-FnCpf1-RecJ 간의 Knock-out 유전자 편집 효율을 나타낸 것이다. 도 33C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 SSB-FnCpf1-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
도 34A는 SSB-FnCpf1-RecJ의 구조를 나타낸 것이다. 도 34B는 대조군, FnCpf1 및 SSB-FnCpf1-RecJ 간의 Knock-in 유전자 편집 효율을 나타낸 것이다. 도 34C는 CCR5 및 DNMT1 유전자의 편집 정도를 확인하여, FnCpf1 대비 SSB-FnCpf1-RecJ의 편집 효율 변화를 백분율로 나타낸 것이다.
실험예 11. 변형된 크리스퍼 연관 단백질의 편집 효율에 미치는 조건 확인
ssODN 및 노코다졸(Nocodazole)의 존재 유무에 따른 변형된 Cas9 단백질의 활성 변화를 확인하기 위한 실험을 수행하였다.
크리스퍼 플러스 매개 녹아웃을 확인하기 위하여, 노코다졸이 처리되지 않은 세포를 사용하였다. 녹인의 효율을 증가시키기 위하여, HEK293T 세포를 10-cm 배양 접시에 3 x 106 세포 밀도로 접종하였다. 상기 세포를 노코다졸(200 ng/ml)로 16시간 배양한 후, 전기 영동을 수행하였다. 하기 표 6에 기재한 ssODN 주형을 녹인을 위해 사용하였다:
크리스퍼 플러스 단백질을 세포에 트랜스펙션하기 전에, 분리정제된 SpyCas9 또는 FnCpf1 변이체 단백질(33 pmol) 및 sgRNA 또는 crRNA(66 pmol)을 상온에서 20분간 인큐베이션시켜 RNP를 복합체를 형성하였다. HDR 주형(20 pmol ssODN 또는 2 pmol dsDNA)을 RNP 복합체에 첨가하였다. Lonza를 이용하여 HEK293T 세포에 뉴클레오펙션(Nucleofection)을 수행하였다. 그 후, 각각의 뉴클레오펙션은 20 μl의 뉴클레오펙션 시약에 있는 2 x 105 세포와 RNP:DNA 10 μl을 혼합였다.
Proteins Genes ssODN donor sequences (5’-3’)
SpyCas9 CCR5 TGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTAT (서열번호 94)
ACATATGCATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCC (서열번호 95)
DHCR7 ATGGGCCCCAGTGTGACTGCCTGCATCCGTCCTCGCAGGGAGGTGGACTGGT (서열번호 96)
TTTGAATTCCACTGGCGAGCGTCATCTTCCTACTGCTGTTCGCCCCCTTCATCG (서열번호 97)
FnCpf1 CCR5 AATTCTCTGAGGCTTTCTTTTAAATATACATAAGGAACTTTCGGAGTGAAGGG (서열번호 98)
AGAGTTTCATATGGTCAATAACTTGATGCATGTGAAGGGGAGATAAAAAGGTT (서열번호 99)
DNMT1 TGGCCCTGGGGCCGTTTCCCTCACTCCTGCTCGGTGAATTTGGCTCAGCAGGC (서열번호 100)
ACCTGCCGAATTCTCAGCTGCTCACTTGAGCCTCTGGGTCTAGAACCCTCTGG (서열번호 101)
실험예 11.1. 변형된 Cas9의 조건 확인
도 35a 및 도 36a는 ssODN 및 노코다졸이 처리되지 않았을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 35b 및 도 36b는 ssODN 및 노코다졸이 처리되었을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 35c 및 도 36c는 ssODN 및 노코다졸의 존재 유무에 따른 효율을 비교한 것이다. ssODN 및 노코다졸은 Knock-out의 경우, SpyCas9 및 변형된 SpyCas9의 효율에는 큰 영향이 없음을 확인하였다.
실험예 11.2. 변형된 Cpf1의 조건 확인
크리스퍼 플러스 매개 녹아웃을 확인하기 위하여, 노코다졸이 처리되지 않은 세포를 사용하였다. 녹인의 효율을 증가시키기 위하여, HEK293T 세포를 10-cm 배양 접시에 3 x 106 세포 밀도로 접종하였다. 상기 세포를 노코다졸(200 ng/ml)로 16시간 배양한 후, 전기 영동을 수행하였다. 상기 표 6에 기재한 ssODN 주형을 녹인을 위해 사용하였다:
크리스퍼 플러스 단백질을 세포에 트랜스펙션하기 전에, 분리정제된 SpyCas9 또는 FnCpf1 변이체 단백질(33 pmol) 및 sgRNA 또는 crRNA(66 pmol)을 상온에서 20분간 인큐베이션시켜 RNP를 복합체를 형성하였다. HDR 주형(20 pmol ssODN 또는 2 pmol dsDNA)을 RNP 복합체에 첨가하였다. Lonza를 이용하여 HEK293T 세포에 뉴클레오펙션(Nucleofection)을 수행하였다. 그 후, 각각의 뉴클레오펙션은 20 μl의 뉴클레오펙션 시약에 있는 2 x 105 세포와 RNP:DNA 10 μl을 혼합하였다.
FnCpf1 및 FnCpf1의 C 말단에 단백질이 결합된 변형된 FnCpf1에 유도되는 비상동적인 말단 연결에 있어서, ssODN 및 노코다졸(nocodazole)이 미치는 영향을 확인한 것을 도 37에 나타내었다. 도 37a는 ssODN 및 노코다졸이 처리되지 않았을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 37b는 ssODN 및 노코다졸이 처리되었을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 37c는 ssODN 및 노코다졸의 존재 유무에 따른 효율을 비교한 것이다. ssODN 및 노코다졸은 Knock-out의 경우, FnCpf1 및 변형된 FnCpf1의 효율을 향상 시킨다는 점을 확인하였다.
FnCpf1 및 FnCpf1의 N 말단 또는 N/C 양말단에 단백질이 결합된 변형된 FnCpf1에 유도되는 비상동적인 말단 연결에 있어서, ssODN 및 노코다졸이 미치는 영향을 도 38에 나타내었다. 도 38a는 ssODN 및 노코다졸이 처리되지 않았을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 38b는 ssODN 및 노코다졸이 처리되었을 때의 비상동적인 말단 연결 효율을 나타낸다. 도 38c는 ssODN 및 노코다졸의 존재 유무에 따른 효율을 비교한 것이다. ssODN 및 노코다졸은 Knock-out의 경우, FnCpf1 및 변형된 FnCpf1의 효율에는 큰 영향이 없음을 확인하였다.
실험예 12. 크리스퍼 플러스 단백질의 유전자 편집 효능 검증
제조예 1. 식물 유전자 편집을 위한 용액 조성물 제조
종자 파종 배지의 조성은 다음과 같다.
종자 파종 배지 1 L
1/2 MS Powder 2.2 g
Sucrose 10 g
Plant agar 7 g
pH 5.7 (1N NaOH 또는 1N HCl 로 측정함)
Enzyme solution의 조성은 다음과 같다. 단, 사용 직전에 만들어 사용함을 원칙으로 한다.
Enzyme Solution 20 mL
1.0% Cellulase R10 200 mg
0.5% Macerozyme R10 100 mg
0.4 M Mannitol 10 mL (0.8 M Mannitol Stock solution)
20 mM MES, pH 5.7 4 mL (100 mM MES Stock solution, pH 5.7)
20 mM KCl 200 μL (2 M KCl Stock solution)
위 시약들 조합한 후 60도에서 10분간 배양한 후 아래 시약을 조합한다.
10 mM CaCl2·2H2O 200 μL (1 M CaCl2·2H2O Stock solution)
0.1 % BSA 200 μL (10% BSA Stock solution)
MMG solution의 조성은 다음과 같다. 단, 사용 직전에 만들어 사용함을 원칙으로 한다.
MMG Solution 10 mL
0.4 M Mannitol 5 mL (0.8 M Mannitol Stock solution)
4 mM MES, pH 5.7 400 μL (0.1 M MES Stock solution, pH5.7)
15 mM MgCl2 150 μL (1 M MgCl2 Stock solution)
Nuclease-free water 4.45 mL
PEG4000 solution의 조성은 다음과 같다. 단, 사용 직전에 만드는 것을 원칙으로 한다.
PEG Solution 5 mL
0.2 M Mannitol 1.25 mL (0.8 M Mannitol Stock solution)
40%W/V PEG-4000 2 g (Polyethylene glycol 4000)
100 mM CaCl2·2H2O 500 μL (1 M CaCl2*?*2H2O Stock solution)
Nuclease-free water 1.5 mL
W5 solution의 조성은 다음과 같다.
W5 Solution 50 mL
154 mM NaCl 3.85 mL (2 M NaCl Stock solution)
125 mM CaCl2·2H2O 6.25 mL (1 M CaCl2·2H2O Stock solution)
5 mM KCI 125 μL (2 M KCl Stock solution)
2 mM MES, pH 5.7 500 μL (0.1 M MES Stock solution)
Nuclease-free water 39.275 mL
상기 Enzyme Solution, MMG Solution, PEG Solution, W5 Solution은 모두 0.45 μm Syringe-filter로 필터링하였다..
실험예 12.1. 실험 식물체의 준비
도 51에 나타낸 바와 같이 실험을 수행하였다. 구체적으로, 담배 종자 파종 및 원형질체 분리를 다음과 같이 수행하였다. 먼저, 담배 종자를 50% Clorox로 1분간 처리하였다. 그 후, Clean bench로 옮겨 멸균수로 5번 씻어, 필터페이퍼에서 20분간 건조하였다. 그 후, 멸균된 종자를 종자파종 배지에 올려 일주일간 배양한 후 마젠타박스에 옮겨 3주동안 키웠다. 배양 빛조건은 광배양 16시간, 암배양 8시간이며, 온도는 25℃ 내지 28℃에서 키웠다. 이때, 식물은 4~6주 자란 잎을 사용해야하며, 8주 이상 자란 잎은 사용하지 않음을 원칙으로 한다.
4~6주 자란 후, 담배 어린잎을 유리판에 올려 잎병과 잎끝부분을 자른 후 잎 안쪽부분만을 잘랐다. 0.5 mm 이하로 자르고, 자른 잎은 Enzyme solution 10 mL에 올려 상온에서 암 상태로 3~4시간 orbital shaker(50 rpm) 위에 올려서 배양하였다(도 52).
배양 후 W5 solution 10mL을 첨가한 후 조심스럽게 잘 섞어주었다. 그 후, Cell strainer(70 μm)를 이용해서 enzyme solution 내에 존재하는 원형질체를 걸러주었다. 그 후, 걸러진 원형질체를 100 g으로 6분 원심분리하였다. 상층액을 버리고 MMG solution을 첨가해서 조심스럽게 원형질체 pellet을 풀어 주었다. 그 후, Ice에서 10 ~ 30분 정도 방치하였다. 이 중 일부를 hemocytometer 이용해 현미경으로 원형질체의 수를 계수하였다. MMG solution을 더 첨가하여 원형질체의 농도가 2 X 106 cells/mL이 되도록 희석하였다.
실험예 12.2. 크리스퍼 플러스 단백질을 이용한 식물체 유전자 편집
2 mL e-tube에 sgRNA와 Cas9 protein 그리고 NEB buffer 3.1을 최종 volume 20 μL 가 되게 한 후 상온에서10분간 반응시켰다. Folding을 방지함으로써 DNA binding 효율을 높여 주기 위하여, gRNA는 사용 전에 95℃에서 3분, ice에서 3분 놓아준다.
SgRNA 2.5ug 5ug 10ug
Cas9 10ug 20ug 30ug
10x NEB buffer (3.1) 2 μL 2 μL 2 μL
Nuclease-free water Up to 20 μL
E-tube (2 mL)에 원형질체 200 μL (5 X 105 cells)와 PFT3 sgRNA 서열은 CAAGGGCTTCTAAAGCTTGCAAA과 PFT1 sgRNA 서열 GGCTTTTGAGAATTCTAATG 이 사용되었다. PFT3은 담배의 알파 1,3-푸코스트랜스퍼래이즈의 3번 도메인을 목표하고 PFT1은 담배의 알파 1,3-푸코스트랜스퍼래이즈의 4번 도메인을 목표하였다. Cas9 protein (volume 20 μL)을 넣고 잘 섞은 후, Clean bench에서 10분간 배양하였다. 앞에서 배양한 volume과 동일한 PEG solution 220 μL를 넣고 조심스럽게 섞어주었다. 상온에서 15분간 배양 후, W5 solution 840 μL을 첨가하여 잘 혼합하였다. 100 g에서 2분간 원심분리한 후, 상등액을 제거하였다. 형질전환한 원형질체에 W5 Solution 1000 μL을 넣어 재현탁하였다. 100g에서 2분간 원심분리한 후, 상등액을 제거하였다. W5 Solution 1000 μL을 넣어 Resuspension한 후, 6 또는 12 well plate에 넣어 30℃ 암배양기에서 48시간 배양 후 수거하였다. 수거된 세포는 DNA를 추출 후 타겟 시퀀스를 PCR 한 후 NGS 분석에 들어간다.
실험예 12.3. NGS 분석
분석장비(일루미나제품, MiniSeq (SY-420-1001), Miniseq mid output kit (300 cycles, FC-420-1004))를 이용하여 분석하였다. Amplicon primers 조건은 아래와 같으며, 타겟 specific 시퀀스 앞 뒤로 붙여서 제작하였다.
타겟 PFT1에 사용한 시퀀스 정보는 다음과 같다:
F:5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-ACTTCTCTTGGGCTGAGTATGA
R:5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-CAGTAAGTTTGGATATTTGAAA
타겟 PFT3에 사용한 시퀀스 정보는 다음과 같다:
F:5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-GCATTTGGTGTAGGTTTAGGCT
R:5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-AATTCTGAAAATCCAAGTCTAT
타겟 영역을 amplicon primer를 이용해 1차 PCR을 수행하였다. 생산된 PCR product는 반드시 clean-up을 수행하였다. 타겟 영역에 amplicon이 포함된 DNA에 index를 붙여서 library를 만들었다. 수행한 2차 PCR을 Bioanalyzer로 QC를 수행 후 시퀀싱을 수행하였다.
<110> g-flas life sciences <120> MODIFIED CRISPR ASSOCIATED PROTEIN COMPRISING CRISPR ASSOCIATED PROTEIN AND EXONUCLEASE AND USE THEREOF <130> FPD201811-0042c <160> 102 <170> KoPatentIn 3.0 <210> 1 <211> 577 <212> PRT <213> Artificial Sequence <220> <223> Escherichia coli str. K-12 substr. MG1655 <400> 1 Val Lys Gln Gln Ile Gln Leu Arg Arg Arg Glu Val Asp Glu Thr Ala 1 5 10 15 Asp Leu Pro Ala Glu Leu Pro Pro Leu Leu Arg Arg Leu Tyr Ala Ser 20 25 30 Arg Gly Val Arg Ser Ala Gln Glu Leu Glu Arg Ser Val Lys Gly Met 35 40 45 Leu Pro Trp Gln Gln Leu Ser Gly Val Glu Lys Ala Val Glu Ile Leu 50 55 60 Tyr Asn Ala Phe Arg Glu Gly Thr Arg Ile Ile Val Val Gly Asp Phe 65 70 75 80 Asp Ala Asp Gly Ala Thr Ser Thr Ala Leu Ser Val Leu Ala Met Arg 85 90 95 Ser Leu Gly Cys Ser Asn Ile Asp Tyr Leu Val Pro Asn Arg Phe Glu 100 105 110 Asp Gly Tyr Gly Leu Ser Pro Glu Val Val Asp Gln Ala His Ala Arg 115 120 125 Gly Ala Gln Leu Ile Val Thr Val Asp Asn Gly Ile Ser Ser His Ala 130 135 140 Gly Val Glu His Ala Arg Ser Leu Gly Ile Pro Val Ile Val Thr Asp 145 150 155 160 His His Leu Pro Gly Asp Thr Leu Pro Ala Ala Glu Ala Ile Ile Asn 165 170 175 Pro Asn Leu Arg Asp Cys Asn Phe Pro Ser Lys Ser Leu Ala Gly Val 180 185 190 Gly Val Ala Phe Tyr Leu Met Leu Ala Leu Arg Thr Phe Leu Arg Asp 195 200 205 Gln Gly Trp Phe Asp Glu Arg Asn Ile Ala Ile Pro Asn Leu Ala Glu 210 215 220 Leu Leu Asp Leu Val Ala Leu Gly Thr Val Ala Asp Val Val Pro Leu 225 230 235 240 Asp Ala Asn Asn Arg Ile Leu Thr Trp Gln Gly Met Ser Arg Ile Arg 245 250 255 Ala Gly Lys Cys Arg Pro Gly Ile Lys Ala Leu Leu Glu Val Ala Asn 260 265 270 Arg Asp Ala Gln Lys Leu Ala Ala Ser Asp Leu Gly Phe Ala Leu Gly 275 280 285 Pro Arg Leu Asn Ala Ala Gly Arg Leu Asp Asp Met Ser Val Gly Val 290 295 300 Ala Leu Leu Leu Cys Asp Asn Ile Gly Glu Ala Arg Val Leu Ala Asn 305 310 315 320 Glu Leu Asp Ala Leu Asn Gln Thr Arg Lys Glu Ile Glu Gln Gly Met 325 330 335 Gln Ile Glu Ala Leu Thr Leu Cys Glu Lys Leu Glu Arg Ser Arg Asp 340 345 350 Thr Leu Pro Gly Gly Leu Ala Met Tyr His Pro Glu Trp His Gln Gly 355 360 365 Val Val Gly Ile Leu Ala Ser Arg Ile Lys Glu Arg Phe His Arg Pro 370 375 380 Val Ile Ala Phe Ala Pro Ala Gly Asp Gly Thr Leu Lys Gly Ser Gly 385 390 395 400 Arg Ser Ile Gln Gly Leu His Met Arg Asp Ala Leu Glu Arg Leu Asp 405 410 415 Thr Leu Tyr Pro Gly Met Met Leu Lys Phe Gly Gly His Ala Met Ala 420 425 430 Ala Gly Leu Ser Leu Glu Glu Asp Lys Phe Lys Leu Phe Gln Gln Arg 435 440 445 Phe Gly Glu Leu Val Thr Glu Trp Leu Asp Pro Ser Leu Leu Gln Gly 450 455 460 Glu Val Val Ser Asp Gly Pro Leu Ser Pro Ala Glu Met Thr Met Glu 465 470 475 480 Val Ala Gln Leu Leu Arg Asp Ala Gly Pro Trp Gly Gln Met Phe Pro 485 490 495 Glu Pro Leu Phe Asp Gly His Phe Arg Leu Leu Gln Gln Arg Leu Val 500 505 510 Gly Glu Arg His Leu Lys Val Met Val Glu Pro Val Gly Gly Gly Pro 515 520 525 Leu Leu Asp Gly Ile Ala Phe Asn Val Asp Thr Ala Leu Trp Pro Asp 530 535 540 Asn Gly Val Arg Glu Val Gln Leu Ala Tyr Lys Leu Asp Ile Asn Glu 545 550 555 560 Phe Arg Gly Asn Arg Ser Leu Gln Ile Ile Ile Asp Asn Ile Trp Pro 565 570 575 Ile <210> 2 <211> 1734 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli str. K-12 substr. MG1655 <400> 2 gtgaaacaac agatacaact tcgtcgccgt gaagtcgatg aaacggcaga cttgcccgct 60 gaattgcctc ccttgctgcg ccgtttatac gccagccggg gagtacgcag tgcgcaagaa 120 ctggaacgca gtgttaaagg tatgctgccc tggcagcaac tgagcggcgt cgaaaaggcc 180 gttgagatcc tttacaacgc ttttcgcgaa ggaacgcgga ttattgtggt cggtgatttc 240 gacgccgacg gcgcgaccag cacggctcta agcgtgctgg cgatgcgctc gcttggttgc 300 agcaatatcg actacctggt accaaaccgt ttcgaagacg gttacggctt aagcccggaa 360 gtggtcgatc aggcccatgc ccgtggcgcg cagttaattg tcacggtgga taacggtatt 420 tcctcccatg cgggggttga gcacgctcgc tcgttgggca tcccggttat tgttaccgat 480 caccatttgc caggcgacac attacccgca gcggaagcga tcattaaccc taacttgcgc 540 gactgtaatt tcccgtcgaa atcactggca ggcgtgggtg tggcgtttta tctgatgctg 600 gcgctgcgca cctttttgcg cgatcagggc tggtttgatg agcgtaacat cgcaattcct 660 aacctggcag aactgctgga tctggtcgcg ctggggacag tggcggacgt cgtgccgctg 720 gacgctaata atcgcattct gacctggcag gggatgagtc gcatccgagc cggaaagtgc 780 cgtccgggga ttaaagcgct gcttgaagtg gcaaaccgtg atgcacaaaa actcgccgcc 840 agcgatttag gttttgcgct ggggccacgt ctcaatgctg ccggacgact ggacgatatg 900 tccgtcggtg tggcgctgtt gttgtgcgac aacatcggcg aagcgcgcgt gctggcaaat 960 gaactcgatg cgctaaacca gacgcgaaaa gagatcgaac aaggaatgca aattgaagcc 1020 ctgaccctgt gcgagaaact ggagcgcagc cgtgacacgc tacccggcgg gctggcaatg 1080 tatcaccccg aatggcatca gggcgttgtc ggtattctgg cttcgcgcat caaagagcgt 1140 tttcaccgtc cggttatcgc gtttgcgcca gcaggtgacg gtacgctgaa aggttccggt 1200 cgctccattc aggggctgca tatgcgtgat gcgctggagc gattagacac actctaccct 1260 ggcatgatgc tgaagtttgg cggtcatgcg atggcggcgg gtttgtcgct ggaagaggat 1320 aaattcaaac tctttcaaca acggtttggc gaactggtta ctgagtggct ggacccttcg 1380 ctattgcaag gcgaagtggt atcagacggt ccgttaagcc cggccgaaat gaccatggaa 1440 gtggcgcagc tgctgcgcga tgctggcccg tgggggcaga tgttcccgga gccgctgttt 1500 gacggtcatt tccgtctgct gcaacagcgg ctggtgggcg aacgtcattt gaaggtgatg 1560 gtcgaaccgg tcggcggcgg tccactgctg gatggtattg cttttaatgt cgataccgcc 1620 ctctggccgg ataacggcgt gcgcgaagtg caactggctt ataagctcga tatcaacgag 1680 tttcgcggca accgcagcct gcaaattatc atcgacaata tctggccaat ttag 1734 <210> 3 <211> 744 <212> PRT <213> Artificial Sequence <220> <223> Streptococcus pneumoniae R6 chromosome <400> 3 Val Asp Val Phe Leu Ile Thr Pro Thr Tyr Glu Trp Gln Phe Ala Leu 1 5 10 15 Gln Val Glu Asp Ala Asp Phe Thr Lys Ile Ala Lys Lys Ala Gly Leu 20 25 30 Gly Pro Glu Val Ala Arg Leu Leu Phe Glu Arg Gly Ile Gln Asp Gln 35 40 45 Glu Ser Leu Lys Lys Phe Leu Glu Pro Ser Leu Glu Asp Leu His Asp 50 55 60 Ala Tyr Leu Leu His Asp Met Asp Lys Ala Val Glu Arg Ile Arg Gln 65 70 75 80 Ala Ile Glu Glu Gly Glu Asn Ile Leu Val Tyr Gly Asp Tyr Asp Ala 85 90 95 Asp Gly Met Thr Ser Ala Ser Ile Val Lys Glu Ser Leu Glu Gln Leu 100 105 110 Gly Ala Glu Cys Arg Val Tyr Leu Pro Asn Arg Phe Thr Asp Gly Tyr 115 120 125 Gly Pro Asn Ala Ser Val Tyr Lys Tyr Phe Ile Glu Gln Glu Gly Ile 130 135 140 Ser Leu Ile Val Thr Val Asp Asn Gly Val Ala Gly His Glu Ala Ile 145 150 155 160 Ala Leu Ala Gln Ser Met Gly Val Asp Val Ile Val Thr Asp His His 165 170 175 Ser Met Pro Glu Thr Leu Pro Asp Ala Tyr Ala Ile Val His Pro Glu 180 185 190 His Pro Asp Ala Asp Tyr Pro Phe Lys Tyr Leu Ala Gly Cys Gly Val 195 200 205 Ala Phe Lys Leu Ala Cys Ala Leu Leu Glu Glu Val Gln Val Glu Leu 210 215 220 Leu Asp Leu Val Ala Ile Gly Thr Ile Ala Asp Met Val Ser Leu Thr 225 230 235 240 Asp Glu Asn Arg Ile Leu Val Gln Tyr Gly Leu Glu Met Leu Gly His 245 250 255 Thr Gln Arg Ile Gly Leu Gln Glu Met Leu Asp Met Ala Gly Ile Ala 260 265 270 Ala Asn Glu Val Thr Glu Glu Thr Val Gly Phe Gln Ile Ala Pro Arg 275 280 285 Leu Asn Ala Leu Gly Arg Leu Asp Asp Pro Asn Pro Ala Ile Asp Leu 290 295 300 Leu Thr Gly Phe Asp Asp Glu Glu Ala His Glu Ile Ala Leu Met Ile 305 310 315 320 His Gln Lys Asn Glu Glu Arg Lys Glu Ile Val Gln Ser Ile Tyr Glu 325 330 335 Glu Ala Lys Thr Met Val Asp Pro Glu Lys Lys Val Gln Val Leu Ala 340 345 350 Lys Glu Gly Trp Asn Pro Gly Val Leu Gly Ile Val Ala Gly Arg Leu 355 360 365 Leu Glu Glu Leu Gly Gln Thr Val Ile Val Leu Asn Ile Glu Asp Gly 370 375 380 Arg Ala Lys Gly Ser Ala Arg Ser Val Glu Ala Val Asp Ile Phe Glu 385 390 395 400 Ala Leu Asp Pro His Arg Asp Leu Phe Ile Ala Phe Gly Gly His Ala 405 410 415 Gly Ala Ala Gly Met Thr Leu Glu Val Glu Gln Leu Ser Asp Leu Ser 420 425 430 Gln Val Leu Glu Asp Tyr Val Arg Glu Lys Gly Ala Asp Ala Gly Gly 435 440 445 Lys Asn Lys Leu Asn Leu Asp Glu Glu Leu Asp Leu Glu Ala Leu Ser 450 455 460 Leu Glu Thr Val Lys Ser Phe Glu Arg Leu Ala Pro Phe Gly Met Asp 465 470 475 480 Asn Gln Lys Pro Ile Phe Tyr Ile Lys Asn Phe Gln Val Glu Ser Ala 485 490 495 Arg Thr Met Gly Ala Gly Asn Ala His Leu Lys Leu Lys Ile Ser Lys 500 505 510 Gly Glu Ala Ser Phe Glu Val Val Ala Phe Gly Gln Gly Arg Trp Ala 515 520 525 Thr Glu Phe Ser Gln Thr Lys Asn Leu Glu Leu Ala Val Lys Leu Ser 530 535 540 Val Asn Gln Trp Asn Gly Gln Thr Ala Leu Gln Leu Met Met Val Asp 545 550 555 560 Ala Arg Val Glu Gly Val Gln Leu Phe Asn Ile Arg Gly Lys Asn Ala 565 570 575 Val Leu Pro Glu Gly Val Pro Val Leu Asp Phe Pro Gly Glu Leu Pro 580 585 590 Asn Leu Ala Ala Ser Glu Ala Val Val Val Lys Asn Ile Pro Glu Asp 595 600 605 Ile Thr Gln Leu Lys Thr Ile Phe Gln Glu Gln His Phe Ser Ala Val 610 615 620 Tyr Phe Lys Asn Asp Ile Asp Lys Ala Tyr Tyr Leu Thr Gly Tyr Gly 625 630 635 640 Thr Arg Asp Gln Phe Ala Lys Leu Tyr Lys Thr Ile Tyr Gln Phe Pro 645 650 655 Glu Phe Asp Ile Arg Tyr Lys Leu Lys Asp Leu Ala Ala Tyr Leu Asn 660 665 670 Ile Gln Gln Ile Leu Leu Val Lys Met Ile Gln Val Phe Glu Glu Leu 675 680 685 Gly Phe Val Thr Ile Lys Asp Gly Val Met Thr Val Asn Lys Glu Ala 690 695 700 Pro Lys Arg Glu Ile Gly Glu Ser Gln Ile Tyr Gln Asn Leu Lys Gln 705 710 715 720 Thr Val Lys Asp Gln Glu Met Met Ala Leu Gly Thr Val Gln Glu Ile 725 730 735 Tyr Asp Phe Leu Met Glu Lys Glu 740 <210> 4 <211> 2235 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus pneumoniae R6 chromosome <400> 4 gtggatgtct ttttgataac acctacttat gaatggcagt ttgccctgca ggtagaagat 60 gcggatttta caaagatagc caagaaggct ggactgggtc ctgaggtggc tcggttattg 120 tttgagagag ggattcagga ccaagaaagt ctgaagaagt ttttagaacc ttccttggag 180 gacttacatg atgcttatct gctccatgat atggacaagg cagtggagcg gattcgtcag 240 gctattgaag aaggggaaaa tattctcgtt tatggagact atgatgcgga tggcatgact 300 tcggcttcta ttgtgaagga aagtttggaa caacttggtg ctgagtgccg agtttacctg 360 ccaaatcgtt ttaccgatgg ctatggccct aatgctagtg tttataaata ctttatcgag 420 caagaaggaa tttccttgat tgtgacggtg gacaatgggg ttgctggtca tgaggctatt 480 gcattggctc agtctatggg agtagatgtc attgtgacag accatcattc catgcctgaa 540 accctgccag atgcttatgc tattgtccat cctgaacatc cagatgcgga ttatcctttt 600 aaatatttgg ctggttgtgg agttgctttc aagttggctt gtgccctgtt agaagaagtg 660 caagtggaat tgcttgattt ggtcgctatt ggaactattg cagatatggt gagtctgacg 720 gatgaaaatc gtatcttagt tcaatatggt ctggaaatgt tgggtcatac ccagcgcatt 780 ggtctgcaag aaatgctgga catggctggg attgctgcca acgaagtaac agaagaaacg 840 gttggtttcc agattgctcc tcgtttgaat gccttgggtc gcttggatga tcccaatcct 900 gccattgatt tgttgactgg atttgatgat gaggaagcgc atgagattgc ccttatgatt 960 caccagaaaa acgaagagcg caaggaaatc gttcagtcta tctatgaaga agccaagacc 1020 atggtggatc ctgagaagaa ggttcaggtc ttggccaagg aaggctggaa tcctggggtt 1080 ctaggaatcg tggctggtcg tttattggaa gaattgggac agacagtcat tgttcttaat 1140 atagaagacg gtcgtgccaa gggcagtgct cgtagtgtgg aagcggtcga tatttttgaa 1200 gctctggatc cccatcgaga cctcttcatc gcctttggag gtcatgcagg tgcagcgggt 1260 atgacgctgg aagttgagca actctcagat ttatctcagg ttttggaaga ttatgttcgt 1320 gaaaaaggtg cagatgctgg tggcaagaat aagttaaacc tagatgaaga gttggatttg 1380 gaggcactta gcttggaaac ggtcaaaagt tttgaacgtt tagctccttt tggaatggat 1440 aatcagaaac ctatttttta tatcaagaat tttcaggtcg aaagtgctcg tactatgggg 1500 gcaggtaatg cccatctaaa gctgaaaatt tccaagggtg aggcgagttt tgaagtggta 1560 gcctttggtc aaggcagatg ggcgacagag ttttctcaaa ccaagaatct agagttagcg 1620 gttaaattgt ctgtcaacca atggaatggc caaactgctc tccagttgat gatggtggat 1680 gcgcgagtgg aaggtgttca actttttaac attcgtggaa aaaatgcagt cttgccagaa 1740 ggtgttccag tcttggattt tcctggagaa ctgccaaatc ttgcggctag tgaagctgtt 1800 gtcgtaaaaa acattccaga ggatattact cagctgaaga ccatttttca ggaacagcat 1860 ttctctgctg tctatttcaa aaatgatatt gacaaggctt attatctgac aggttatggg 1920 actagagatc agtttgccaa attgtacaag actatttacc agttcccaga gtttgatatt 1980 cgctacaagc tgaaagattt ggctgcatat cttaatattc aacaaatctt gctggtcaag 2040 atgattcaag tatttgaaga actaggcttt gtgacgataa aagatggtgt gatgacagtc 2100 aataaagagg cgccaaagcg ggagatagga gaaagtcaaa tttaccaaaa tctcaaacaa 2160 accgttaaag accaagaaat gatggcgctg ggtacggtgc aagaaattta tgattttttg 2220 atggaaaaag agtag 2235 <210> 5 <211> 742 <212> PRT <213> Artificial Sequence <220> <223> Lactococcus lactis subsp. lactis Il1403 <400> 5 Met Ile Lys Ala Lys Tyr Asp Trp Lys Val Ala Asp Thr Ala Ile Ser 1 5 10 15 Glu Asp Phe Leu Lys Ile Ala Lys Lys His Lys Leu Asp Glu Leu Thr 20 25 30 Ser Arg Val Leu Tyr Gln Arg Gly Ile His Ala Glu Ala Glu Ile Glu 35 40 45 Gln Phe Leu Lys Pro Ser Leu Glu Asn Leu His Asp Pro Phe Leu Leu 50 55 60 His Asp Met Glu Lys Ala Thr Gly Arg Ile Leu Ser Ala Ile Glu Gln 65 70 75 80 Gly Glu Asn Ile Leu Ile Tyr Gly Asp Tyr Asp Ala Asp Gly Met Thr 85 90 95 Ala Ser Ser Val Met Lys Ser Ala Leu Asp Glu Leu Gly Ala Glu Ala 100 105 110 Gln Val Tyr Leu Pro Asn Arg Phe Thr Asp Gly Tyr Gly Pro Asn Leu 115 120 125 Asp Val Tyr Gln Tyr Tyr Ile Lys Asn Glu Asn Ile Asn Leu Ile Ile 130 135 140 Thr Val Asp Asn Gly Val Ala Gly Leu Glu Ala Ile Thr Trp Ala Gln 145 150 155 160 Glu Asn Gly Val Asp Val Ile Val Thr Asp His His Ser Ile Pro Asp 165 170 175 Gln Leu Pro Pro Ala Tyr Ala Ile Val His Pro Glu His Pro Asp Ser 180 185 190 Gln Tyr Pro Phe Lys Tyr Leu Ala Gly Val Gly Val Ala Phe Lys Val 195 200 205 Ala Cys Ala Leu Leu Glu Tyr Ala Pro Ser Glu Met Leu Asp Leu Val 210 215 220 Ala Ile Gly Thr Ile Ala Asp Met Val Ser Leu Thr Asp Glu Asn Arg 225 230 235 240 Ile Leu Val Ala His Gly Leu Lys Val Leu Ala Gln Thr Glu Arg Ala 245 250 255 Gly Leu Gln Glu Leu Met Lys Tyr Ala Gly Val Asp Phe Asp Lys Ile 260 265 270 Thr Glu Glu Thr Val Gly Phe Gln Ile Ala Pro Arg Leu Asn Ala Leu 275 280 285 Gly Arg Leu Asp Asp Pro Asn Pro Ala Ile Glu Leu Leu Thr Gly Trp 290 295 300 Asp Glu Asp Glu Ala His Glu Ile Ala Lys Met Ile Asp Gln Lys Asn 305 310 315 320 Ser Glu Arg Lys Glu Ile Val Glu Lys Ile His Asn Glu Ala Leu Ser 325 330 335 Met Leu Thr Asp Glu Pro Val Gln Ile Leu Tyr His Lys Asp Trp His 340 345 350 Lys Gly Val Leu Gly Ile Val Ala Gly Arg Leu Leu Glu Ala Ile His 355 360 365 Lys Pro Val Ile Met Leu Ala Gln Glu Asp Gly Ile Leu Arg Gly Ser 370 375 380 Ala Arg Ser Ile Glu Asn Phe Asp Ile Phe Lys Ala Leu Asn Ala His 385 390 395 400 Arg Glu Leu Phe Ile Ala Phe Gly Gly His Lys Gln Ala Ala Gly Met 405 410 415 Thr Leu Ser Leu Glu Asn Val Glu Ala Val Lys Lys Ala Met Ile Asp 420 425 430 Tyr Ile Val Asp Asn His Leu Asp Met Ser Lys Lys Ser Pro Leu Glu 435 440 445 Ile Ala Asp Arg Cys His Leu Asp Asp Ile Ser Leu Ser Thr Ile Thr 450 455 460 Asn Leu Ala Lys Leu Ala Pro Phe Gly Met Asp Asn Pro Lys Pro Arg 465 470 475 480 Phe Leu Ile Glu Asp Tyr Lys Val Ile Gln Ser Arg Ser Met Gly Lys 485 490 495 Asp Asn Ala His Leu Lys Leu Lys Ile Gln Glu Glu Lys Gln Gln Ile 500 505 510 Asp Ala Val Tyr Phe Gln His Gly Ser Glu Glu Leu Glu Phe Glu Gln 515 520 525 Ala Gln Thr Lys Leu Val Ala Thr Leu Ser Ser Asn Ser Trp Asn Gly 530 535 540 Asn Thr Ser Leu Gln Leu Met Ile Glu Asp Ala Asp Ser Val Gly Val 545 550 555 560 Glu Leu Leu Asp Ile Arg Ser Lys Gln Ile Pro Ile Pro Lys Glu Ala 565 570 575 Asn Ile Phe Ser Gln Asn Gln Leu Lys His Gly Ile Met Glu Asp Val 580 585 590 Leu Val Ile Glu Glu Ile Pro Gly Asp Leu Ala Gly Leu Ser Val Leu 595 600 605 Lys Glu Ala Val Ser Lys Ala Ala Ser Asp Asp Phe Lys Met Ile Tyr 610 615 620 Phe Lys Asn Lys Ile Thr Asp Ser Tyr Tyr Leu Thr Gly Ser Gly Thr 625 630 635 640 Arg Glu Glu Phe Ala Arg Leu Tyr Lys Ala Ile Tyr Gln Phe Pro Glu 645 650 655 Phe Asp Ile Arg Tyr Lys Leu Lys Ser Leu Ala Asp Tyr Leu Lys Ile 660 665 670 Pro His Leu Leu Leu Val Lys Met Ile Lys Ile Phe Glu Glu Leu Glu 675 680 685 Phe Val Ser Ile Asp Asn Gly Leu Met Thr Val Asn Lys Thr Ala Asp 690 695 700 Lys Arg Glu Ile Ser Glu Ser Thr Ile Tyr Gln Glu Leu Glu Lys Ile 705 710 715 720 Val Lys Met Gln Glu Leu Phe Ala Leu Ala Pro Val Lys Glu Ile Tyr 725 730 735 Gln Asn Leu Leu Glu Lys 740 <210> 6 <211> 2229 <212> DNA <213> Artificial Sequence <220> <223> Lactococcus lactis subsp. lactis Il1403 <400> 6 atgataaaag caaaatatga ttggaaagtg gctgatacag ccatttccga agattttcta 60 aagatagcga aaaaacataa attagatgaa ttgacaagtc gcgtgcttta tcaaagagga 120 attcatgcag aagctgaaat tgagcaattt ttaaagccaa gtttagaaaa tttacatgac 180 ccttttctac ttcatgatat ggaaaaagca acagggcgta ttttatcagc aattgagcaa 240 ggtgaaaaca tcttaattta tggtgactat gatgctgacg gaatgactgc atcatcagtc 300 atgaaatccg ctctggatga attaggagct gaggctcaag tttatttacc caatcgtttt 360 actgacggtt acgggccaaa tcttgacgtt tatcagtact atattaagaa tgaaaatatc 420 aatcttatta ttactgtaga taatggggtt gctggcttgg aggcaattac ctgggcacag 480 gaaaatggag tggatgtaat tgttaccgac catcactcaa ttcctgacca acttccccct 540 gcttatgcca ttgtccaccc cgaacatcct gacagtcaat atcctttcaa atatttggca 600 ggagttggtg tcgccttcaa agttgcctgt gctcttttag aatatgcacc cagtgaaatg 660 cttgatttag ttgcaattgg tacaattgct gacatggtca gtttgactga tgaaaaccga 720 attttagtgg ctcatggtct aaaagttttg gcccaaacag agcgtgccgg cctacaagaa 780 ttaatgaagt atgccggggt tgattttgat aaaatcactg aggaaacggt tggttttcaa 840 attgctcccc gtttaaacgc tttgggacgt ctcgatgacc caaacccagc cattgaattg 900 ctgacaggtt gggatgaaga tgaagcgcat gaaattgcca aaatgattga ccaaaaaaat 960 tcagagcgaa aagaaattgt ggaaaaaatt cacaatgaag ctctgagtat gctgacagat 1020 gaacctgttc agattttata tcataaggac tggcataaag gtgtcttagg aattgttgcg 1080 ggtcgcttgc tagaagccat acataagcca gtaatcatgc tggcccaaga agatgggatt 1140 ttgcgtgggt ctgctcggtc aatcgaaaat tttgatattt tcaaggcatt aaatgcccat 1200 cgtgaacttt ttattgcttt tggtggtcat aaacaagccg cagggatgac tttaagtctg 1260 gaaaacgtag aagcagttaa aaaggcgatg attgattata tcgtggacaa tcatttagat 1320 atgtctaaaa aaagtccctt agaaattgct gaccgttgcc acttagacga tatttcgctc 1380 tcaacaatca ctaatttagc aaaattggct ccctttggca tggataatcc taagcctcga 1440 tttttaatcg aagattataa ggtgattcaa agccgaagta tggggaaaga caatgctcat 1500 cttaaattaa aaattcagga agaaaagcaa caaattgatg ccgtttattt ccaacatggt 1560 tcagaagaat tggaattcga acaggcacaa acaaaattag tcgcaacact ttcaagtaat 1620 agttggaatg gaaatacgag tcttcaatta atgattgaag atgctgattc agttggtgtc 1680 gaacttcttg atataagaag taaacaaatt cctatcccaa aagaggctaa tattttttct 1740 caaaatcagc taaaacatgg tataatggaa gatgtgcttg tcatcgaaga gatacctgga 1800 gatttagcag gtttgtcagt attaaaagaa gctgtcagta aagcagcgtc ggatgatttt 1860 aaaatgattt attttaaaaa taaaatcact gatagctatt atttaacagg aagtggaaca 1920 cgagaagaat ttgcaagact ttataaagca atttatcaat ttccagaatt tgatattcgc 1980 tataaattaa aatcacttgc ggattatttg aaaatccctc atctcttgct tgtcaagatg 2040 ataaaaatct ttgaggaact tgaatttgtc agcattgaca atggcttaat gaccgttaat 2100 aagacggccg ataaacgcga aatttctgag agtacaattt atcaagaact agaaaaaatt 2160 gtaaaaatgc aagaactttt tgctttagca cccgtcaaag aaatttatca aaatttatta 2220 gaaaaataa 2229 <210> 7 <211> 785 <212> PRT <213> Unknown <220> <223> Bacillus clausii <400> 7 Met Leu Gln Ser Lys Thr Arg Trp Arg Ile Leu Glu Gln Asn Glu Leu 1 5 10 15 Leu Ala Gln Glu Leu Ala Asp Glu Leu Arg Val Ser Met Leu Thr Ala 20 25 30 Arg Leu Leu Val Arg Arg Gly Ile Gln Thr Val Gln Ala Ala Lys Arg 35 40 45 Phe Leu His Tyr Glu Glu Pro Thr Phe Tyr Asp Pro Phe Leu Leu Lys 50 55 60 Gly Met Glu Glu Thr Ile Glu Arg Ile Ala Leu Ala Val Lys Arg Lys 65 70 75 80 Glu Arg Ile Leu Val Phe Gly Asp Tyr Asp Ala Asp Gly Val Ser Ser 85 90 95 Thr Ser Val Met Leu Thr Ala Leu Gln Thr Tyr Gly Ala Asp Cys Asp 100 105 110 Tyr Tyr Ile Pro Asn Arg Phe Thr Glu Gly Tyr Gly Pro Asn Cys Pro 115 120 125 Ala Leu Asp Phe Ala Lys Arg Gln Gly Tyr His Leu Val Ile Thr Val 130 135 140 Asp Thr Gly Ile Ser Ala Leu Asn Glu Ala Ala His Ala Lys Asp Ile 145 150 155 160 Gly Leu Asp Phe Ile Ile Thr Asp His His Glu Pro Pro Pro Val Leu 165 170 175 Pro Glu Ala Leu Ala Ile Ile Asn Pro Lys Gln Pro Gly Cys Pro Tyr 180 185 190 Pro Phe Lys Glu Leu Ala Gly Val Gly Val Ala Phe Lys Val Ala His 195 200 205 Ala Leu Leu Gly Arg Leu Pro Glu Glu Leu Leu Asp Tyr Ala Val Ile 210 215 220 Gly Thr Ile Ala Asp Leu Val Pro Leu Ile Asp Glu Asn Arg Leu Leu 225 230 235 240 Ala Lys Lys Gly Leu Arg Ala Ile Glu Ser Ser Gly Arg Pro Gly Ile 245 250 255 Arg Ala Leu Lys Glu Val Cys Gly Met Lys Gln Glu Ala Met Asp Ala 260 265 270 Asp His Ile Gly Phe Ala Ile Gly Pro Arg Leu Asn Ala Ala Gly Arg 275 280 285 Leu Asp Ser Ala Asn Pro Ala Val Glu Leu Leu Leu Ala Asp Asp Glu 290 295 300 Glu Glu Ala Lys Ala Leu Ala Thr Glu Ile Asp Ser Leu Asn Lys Glu 305 310 315 320 Arg Gln Ala Ile Val Ser Lys Met Thr Glu Glu Ala Ile Arg Leu Val 325 330 335 Glu Thr Thr Tyr Gly Thr Lys Ile Pro His Ala Ile Val Val Ala Lys 340 345 350 Glu Gly Trp Asn Pro Gly Val Ile Gly Ile Val Ala Ser Arg Leu Val 355 360 365 Glu Gln Phe Tyr Arg Pro Thr Ile Val Met Ser Ile Asp Glu Ser Ser 370 375 380 Gly Leu Ala Lys Gly Ser Ala Arg Ser Ile Glu Gly Phe Asp Met Tyr 385 390 395 400 Gln Glu Leu Ala Asn Asn Arg Asp Ile Leu Pro His Phe Gly Gly His 405 410 415 Pro Met Ala Ala Gly Met Thr Leu Lys Thr Glu Asp Ile Asp Asp Leu 420 425 430 Arg Ser Arg Leu Ile Lys Gln Ala Lys Glu Thr Leu Thr Asp Asp Met 435 440 445 Leu Thr Pro Ala Thr Asp Ile Asp Leu Val Ala Glu Val Glu Asp Val 450 455 460 Thr Val Gln Val Ile Gly Glu Leu Gln Ala Leu Ala Pro Phe Gly Val 465 470 475 480 Ala Asn Arg Lys Pro Ile Val Leu Val Glu Gly Ala His Ile Ser Asp 485 490 495 Met Arg Arg Ile Gly Ser Asn Gln Asn His Leu Lys Ile Gln Phe Thr 500 505 510 Gly Ala Gln Lys Pro Leu Asp Gly Ile Ala Phe Arg Met Gly His Leu 515 520 525 Phe Glu Glu Ile Thr Pro His Ala Lys Leu Ser Ala Ile Gly Thr Val 530 535 540 Ser Leu Asn Glu Trp Asn Gly Lys Val Lys Pro Gln Leu Ile Ile Asp 545 550 555 560 Asp Leu Ala Val Leu Glu Trp Gln Leu Phe Asp Trp Arg Ser Ile Gln 565 570 575 Pro Asn Arg Leu Asn Ser Arg Leu Leu Asp Leu Pro Arg Glu Lys Leu 580 585 590 Val Ala Ile Ser Phe Gln Glu Gly Thr Lys Glu Arg Leu Gly Leu Asp 595 600 605 Val Pro Val Tyr Asp Tyr Arg Gln Ala Pro Ser Phe Phe Glu Ala Tyr 610 615 620 Val Val Leu Leu Asp Leu Pro Ser Asn Arg Val Glu Leu Glu Ser Leu 625 630 635 640 Phe Ser Lys Lys Gly Glu Pro Ser Arg Val Tyr Val Val Phe Ser Glu 645 650 655 Glu Glu Glu Ser Phe Phe Gln Thr Asn Pro Asn Arg Glu Gln Phe Lys 660 665 670 Trp Tyr Tyr Gly Phe Ile Lys Lys Asn Gln Arg Phe Ser Leu Asn Gln 675 680 685 Leu Gly Ala Lys Leu Glu Lys His Lys Gly Trp Ser Ala Arg Thr Val 690 695 700 Glu Phe Met Thr Thr Val Phe Leu Glu Leu Gly Phe Ile Lys Leu Asp 705 710 715 720 Asp Ser Ile Val Glu Ala Val Glu Asn Pro Glu Lys Lys Ala Leu Thr 725 730 735 Ala Ser Pro Thr Tyr Gln Ala Lys Gln Glu Lys Ala Trp Leu Glu Asn 740 745 750 Glu Phe Val Phe Ala Ser Tyr Gln Gln Leu Lys Glu Trp Phe Gln Ala 755 760 765 Ala Ile Glu Gly Ser Ala Glu Thr Lys Glu Glu Ser Ile Leu Asn Gly 770 775 780 Leu 785 <210> 8 <211> 2358 <212> DNA <213> Unknown <220> <223> Bacillus clausii <400> 8 atgttgcaat caaaaacaag atggcgcatt ttagaacaaa acgagctgtt ggcgcaagag 60 ctcgcagacg agctgcgtgt atcgatgtta acggcccgtc ttcttgtacg ccggggaatc 120 caaaccgtcc aggcggccaa gcgcttttta cactacgaag agccgacgtt ttacgatccc 180 tttttgttga aagggatgga ggaaacgatt gaacgaattg ccctcgcagt caaacgaaaa 240 gaacgcattc ttgtttttgg ggactatgat gctgatggcg ttagttcgac ttccgtgatg 300 ctaacagcgt tacaaacgta tggcgccgac tgtgattact atataccaaa ccgttttaca 360 gaaggatacg ggcccaattg cccagcgcta gactttgcaa aaaggcaagg ttatcactta 420 gtgataacag tcgatacagg catttctgct ttgaatgaag ctgcccatgc caaagacatc 480 ggtctggatt ttattatcac tgaccaccat gagccgccgc cggttttgcc agaagcattg 540 gcgatcatta atcctaagca gcctggttgc ccgtatccat ttaaagagct tgccggcgta 600 ggcgttgcct ttaaagttgc acacgctttg ctcgggcgcc tgcctgaaga gctgctcgat 660 tacgctgtga tcggcacgat cgccgacttg gtgcctctca ttgatgaaaa tcggctgctg 720 gccaaaaagg ggcttcgcgc gattgaatca agcggccgcc ctgggatccg tgccttaaag 780 gaagtatgcg gcatgaagca ggaagcaatg gatgccgacc atatcggctt tgcgattggc 840 cctcgtctaa atgcagcagg gcggctcgat tctgctaatc cagctgttga gctgctcctt 900 gctgatgatg aagaagaagc aaaagcgcta gccacagaaa tcgatagcct caataaagaa 960 cggcaagcaa ttgttagcaa aatgacagaa gaagcgattc gccttgtgga aacaacctat 1020 ggcacaaaga tcccccatgc catcgttgta gcgaaggaag gctggaatcc aggggtcatc 1080 gggattgtcg cttctcgcct tgtagagcag ttttaccgtc cgacaattgt catgagcatt 1140 gacgaatctt caggacttgc caaaggctcg gccagaagca tcgaagggtt tgacatgtac 1200 caagaattgg cgaataaccg ggatatactt cctcattttg gcggacaccc gatggcggca 1260 gggatgacac ttaaaaccga ggacattgac gacttacgtt ctcgtttaat caagcaagcc 1320 aaggagacac taacggacga catgcttacg ccagccacgg atatagacct cgttgcagaa 1380 gtggaggatg taaccgttca agtgattggc gagttgcagg cacttgcccc gtttggcgtt 1440 gccaaccgta aaccgattgt acttgttgag ggtgcgcata tttccgatat gcgccggatt 1500 ggcagcaacc aaaaccattt aaaaatccaa tttactggcg cccaaaagcc gcttgacgga 1560 attgccttta gaatggggca tttgtttgaa gagatcacgc cacatgcaaa gttatcggcc 1620 atcggaacgg tatcgttgaa tgaatggaac ggaaaagtaa agccacaact tattattgac 1680 gatcttgccg tattggagtg gcaactattt gattggcgca gcatccagcc aaaccgctta 1740 aacagccggt tgcttgactt gcctcgtgaa aaactcgtcg ctatttcctt tcaagaaggg 1800 acaaaagaaa gacttgggtt ggacgtaccc gtatatgatt accggcaagc gccttccttt 1860 tttgaagctt acgtcgtgtt gctcgatttg cctagcaacc gggttgaact ggaaagtctt 1920 ttttctaaga aaggggagcc aagccgtgtt tatgttgtgt tctctgagga agaagaatcg 1980 ttttttcaaa cgaacccaaa ccgcgagcag ttcaaatggt attatggttt tataaagaaa 2040 aaccaacgct tttcactgaa tcaactcggc gctaagcttg aaaagcacaa aggctggtca 2100 gcccgcacgg tcgaatttat gaccactgta tttttagaac ttggttttat caagcttgac 2160 gacagcatcg ttgaagcagt agaaaaccct gaaaaaaaag cgttgactgc atcgccgacc 2220 taccaagcca agcaagaaaa agcatggctt gagaatgaat ttgtttttgc ttcttatcaa 2280 cagttaaaag aatggttcca ggctgccatc gaagggagcg ctgaaacaaa agaggagtcg 2340 atcctaaatg gattataa 2358 <210> 9 <211> 590 <212> PRT <213> Unknown <220> <223> Ehrlichia ruminantium strain Welgevonden <400> 9 Leu Asn Ile Cys Ser Met Leu Asn Cys Asn Leu Glu Asn Ser Glu Tyr 1 5 10 15 Ile Gly Val Thr Gly Ala Leu Trp Lys Pro Tyr Asp Val Asn Leu Arg 20 25 30 Asp Ile Leu Thr Ile Lys Gln Lys Phe Tyr Leu Ser Glu Ile Val Ala 35 40 45 Arg Ile Leu Ser Ala Arg Lys Ile Asn Ile Glu Glu Ile Ser Asn Phe 50 55 60 Leu Tyr Pro Thr Leu Lys Ser Ser Leu Pro Asn Pro Phe His Met Leu 65 70 75 80 Asp Met Asp Lys Ala Val Tyr Arg Ile Cys His Ala Ile His Asn Arg 85 90 95 Glu Asn Ile Val Ile Phe Gly Asp Tyr Asp Val Asp Gly Ala Thr Ser 100 105 110 Ser Ala Leu Ile Lys Gln Tyr Leu Ser Gln Ile Gly Val Pro Thr Thr 115 120 125 Ile Tyr Ile Pro Asp Arg Ile Cys Glu Gly Tyr Gly Pro Asn Thr Gln 130 135 140 Ala Leu Leu Lys Leu Lys Glu Ile Gly Asn Ser Leu Cys Ile Thr Val 145 150 155 160 Asp Cys Gly Thr Ile Ala His Glu Pro Ile Ser Ala Ala Lys Ser Val 165 170 175 Asn Leu Asp Val Ile Val Ile Asp His His Ile Gly Ile Asn Thr Leu 180 185 190 Pro Asp Ala Val Ala Val Ile Asn Pro Asn Arg Leu Asp Glu Thr Ser 195 200 205 Pro Tyr Thr Tyr Leu Ala Gly Val Gly Val Ser Phe Leu Met Leu Val 210 215 220 Ala Leu His Lys Thr Leu Lys Glu Gln Gly Phe Phe Lys Asn Asn Gln 225 230 235 240 Glu Pro Asn Leu Ile Asn Tyr Leu Asp Leu Val Ala Leu Gly Thr Val 245 250 255 Cys Asp Val Met Pro Ile Ile Gly Leu Asn Arg Thr Phe Val Lys Gln 260 265 270 Gly Leu Lys Ile Met Thr Thr Arg Gln Asn Leu Gly Leu Lys Thr Leu 275 280 285 Ser Asp Val Ile Gly Leu Glu Glu Lys Pro Asn Ile Tyr Gln Leu Gly 290 295 300 Phe Asn Ile Gly Pro His Ile Asn Ala Gly Gly Arg Val Gly Asn Ala 305 310 315 320 Ser Leu Gly Ala Arg Leu Leu Ser Thr Asn Asn Glu Glu Glu Ala Leu 325 330 335 Glu Ile Ser Glu Lys Leu Gln Asn Phe Asn Leu Glu Arg Lys Thr Leu 340 345 350 Glu Asn Gln Ser Phe Asn Glu Ala Val Glu Gln Ile Glu Ala Thr Ile 355 360 365 Ile Ser Ser Asn Ile Ile Ile Ala Thr Gly Asn Trp His Pro Gly Ile 370 375 380 Ile Gly Ile Val Ala Gly Arg Leu Lys Asp Lys Phe Phe Leu Pro Ser 385 390 395 400 Ile Val Ile Ser Leu Glu Asn Gly Ile Gly Lys Ala Ser Ala Arg Ser 405 410 415 Ile Pro Asp Val Asp Leu Gly Ala Ala Ile Leu Glu Ala Lys Ala Leu 420 425 430 Gly Ile Ile Ile Glu Gly Gly Gly His Ala Met Ala Ala Gly Phe Ser 435 440 445 Ile Gln Glu Asn Lys Ile Lys Ile Leu His Glu Phe Leu Ser Gln Lys 450 455 460 Phe His Asn Ile Asn Thr Arg Lys Val Phe Lys Val Asp Gly Ile Val 465 470 475 480 Thr Ala Glu Ala Ile Asn Leu Lys Leu Trp Lys Glu Leu Gln Phe Leu 485 490 495 Glu Pro Phe Gly Ile Gly Asn Pro Glu Pro Arg Phe Ile Leu Thr Asn 500 505 510 Ile Arg Ile Lys Asn Ser Glu Val Ile Gly Glu Ser His Ile Arg Cys 515 520 525 Leu Ile Tyr Asp Asn Lys Thr Phe Ile Lys Gly Ile Cys Phe Arg Cys 530 535 540 Ile Gly Thr Glu Leu Gly Thr Thr Leu Leu Glu Cys Thr Thr Thr Thr 545 550 555 560 Leu Leu Gly Lys Ile Ser Ile Asn Tyr Trp Arg Gly Asn Glu Asn Ile 565 570 575 Gln Phe Ile Ile Glu Asp Ala Leu Gln His Asn Gln Ile Arg 580 585 590 <210> 10 <211> 1773 <212> DNA <213> Unknown <220> <223> Ehrlichia ruminantium strain Welgevonden <400> 10 ttgaatattt gtagcatgtt aaactgtaat ctagaaaact cggaatatat cggagtaaca 60 ggagcattat ggaaaccata cgatgtaaat ctgagagata ttttaacaat caagcaaaaa 120 ttttaccttt ctgaaattgt tgcaagaata ttatctgcaa gaaaaattaa tatagaagaa 180 attagtaatt ttttatatcc aactctaaaa tcatctctac ctaatccttt tcacatgtta 240 gatatggata aagcagtata cagaatttgt catgcaatac ataatcgaga aaacatagta 300 atctttggag actatgatgt agatggagca acatcatctg cattaataaa acaatatcta 360 tcacagatag gagtaccaac aacgatatat attccagatc gtatatgtga aggatatgga 420 ccaaatacac aggccttact aaaattaaaa gaaataggaa atagtctatg tataaccgta 480 gactgcggta ctatagcaca tgaaccaata tccgcagcaa agtcagtaaa tcttgatgtt 540 atagttatag atcatcatat aggaattaat actttaccag atgcagttgc agtaatcaat 600 cctaatcgct tagacgaaac atccccctac acatatctag caggagtagg cgtatctttt 660 ttaatgttag tagcactaca taaaacttta aaagagcaag gattttttaa aaataatcaa 720 gaaccaaatt taataaatta tttagattta gtagcactag gaactgtatg tgatgttatg 780 cctattatag gactaaatag gacttttgta aaacaaggtc taaagattat gacaacaaga 840 caaaatctag gccttaaaac attatctgat gtaattggat tagaggaaaa gccaaatatt 900 tatcaactgg gatttaatat aggaccacat ataaatgcag gaggaagagt aggtaatgct 960 agcctaggag caagattatt atcaaccaac aatgaagaag aagcattaga aatttcagaa 1020 aaactgcaaa atttcaattt agaaagaaaa acattagaaa atcaaagctt caatgaagca 1080 gtagaacaaa tagaagctac tataatatca agcaatataa ttattgctac aggaaattgg 1140 catccaggaa taataggtat tgttgcagga agattaaaag ataaattttt cttaccaagt 1200 atagtaatat cacttgaaaa tggtattggt aaagcaagtg ctaggtcaat tccagatgta 1260 gatttaggcg cagctatttt agaagcaaaa gccctcggaa taataataga aggaggagga 1320 catgctatgg ctgcaggatt ttcaatacaa gaaaataaaa tcaaaatatt acacgagttt 1380 ctatcacaaa agttccataa tatcaataca cgaaaagtat ttaaagtaga cggtattgtc 1440 actgcagaag ctatcaattt aaaattatgg aaagaattac aatttctaga accatttggc 1500 ataggtaatc ctgaaccaag attcattctc acaaatatca gaataaaaaa ttcagaagtt 1560 ataggagaaa gtcatatcag atgtttaatt tacgataaca aaacatttat aaaaggaatt 1620 tgttttagat gtatcggcac agaattgggt accactttac tagaatgtac tactaccaca 1680 ttactaggaa aaataagtat aaattactgg agaggtaatg agaatataca atttataata 1740 gaagatgctt tacagcataa ccaaataagg taa 1773 <210> 11 <211> 589 <212> PRT <213> Unknown <220> <223> Chlamydia psittaci 6BC <400> 11 Met Ala Ser Lys Asp Asn Ser Ser Val Ser Asn Pro Thr Trp Ile Tyr 1 5 10 15 Pro Lys Tyr Asp Pro Ala Leu Leu Ser Ser Ile Ile Lys Glu Leu His 20 25 30 Leu His Pro Val Ala Ala Gln Thr Phe Ile Ser Arg Gly Phe Gln Thr 35 40 45 Val Asp Glu Val Arg Asp Phe Leu Tyr Val His Leu Ser Asn Leu His 50 55 60 Asp Pro Glu Leu Leu Leu Asp Met Ser Lys Ala Val Gln Arg Leu Leu 65 70 75 80 Leu Ala Lys Glu Arg Gly Glu His Val Met Val Tyr Gly Asp Ser Asp 85 90 95 Val Asp Gly Ile Thr Gly Val Ala Leu Leu Val Glu Phe Leu Arg Ser 100 105 110 Ile Glu Met Lys Val Ser Tyr Cys Phe Leu Gly Ala Phe Leu Lys His 115 120 125 Tyr Gly Glu Pro Ser Leu Leu Ile Ala Lys Met Lys Glu Glu Gly Val 130 135 140 Thr Leu Leu Ile Thr Val Asp Cys Gly Ile Thr Ala Gly Lys Glu Val 145 150 155 160 Ser Asp Ile Asn Lys Gln Gly Ile Asp Val Ile Val Thr Asp His His 165 170 175 Met Pro Thr Gly Lys Ile Pro His Cys Ile Ala Thr Leu Asn Pro Lys 180 185 190 Leu Arg Asp His Thr Tyr Pro Asn Lys Asp Leu Thr Gly Val Gly Val 195 200 205 Ala Phe Lys Leu Ala Arg Gly Val Val Asn Ala Leu Gln Lys Asn Asn 210 215 220 Pro Lys Leu Lys Leu Asp Ile Lys His Leu Leu Asp Leu Val Thr Leu 225 230 235 240 Gly Thr Val Thr Asp Val Gly Thr Leu Leu Gly Glu Asn Arg Thr Met 245 250 255 Val Arg His Gly Ile Lys Glu Ile Ala Lys Gly Ser Arg Leu Gly Leu 260 265 270 Arg Lys Leu Cys Ile Phe Ser Gly Val Lys Pro Ser Glu Val Thr Ser 275 280 285 Thr Asp Ile Val Leu Lys Ile Ser Pro Lys Leu Asn Ser Leu Gly Arg 290 295 300 Leu Ala Asp Ala Ser Lys Gly Val Glu Leu Leu Leu Thr Lys Asp Pro 305 310 315 320 Glu Val Ala Asp Asp Leu Ile Gln Tyr Leu Asp Lys Ile Asn Arg Glu 325 330 335 Arg Gln Lys Ile Glu Ala Asp Val Phe His Asp Val Gln Lys Ile Leu 340 345 350 Lys Asn Gln Pro Asp Ile Val Lys Gln Ala Ala Ile Val Leu Ser Ser 355 360 365 Gln Asp Trp His Ser Arg Val Ile Pro Ile Ile Ser Ala Arg Leu Ala 370 375 380 Lys Ala Tyr Asn Lys Pro Val Ala Ile Ile Ser Asn Gln Gly Gly Ile 385 390 395 400 Gly Lys Gly Ser Leu Arg Thr Ile Gly Ser Phe Pro Leu Leu Gly Ile 405 410 415 Leu Gln Lys Cys Ser Pro Met Phe Ile Ser Tyr Gly Gly His Asp Phe 420 425 430 Ala Ala Gly Ile Ile Ile Asn Glu Asp Arg Ile Glu Ala Phe Arg Lys 435 440 445 Lys Phe Ile His Leu Val Asn Ser Ser Leu Lys Lys Glu Lys Ala Val 450 455 460 Val Thr Leu Pro Leu Asp Ala Arg Ala Asp Phe Asp Glu Ile Asp His 465 470 475 480 Asp Leu Leu Ser Ser Ile Asp Leu Phe Glu Pro Phe Gly Lys Gly Asn 485 490 495 Pro Val Pro Ile Phe Tyr Thr Ile Val His Gln Val Arg Tyr Pro Lys 500 505 510 Leu Leu Pro Gly Asn His Leu Lys Leu Tyr Leu Asn Tyr Gly Glu Arg 515 520 525 Asn Leu Glu Gly Ile Ala Phe Gly Leu Gly Asp Arg Ile Gly Ala Leu 530 535 540 Lys Ala Ser Trp Asn Gln Pro Leu Glu Leu Ala Tyr Thr Pro Arg Leu 545 550 555 560 Ser Gln Ser Ala Asn Gly Gly Val Ile His Leu Leu Val Arg Asp Phe 565 570 575 Arg Ile Leu Pro Leu Asn Tyr Lys Asp Thr Thr Ala Arg 580 585 <210> 12 <211> 1773 <212> DNA <213> Unknown <220> <223> Chlamydia psittaci 6BC <400> 12 atggcaagta aagataattc ttctgtatct aatcctactt ggatatatcc taagtacgat 60 cccgctctac tttcttctat tataaaggag ttacatcttc atccggttgc tgcacagact 120 tttatttctc gaggatttca aacggtagat gaagttcgtg attttcttta tgtacatcta 180 tccaacctgc atgatcccga acttttactc gacatgtcaa aagctgtaca acgtttgctt 240 ctcgcaaaag aacgtggtga gcatgtcatg gtctatggag atagtgatgt tgatgggata 300 acaggcgtgg ctcttcttgt agagtttttg agatctatag aaatgaaggt tagctattgt 360 tttttaggag cattcctgaa acattatggc gaaccttctc tgttgattgc caagatgaag 420 gaagaaggcg tcactttgct gattaccgta gattgcggga ttactgcagg gaaagaagtc 480 agtgatatca ataagcaagg cattgatgtc attgttacag atcatcatat gcctacaggt 540 aaaatccctc attgcatagc aacattaaat ccaaagctca gagatcatac ttatccaaat 600 aaagatctca ctggtgtagg cgtcgctttt aaactcgccc gtggtgttgt taacgcgtta 660 cagaaaaaca atccaaaact caaattagac attaagcatt tattagattt agtaactcta 720 gggacagtga cggatgtggg aaccctttta ggagagaatc gcaccatggt tcgccacggc 780 attaaagaaa tcgccaaagg gtcacggtta gggttgcgta aattatgcat tttttcaggg 840 gtaaaacctt cagaggttac ctctacagat attgttttga aaatttctcc aaaactgaat 900 agtttaggaa gacttgccga cgcttctaaa ggcgttgaat tgttgctgac caaagatcca 960 gaagttgctg atgacctcat tcaatatctc gataagatca atagagaacg ccaaaaaata 1020 gaggctgacg tattccatga tgtacaaaaa atcttaaaaa atcagcccga tatcgttaaa 1080 caagcagcta ttgtactatc gtcacaagat tggcattcca gagtcattcc tattatttca 1140 gcacgcctag cgaaagctta taataagccc gtagcgatta tttctaatca gggagggata 1200 gggaaaggat cgttaagaac aatagggtct ttccctctcc ttggaatatt acaaaagtgc 1260 tctcctatgt tcatatccta tggaggacat gattttgcag caggcattat cattaatgaa 1320 gaccgaatag aagcttttag gaaaaagttc attcatcttg tgaattcatc gttaaaaaag 1380 gaaaaagctg tagttaccct tcccttagat gctcgagctg attttgatga gatagatcac 1440 gatttactct cttccatcga tctgttcgaa ccttttggca aaggtaatcc tgtacctatc 1500 ttttatacca tagtacatca agtacgttat ccgaagttat taccagggaa tcatctgaaa 1560 ctctatctta attacggaga aagaaactta gaaggcatag cttttggatt aggggataga 1620 ataggagctc taaaagcgag ttggaatcaa cctttagaac tggcttatac accacgttta 1680 tcccaatctg ctaacggagg agtcattcat ttgttagtgc gtgattttcg tattcttcca 1740 ctaaattaca aggatacaac agcaaggttt taa 1773 <210> 13 <211> 580 <212> PRT <213> Unknown <220> <223> Finegoldia magna ATCC 29328 <400> 13 Met Asn Lys Trp Leu Ile Asn Asn Arg Gly Asn Asn Tyr Asn Glu Ile 1 5 10 15 Ser Lys Lys Tyr Asn Ile His Pro Leu Ile Ala Lys Ile Leu Leu Asn 20 25 30 Arg Asn Ile Thr Asp Phe Glu Lys Phe Leu Asn Pro Asn Ala Glu Asn 35 40 45 Ser Tyr His Asn Pro Phe Leu Met Lys Asp Met Asp Lys Ala Val Asp 50 55 60 Ile Ile Ile Ser Thr Ile Asp His Asn Glu Lys Ile Arg Ile Val Gly 65 70 75 80 Asp Tyr Asp Gln Asp Gly Asn Ser Ser Thr Met Thr Leu Leu Asp Gly 85 90 95 Leu Gly Tyr Phe Thr Asp Lys Ile Ser Tyr Asp Ile Pro Asn Arg Met 100 105 110 Thr Asp Gly Tyr Gly Ile Ser Phe Ser Ile Ile Asp Lys Cys Ile Glu 115 120 125 Asp Asn Ile Asp Leu Ile Ile Thr Cys Asp Asn Gly Ile Ser Ala Ile 130 135 140 Glu Gln Cys Asp Tyr Ala Arg Lys Asn Gly Ile Lys Ile Ile Val Thr 145 150 155 160 Asp His His Gln Thr Ile Lys His Asp Gly Lys Glu Ile Ile Pro Asn 165 170 175 Ala Asp Ala Val Val Asn Pro Gln Gln Gln Ser Cys Lys Tyr Pro Phe 180 185 190 Lys Ser Leu Cys Gly Ala Gly Val Ala Tyr Lys Leu Ile Gln Ala Ile 195 200 205 Asn Ile Lys Lys Gly Tyr Gly Met Leu Gln Cys Glu Asn Leu Leu Gln 210 215 220 Tyr Val Ala Met Gly Thr Val Cys Asp Ile Val Asp Leu Lys Asp Glu 225 230 235 240 Asn Arg Tyr Phe Val Thr Lys Gly Leu Glu Glu Ile Asn Asn Thr Asp 245 250 255 Asn Tyr Gly Leu Lys Cys Leu Ile Asn Met Thr Gly Ile Lys Asn Ala 260 265 270 Val Asn Val Tyr Ser Leu Gly Phe Ile Ile Gly Pro Cys Ile Asn Ala 275 280 285 Ala Gly Arg Leu Asp Thr Ala Lys Leu Gly Val Glu Leu Phe Arg Asp 290 295 300 Glu Asn Met Asp Asn Val Glu Ala Tyr Ala Lys Ile Leu Val Asp Leu 305 310 315 320 Asn Glu Lys Arg Lys Lys Leu Thr Glu Asp Gly Phe Asn Lys Ala Val 325 330 335 Glu Ile Ile Glu Asn Thr Ala Leu Lys Asn Asp Asp Ile Leu Ile Cys 340 345 350 Asn Val Glu Gly Ile His Glu Ser Val Cys Gly Ile Ile Ala Gly Arg 355 360 365 Ile Lys Glu Lys Tyr Asn Lys Pro Thr Leu Ile Leu Thr Glu Ser Ala 370 375 380 Asn Lys Asn Leu Leu Lys Gly Ser Gly Arg Ser Ile Ser Glu Tyr Asn 385 390 395 400 Ile Phe Glu Glu Phe Asp Glu Phe Arg Glu Met Phe Val Ser Phe Gly 405 410 415 Gly His Pro Met Ala Cys Gly Leu Ser Ile Glu Lys Asn Lys Leu Asp 420 425 430 Glu Phe Arg Thr Lys Val Asn Lys Asn Ser Lys Leu Thr Glu Glu Asp 435 440 445 Phe Val Lys Lys Ile Leu Ile Asp Ser Ser Phe Tyr Ile Asp Lys Ile 450 455 460 Asp Phe Asp Leu Ile Glu Glu Ile Gly Asn Leu Arg Pro Phe Gly Lys 465 470 475 480 Asp Asn Pro Arg Pro Ile Leu Gly Asp Lys Asp Val Glu Ile Ile Phe 485 490 495 Ala Lys Met Ile Gly Lys Asn Gln Asn Val Leu Lys Met Lys Leu Leu 500 505 510 Lys Asn Asn Lys Ala Ile Asp Ala Ile Leu Phe Thr Asp Ala Ile Glu 515 520 525 Lys Tyr Thr Tyr Leu Leu Gly Lys Phe Gly Glu Asn Val Leu Lys Glu 530 535 540 Leu Glu Asn Ser Ile Ser Cys Asn Ala Tyr Ile Asp Ile Leu Tyr Tyr 545 550 555 560 Pro Glu Ile Asn Asp Phe Asn Asn Val Lys Asn Ile Gln Leu Asn Leu 565 570 575 Ile Asp Leu Arg 580 <210> 14 <211> 1743 <212> DNA <213> Unknown <220> <223> Finegoldia magna ATCC 29328 <400> 14 atgaataaat ggttaattaa taatagagga aataattata acgaaatatc aaaaaaatat 60 aatattcatc cattaatagc aaaaattttg ctaaatagaa atatcactga ttttgaaaaa 120 tttttaaatc ctaatgcaga aaattcatat cataatcctt ttttaatgaa agatatggat 180 aaagcagtag atataattat tagcactata gatcataatg aaaaaataag aatagtagga 240 gattatgatc aagatggaaa ttcttctact atgactctac tagatggact tggatatttt 300 acagataaaa tttcctatga tattcctaat agaatgacag atggatatgg catttctttt 360 agtataatag acaaatgtat cgaagataat atagatttaa taataacttg tgacaatggt 420 attagtgcaa ttgaacagtg tgattatgca aggaaaaatg gaattaaaat aattgtaact 480 gatcatcacc aaacaataaa acatgatgga aaagaaataa ttccaaatgc tgatgcggtc 540 gttaacccac aacaacaatc ttgcaaatat ccttttaaaa gtttatgcgg tgcaggagtt 600 gcttataaat taatacaagc tattaatata aaaaaaggat atggaatgct acagtgtgaa 660 aatttgcttc aatatgtagc aatgggaaca gtatgtgaca tagtcgattt aaaagatgag 720 aataggtatt ttgttacaaa aggtctagaa gaaatcaata atacagataa ttacggttta 780 aaatgcttga tcaacatgac tggaattaag aatgcagtaa atgtctacag tttaggattt 840 ataattggtc cttgtatcaa tgcagcagga agattggata ctgcaaaatt aggtgttgaa 900 ctatttagag atgaaaatat ggataatgtt gaagcatatg caaaaatttt agtcgatctt 960 aatgaaaaaa gaaaaaaact aacagaagat ggatttaata aagctgtcga aattatcgaa 1020 aataccgcac taaaaaacga cgatatttta atttgcaatg tggaaggaat tcatgaaagt 1080 gtttgtggaa tcatagctgg aagaataaaa gaaaaatata ataaaccaac tttaatctta 1140 acggaatctg caaataaaaa tttactcaaa ggatctggaa gaagtatatc agaatataat 1200 atttttgagg agtttgatga gtttagagag atgttcgtat cttttggagg ccatcctatg 1260 gcatgtggat tatcaattga aaaaaataaa ttagatgagt ttagaactaa agttaataaa 1320 aatagcaaac ttacagaaga agattttgtt aaaaaaatct tgatagactc tagcttttat 1380 attgataaaa ttgattttga tttgatagaa gaaattggta atttgcgtcc atttggaaaa 1440 gataatccta ggccaatttt aggagataag gatgttgaaa ttatatttgc caaaatgata 1500 gggaagaatc aaaatgtatt aaaaatgaaa ttactaaaaa acaacaaagc aatagatgca 1560 attttgttta cggatgctat agaaaaatat acctatttat taggaaaatt tggtgaaaat 1620 gtattaaagg aacttgaaaa tagtatttct tgtaatgcat atatagatat tttatactat 1680 ccagaaatta atgattttaa taatgtcaaa aatattcaat taaatttaat tgatttaaga 1740 taa 1743 <210> 15 <211> 739 <212> PRT <213> Unknown <220> <223> Streptococcus cristatus AS 1.3089 <400> 15 Met Ile Ser Ser Lys Tyr Asp Trp Gln Phe Ala Thr Asn Phe Thr Asp 1 5 10 15 Glu Lys Phe Leu Lys Lys Ala Lys Lys Ala Gly Leu Glu Pro Ala Ala 20 25 30 Ala Ser Leu Leu Tyr Gln Arg Gly Val Gln Thr Glu Glu Ala Leu Gln 35 40 45 Glu Phe Leu Glu Pro Ser Leu Asp Gln Leu His Asp Pro Tyr Asp Leu 50 55 60 His Asp Met Glu Arg Ala Val Glu Arg Ile Arg Ala Ala Ile Glu Asn 65 70 75 80 Tyr Glu Gln Ile Leu Ile Tyr Gly Asp Tyr Asp Ala Asp Gly Met Thr 85 90 95 Ser Ala Ser Ile Val Lys Glu Ala Leu Glu Gln Leu Gly Ala Glu Cys 100 105 110 Gln Val Tyr Leu Pro Asn Arg Phe Thr Asp Gly Tyr Gly Pro Asn Ser 115 120 125 Ser Val Tyr Lys Tyr Phe Ile Glu Asn Gln Gly Ile Ser Leu Ile Ile 130 135 140 Thr Val Asp Asn Gly Val Ala Gly Leu Glu Ala Ile Glu Leu Ala Gln 145 150 155 160 Ser Leu Gly Val Asp Val Ile Val Thr Asp His His Ser Met Pro Glu 165 170 175 Glu Leu Pro Asn Ala Tyr Ala Ile Val His Pro Glu His Ser Gly Ala 180 185 190 Asp Tyr Pro Phe Lys His Leu Ala Gly Cys Gly Val Ala Phe Lys Leu 195 200 205 Ala Thr Ala Leu Leu Glu Glu Val Gln Val Glu Leu Leu Asp Leu Val 210 215 220 Ala Ile Gly Thr Ile Ala Asp Met Val Ser Leu Thr Gly Glu Asn Arg 225 230 235 240 Ile Leu Val Lys Tyr Gly Leu Ser Val Leu Lys Asn Thr Gln Arg Val 245 250 255 Gly Leu Gln Glu Leu Phe Lys Ile Ala Gly Ile Gln Pro Asp Glu Leu 260 265 270 Asp Glu Glu Thr Val Gly Phe Gln Leu Ala Pro Arg Leu Asn Ala Leu 275 280 285 Gly Arg Leu Asp Asp Pro Asn Pro Ala Ile Glu Leu Leu Thr Gly Phe 290 295 300 Asp Asp Glu Glu Ala Arg Asp Ile Ala Leu Met Ile Asn Gln Lys Asn 305 310 315 320 Asp Glu Arg Lys Glu Ile Val Gln Gln Ile Tyr Lys Glu Ala Gln Thr 325 330 335 Met Leu Asp Pro Lys Arg Pro Val Gln Val Leu Ala Lys Glu Gly Trp 340 345 350 Asn Pro Gly Val Leu Gly Ile Val Ala Gly Arg Leu Leu Glu Glu Leu 355 360 365 His Gln Pro Val Ile Val Leu Asn Ile Glu Asp Gly Ile Ala Lys Gly 370 375 380 Ser Ala Arg Ser Ile Glu Ala Val Asn Ile Phe Glu Ala Leu Asp Ser 385 390 395 400 His Arg Asp Leu Phe Leu Ala Phe Gly Gly His Ala Gly Ala Ala Gly 405 410 415 Met Thr Leu Glu Ala Asp Lys Leu Ala Glu Leu Ala Asp Ile Leu Thr 420 425 430 Glu Tyr Ile Leu Glu Asn Asp Leu Asp Leu Thr Gly Lys Thr Ala Leu 435 440 445 Tyr Leu Asp Glu Glu Leu His Leu Pro Glu Leu Thr Leu Asp Thr Leu 450 455 460 Lys Ser Phe Glu Lys Leu Ala Pro Phe Gly Met Asp Asn Lys Lys Pro 465 470 475 480 Leu Phe Tyr Leu Lys Asp Phe Lys Val Asp Asn Ala Arg Thr Met Gly 485 490 495 Ala Gly Asn Ser His Leu Lys Leu Lys Ile Ser Gln Ala Asp Ala Ser 500 505 510 Phe Glu Val Val Ala Phe Gly Gln Gly Ser Leu Ala Thr Glu Phe Ala 515 520 525 Gln Thr Lys Asn Leu Glu Leu Ala Val Ser Leu Ser Val Asn Lys Trp 530 535 540 Asn Gly Gln Thr Ser Leu Gln Leu Met Leu Val Asp Ala Arg Val Asp 545 550 555 560 Gly Val Gln Leu Phe Asn Ile Arg Gly Lys Asn Ala Thr Leu Pro Asp 565 570 575 Lys Val Pro Val Leu Arg Phe Thr Glu Glu Leu Pro Asp Leu Thr Asn 580 585 590 Ser Arg Ala Val Val Val Tyr Asp Leu Pro Asp Asn Leu Gln Val Leu 595 600 605 Lys Lys Ile Leu Gln Tyr Gln Asp Phe Glu Ala Ile Tyr Phe Lys Asn 610 615 620 Glu Ile Ala Lys Pro Tyr Tyr Leu Thr Gly Tyr Gly Thr Arg Glu Gln 625 630 635 640 Phe Ala Lys Leu Tyr Lys Thr Ile Tyr Gln Phe Pro Glu Phe Asp Val 645 650 655 Arg Tyr Lys Leu Lys Glu Leu Ala Ala Tyr Leu Lys Ile Asp Pro Ile 660 665 670 Leu Leu Val Lys Met Ile Gln Ile Phe Glu Glu Leu Gly Phe Val Ser 675 680 685 Ile Thr Glu Gly Val Met Thr Val Asn Lys Glu Ala Glu Lys Lys Glu 690 695 700 Ile Asp Ser Ser His Ile Tyr Gln Asp Leu Lys Arg Leu Val Lys Glu 705 710 715 720 Gln Glu Leu Met Ala Leu Gly Thr Val Gln Glu Ile Tyr Asp Tyr Leu 725 730 735 Met Asp Cys <210> 16 <211> 2220 <212> DNA <213> Unknown <220> <223> Streptococcus cristatus AS 1.3089 <400> 16 atgattagct caaaatatga ctggcagttt gccactaatt ttacagatga aaaattttta 60 aaaaaggcta agaaagctgg gttagaaccc gcagccgcca gtcttcttta ccaaagaggt 120 gtgcagactg aagaggcctt gcaggaattt ttggagccta gtttagacca gcttcacgac 180 ccttatgacc ttcatgatat ggagcgggca gtggagcgca ttcgcgcagc gattgagaac 240 tacgaacaga ttttgatcta tggcgattat gatgcagatg gcatgacctc ggcatctatt 300 gtcaaggaag ccttggagca gctgggggct gagtgtcagg tctatctgcc caatcgcttt 360 acggatggct atggtcctaa tagcagtgtt tacaagtatt ttattgaaaa tcaaggcatc 420 tcgctcatta ttacagtgga taatggagtg gctggacttg aagctatcga gctggcccag 480 tctctcggcg tcgatgtcat cgtgacggat caccactcca tgcccgagga gctgccaaat 540 gcctatgcga tcgtccatcc ggaacatagc ggagcggatt accctttcaa acatctggct 600 ggctgtgggg tggcttttaa gctggcaaca gccttgttgg aagaagtcca agtcgaactt 660 ttggacttag ttgccattgg caccattgcc gatatggtca gtctgacggg ggaaaatcgg 720 atcttagtca aatatggtct ttcagttctc aaaaataccc agcgagtggg acttcaggag 780 ctcttcaaaa tcgctggtat tcagccagat gaactggatg aagaaacagt tggtttccag 840 cttgccccta gactcaatgc cttgggacgg ttggatgatc ctaatccagc tattgagctt 900 ttgacaggct ttgacgacga agaagctcgt gacattgctc tcatgattaa ccagaaaaat 960 gacgagcgca aggaaatcgt ccagcagatt tataaagaag cacaaaccat gctggaccct 1020 aagagaccag tgcaggtgtt ggccaaggaa ggctggaatc ccggtgtgct agggattgtc 1080 gctgggcgct tgctggaaga gctgcatcag ccagttatag tgctcaatat tgaggatgga 1140 atcgctaagg gcagcgcccg cagtattgaa gcggtcaata tttttgaggc cttggacagt 1200 caccgtgatc tctttctagc ctttggtggg cacgctggag cggctggaat gactcttgaa 1260 gcagacaagc tggcggagct tgctgatatc ttgacagagt atatcttaga aaatgatttg 1320 gatttaactg gcaaaacagc tctgtattta gatgaggagc tgcatctgcc agaactgacc 1380 ctggatacgc tcaaaagctt cgaaaaactg gcgccttttg gtatggataa taagaagcca 1440 cttttttacc tcaaggactt taaagtggac aatgcacgga ctatgggggc tggcaatagc 1500 catctcaaac tgaaaatttc tcaggcagat gcatcttttg aagtagtagc ttttgggcaa 1560 ggaagcctgg cgacagagtt tgcccagact aagaatctgg agctggctgt cagcctctct 1620 gtcaataaat ggaatggaca gaccagtctc cagctcatgc tggtagatgc ccgtgtggac 1680 ggcgttcagc ttttcaatat ccgcggaaaa aatgcgacac tgcctgacaa ggttccagtc 1740 ttgcgtttca cagaggaatt gccggatttg acaaatagca gagcagttgt agtttatgac 1800 ctgccagata atttgcaggt cttgaagaag attcttcagt atcaggattt tgaagctatt 1860 tactttaaga atgaaattgc caagccatac tatctgaccg gttatggtac tcgtgagcaa 1920 tttgctaaac tttacaagac aatctatcag ttccccgagt ttgatgttcg ctataaactc 1980 aaggagctgg cagcttacct gaaaattgat cctattctct tggtcaaaat gattcagatt 2040 tttgaagagc tgggctttgt cagcatcacg gaaggcgtca tgacggtcaa taaggaagct 2100 gaaaagaaag aaattgacag tagtcacatt taccaagacc tcaagcgcct ggtcaaagaa 2160 caagaactta tggctctggg cactgtgcag gagatttacg actatctcat ggattgctag 2220 2220 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker <400> 17 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker <400> 18 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 19 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Flexible linker <400> 19 Gly Gly Gly Gly Gly Gly 1 5 <210> 20 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Rigid linker <400> 20 Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 <210> 21 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Rigid linker <400> 21 Glu Ala Ala Ala Lys 1 5 <210> 22 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Rigid linker <400> 22 Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 <210> 23 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Rigid linker <400> 23 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu Ala 20 25 30 Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala 35 40 45 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Rigid linker <400> 24 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala 1 5 10 <210> 25 <211> 1442 <212> PRT <213> Artificial Sequence <220> <223> SpyCas9 <400> 25 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Asp Lys 35 40 45 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 50 55 60 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 65 70 75 80 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 85 90 95 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 100 105 110 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 115 120 125 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 130 135 140 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 145 150 155 160 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 165 170 175 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 180 185 190 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 195 200 205 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 210 215 220 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 225 230 235 240 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 245 250 255 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 260 265 270 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 275 280 285 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 290 295 300 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 305 310 315 320 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 325 330 335 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 340 345 350 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 355 360 365 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 370 375 380 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 385 390 395 400 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 405 410 415 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 420 425 430 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 435 440 445 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 450 455 460 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 465 470 475 480 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 485 490 495 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 500 505 510 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 515 520 525 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 530 535 540 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 545 550 555 560 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 565 570 575 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 580 585 590 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 595 600 605 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 610 615 620 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 625 630 635 640 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 645 650 655 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 660 665 670 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 675 680 685 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 690 695 700 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 705 710 715 720 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 725 730 735 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 740 745 750 Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His 755 760 765 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 770 775 780 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 785 790 795 800 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 805 810 815 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 820 825 830 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 835 840 845 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 850 855 860 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 865 870 875 880 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 885 890 895 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 900 905 910 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 915 920 925 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 930 935 940 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 945 950 955 960 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 965 970 975 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 980 985 990 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 995 1000 1005 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1010 1015 1020 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1025 1030 1035 1040 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1045 1050 1055 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1060 1065 1070 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1075 1080 1085 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1090 1095 1100 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1105 1110 1115 1120 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1125 1130 1135 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1140 1145 1150 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1155 1160 1165 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1170 1175 1180 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1185 1190 1195 1200 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1205 1210 1215 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1220 1225 1230 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1235 1240 1245 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1250 1255 1260 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1265 1270 1275 1280 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1285 1290 1295 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1300 1305 1310 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1315 1320 1325 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1330 1335 1340 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1345 1350 1355 1360 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1365 1370 1375 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1380 1385 1390 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1395 1400 1405 Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp Leu Glu Lys Arg Pro Ala 1410 1415 1420 Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Glu His His His His 1425 1430 1435 1440 His His <210> 26 <211> 4329 <212> DNA <213> Artificial Sequence <220> <223> SpyCas9 <400> 26 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgga caagaagtac agcatcggcc tggacatcgg taccaacagc 180 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaagtt caaggtgctg 240 ggcaacaccg accgccacag catcaagaag aacctgatcg gcgccctgct gttcgacagc 300 ggcgagaccg ccgaggccac ccgcctgaag cgcaccgccc gccgccgcta cacccgccgc 360 aagaaccgca tctgctacct gcaggagatc ttcagcaacg agatggccaa ggtggacgac 420 agcttcttcc accgcctgga ggagagcttc ctggtggagg aggacaagaa gcacgagcgc 480 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc 540 taccacctgc gcaagaagct ggtggacagc accgacaagg ccgacctgcg cctgatctac 600 ctggccctgg cccacatgat caagttccgc ggccacttcc tgatcgaggg cgacctgaac 660 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg 720 ttcgaggaga accccatcaa cgccagcggc gtggacgcca aggccatcct gagcgcccgc 780 ctgagcaaga gccgccgcct ggagaacctg atcgcccagc tgcccggcga gaagaagaac 840 ggcctgttcg gcaacctgat cgccctgagc ctgggcctga cccccaactt caagagcaac 900 ttcgacctgg ccgaggacgc caagctgcag ctgagcaagg acacctacga cgacgacctg 960 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgttcctggc cgccaagaac 1020 ctgagcgacg ccatcctgct gagcgacatc ctgcgcgtga acaccgagat caccaaggcc 1080 cccctgagcg ccagcatgat caagcgctac gacgagcacc accaggacct gaccctgctg 1140 aaggccctgg tgcgccagca gctgcccgag aagtacaagg agatcttctt cgaccagagc 1200 aagaacggct acgccggcta catcgacggc ggcgccagcc aggaggagtt ctacaagttc 1260 atcaagccca tcctggagaa gatggacggc accgaggagc tgctggtgaa gctgaaccgc 1320 gaggacctgc tgcgcaagca gcgcaccttc gacaacggca gcatccccca ccagatccac 1380 ctgggcgagc tgcacgccat cctgcgccgc caggaggact tctacccctt cctgaaggac 1440 aaccgcgaga agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccccctg 1500 gcccgcggca acagccgctt cgcctggatg acccgcaaga gcgaggagac catcaccccc 1560 tggaacttcg aggaggtggt ggacaagggc gccagcgccc agagcttcat cgagcgcatg 1620 accaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac 1680 gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga gggcatgcgc 1740 aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc 1800 aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttc 1860 gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg cacctaccac 1920 gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa cgaggacatc 1980 ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat cgaggagcgc 2040 ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa gcgccgccgc 2100 tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga caagcagagc 2160 ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa cttcatgcag 2220 ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca ggtgagcggc 2280 cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc catcaagaag 2340 ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg ccgccacaag 2400 cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa gggccagaag 2460 aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg cagccagatc 2520 ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta cctgtactac 2580 ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg cctgagcgac 2640 tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat cgacaacaag 2700 gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag cgaggaggtg 2760 gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat cacccagcgc 2820 aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga caaggccggc 2880 ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc ccagatcctg 2940 gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga ggtgaaggtg 3000 atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt ctacaaggtg 3060 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt ggtgggcacc 3120 gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga ctacaaggtg 3180 tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc caccgccaag 3240 tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct ggccaacggc 3300 gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat cgtgtgggac 3360 aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt gaacatcgtg 3420 aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc caagcgcaac 3480 agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg cggcttcgac 3540 agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg caagagcaag 3600 aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag cagcttcgag 3660 aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa ggacctgatc 3720 atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg catgctggcc 3780 agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta cgtgaacttc 3840 ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa cgagcagaag 3900 cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca gatcagcgag 3960 ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag cgcctacaac 4020 aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct gttcaccctg 4080 accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga ccgcaagcgc 4140 tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat caccggtctg 4200 tacgagaccc gcatcgacct gagccagctg ggcggcgacg cggccgcact cgacctcgag 4260 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaagagca ccaccaccac 4320 caccactga 4329 <210> 27 <211> 1682 <212> PRT <213> Artificial Sequence <220> <223> SpyCas9-GFP <400> 27 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Asp Lys 35 40 45 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 50 55 60 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 65 70 75 80 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 85 90 95 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 100 105 110 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 115 120 125 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 130 135 140 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 145 150 155 160 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 165 170 175 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 180 185 190 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 195 200 205 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 210 215 220 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 225 230 235 240 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 245 250 255 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 260 265 270 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 275 280 285 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 290 295 300 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 305 310 315 320 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 325 330 335 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 340 345 350 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 355 360 365 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 370 375 380 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 385 390 395 400 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 405 410 415 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 420 425 430 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 435 440 445 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 450 455 460 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 465 470 475 480 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 485 490 495 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 500 505 510 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 515 520 525 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 530 535 540 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 545 550 555 560 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 565 570 575 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 580 585 590 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 595 600 605 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 610 615 620 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 625 630 635 640 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 645 650 655 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 660 665 670 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 675 680 685 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 690 695 700 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 705 710 715 720 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 725 730 735 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 740 745 750 Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His 755 760 765 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 770 775 780 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 785 790 795 800 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 805 810 815 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 820 825 830 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 835 840 845 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 850 855 860 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 865 870 875 880 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 885 890 895 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 900 905 910 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 915 920 925 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 930 935 940 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 945 950 955 960 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 965 970 975 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 980 985 990 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 995 1000 1005 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1010 1015 1020 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1025 1030 1035 1040 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1045 1050 1055 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1060 1065 1070 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1075 1080 1085 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1090 1095 1100 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1105 1110 1115 1120 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1125 1130 1135 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1140 1145 1150 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1155 1160 1165 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1170 1175 1180 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1185 1190 1195 1200 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1205 1210 1215 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1220 1225 1230 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1235 1240 1245 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1250 1255 1260 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1265 1270 1275 1280 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1285 1290 1295 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1300 1305 1310 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1315 1320 1325 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1330 1335 1340 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1345 1350 1355 1360 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1365 1370 1375 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1380 1385 1390 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1395 1400 1405 Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp Leu Glu Met Ser Lys Gly 1410 1415 1420 Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly 1425 1430 1435 1440 Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp 1445 1450 1455 Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys 1460 1465 1470 Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Ser Tyr Gly Val 1475 1480 1485 Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 1490 1495 1500 Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Ser Phe 1505 1510 1515 1520 Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly 1525 1530 1535 Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu 1540 1545 1550 Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His 1555 1560 1565 Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn 1570 1575 1580 Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp 1585 1590 1595 1600 His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro 1605 1610 1615 Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn 1620 1625 1630 Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly 1635 1640 1645 Ile Thr His Gly Met Asp Glu Leu Tyr Lys Leu Glu Lys Arg Pro Ala 1650 1655 1660 Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys His His His His 1665 1670 1675 1680 His His <210> 28 <211> 5049 <212> DNA <213> Artificial Sequence <220> <223> SpyCas9-GFP <400> 28 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgga caagaagtac agcatcggcc tggacatcgg taccaacagc 180 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaagtt caaggtgctg 240 ggcaacaccg accgccacag catcaagaag aacctgatcg gcgccctgct gttcgacagc 300 ggcgagaccg ccgaggccac ccgcctgaag cgcaccgccc gccgccgcta cacccgccgc 360 aagaaccgca tctgctacct gcaggagatc ttcagcaacg agatggccaa ggtggacgac 420 agcttcttcc accgcctgga ggagagcttc ctggtggagg aggacaagaa gcacgagcgc 480 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc 540 taccacctgc gcaagaagct ggtggacagc accgacaagg ccgacctgcg cctgatctac 600 ctggccctgg cccacatgat caagttccgc ggccacttcc tgatcgaggg cgacctgaac 660 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg 720 ttcgaggaga accccatcaa cgccagcggc gtggacgcca aggccatcct gagcgcccgc 780 ctgagcaaga gccgccgcct ggagaacctg atcgcccagc tgcccggcga gaagaagaac 840 ggcctgttcg gcaacctgat cgccctgagc ctgggcctga cccccaactt caagagcaac 900 ttcgacctgg ccgaggacgc caagctgcag ctgagcaagg acacctacga cgacgacctg 960 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgttcctggc cgccaagaac 1020 ctgagcgacg ccatcctgct gagcgacatc ctgcgcgtga acaccgagat caccaaggcc 1080 cccctgagcg ccagcatgat caagcgctac gacgagcacc accaggacct gaccctgctg 1140 aaggccctgg tgcgccagca gctgcccgag aagtacaagg agatcttctt cgaccagagc 1200 aagaacggct acgccggcta catcgacggc ggcgccagcc aggaggagtt ctacaagttc 1260 atcaagccca tcctggagaa gatggacggc accgaggagc tgctggtgaa gctgaaccgc 1320 gaggacctgc tgcgcaagca gcgcaccttc gacaacggca gcatccccca ccagatccac 1380 ctgggcgagc tgcacgccat cctgcgccgc caggaggact tctacccctt cctgaaggac 1440 aaccgcgaga agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccccctg 1500 gcccgcggca acagccgctt cgcctggatg acccgcaaga gcgaggagac catcaccccc 1560 tggaacttcg aggaggtggt ggacaagggc gccagcgccc agagcttcat cgagcgcatg 1620 accaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac 1680 gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga gggcatgcgc 1740 aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc 1800 aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttc 1860 gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg cacctaccac 1920 gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa cgaggacatc 1980 ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat cgaggagcgc 2040 ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa gcgccgccgc 2100 tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga caagcagagc 2160 ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa cttcatgcag 2220 ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca ggtgagcggc 2280 cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc catcaagaag 2340 ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg ccgccacaag 2400 cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa gggccagaag 2460 aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg cagccagatc 2520 ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta cctgtactac 2580 ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg cctgagcgac 2640 tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat cgacaacaag 2700 gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag cgaggaggtg 2760 gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat cacccagcgc 2820 aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga caaggccggc 2880 ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc ccagatcctg 2940 gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga ggtgaaggtg 3000 atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt ctacaaggtg 3060 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt ggtgggcacc 3120 gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga ctacaaggtg 3180 tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc caccgccaag 3240 tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct ggccaacggc 3300 gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat cgtgtgggac 3360 aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt gaacatcgtg 3420 aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc caagcgcaac 3480 agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg cggcttcgac 3540 agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg caagagcaag 3600 aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag cagcttcgag 3660 aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa ggacctgatc 3720 atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg catgctggcc 3780 agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta cgtgaacttc 3840 ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa cgagcagaag 3900 cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca gatcagcgag 3960 ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag cgcctacaac 4020 aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct gttcaccctg 4080 accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga ccgcaagcgc 4140 tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat caccggtctg 4200 tacgagaccc gcatcgacct gagccagctg ggcggcgacg cggccgcact cgacctcgag 4260 atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 4320 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 4380 aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 4440 gtcactactt tctcttatgg tgttcaatgc ttttcaagat acccagatca tatgaagcgg 4500 cacgacttct tcaagagcgc catgcctgag ggatacgtgc aggagaggac catctctttc 4560 aaggacgacg ggaactacaa gacacgtgct gaagtcaagt ttgagggaga caccctcgtc 4620 aacaggatcg agcttaaggg aatcgatttc aaggaggacg gaaacatcct cggccacaag 4680 ttggaataca actacaactc ccacaacgta tacatcacgg cagacaaaca aaagaatgga 4740 atcaaagcta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 4800 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 4860 ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 4920 cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaactcgag 4980 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagca ccaccaccac 5040 caccactga 5049 <210> 29 <211> 1953 <212> PRT <213> Artificial Sequence <220> <223> SpyCas9-hTdT <400> 29 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Asp Lys 35 40 45 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 50 55 60 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 65 70 75 80 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 85 90 95 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 100 105 110 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 115 120 125 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 130 135 140 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 145 150 155 160 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 165 170 175 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 180 185 190 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 195 200 205 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 210 215 220 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 225 230 235 240 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 245 250 255 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 260 265 270 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 275 280 285 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 290 295 300 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 305 310 315 320 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 325 330 335 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 340 345 350 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 355 360 365 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 370 375 380 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 385 390 395 400 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 405 410 415 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 420 425 430 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 435 440 445 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 450 455 460 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 465 470 475 480 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 485 490 495 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 500 505 510 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 515 520 525 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 530 535 540 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 545 550 555 560 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 565 570 575 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 580 585 590 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 595 600 605 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 610 615 620 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 625 630 635 640 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 645 650 655 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 660 665 670 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 675 680 685 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 690 695 700 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 705 710 715 720 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 725 730 735 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 740 745 750 Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His 755 760 765 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 770 775 780 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 785 790 795 800 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 805 810 815 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 820 825 830 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 835 840 845 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 850 855 860 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 865 870 875 880 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 885 890 895 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 900 905 910 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 915 920 925 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 930 935 940 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 945 950 955 960 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 965 970 975 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 980 985 990 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 995 1000 1005 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1010 1015 1020 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1025 1030 1035 1040 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1045 1050 1055 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1060 1065 1070 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1075 1080 1085 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1090 1095 1100 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1105 1110 1115 1120 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1125 1130 1135 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1140 1145 1150 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1155 1160 1165 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1170 1175 1180 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1185 1190 1195 1200 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1205 1210 1215 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1220 1225 1230 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1235 1240 1245 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1250 1255 1260 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1265 1270 1275 1280 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1285 1290 1295 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1300 1305 1310 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1315 1320 1325 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1330 1335 1340 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1345 1350 1355 1360 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1365 1370 1375 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1380 1385 1390 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1395 1400 1405 Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp Leu Gln Met Asp Pro Pro 1410 1415 1420 Arg Ala Ser His Leu Ser Pro Arg Lys Lys Arg Pro Arg Gln Thr Gly 1425 1430 1435 1440 Ala Leu Met Ala Ser Ser Pro Gln Asp Ile Lys Phe Gln Asp Leu Val 1445 1450 1455 Val Phe Ile Leu Glu Lys Lys Met Gly Thr Thr Arg Arg Ala Phe Leu 1460 1465 1470 Met Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu Asn Glu Leu Ser 1475 1480 1485 Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser Gly Ser Asp Val 1490 1495 1500 Leu Glu Trp Leu Gln Ala Gln Lys Val Gln Val Ser Ser Gln Pro Glu 1505 1510 1515 1520 Leu Leu Asp Val Ser Trp Leu Ile Glu Cys Ile Gly Ala Gly Lys Pro 1525 1530 1535 Val Glu Met Thr Gly Lys His Gln Leu Val Val Arg Arg Asp Tyr Ser 1540 1545 1550 Asp Ser Thr Asn Pro Gly Pro Pro Lys Thr Pro Pro Ile Ala Val Gln 1555 1560 1565 Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn Cys 1570 1575 1580 Asn Gln Ile Phe Thr Asp Ala Phe Asp Ile Leu Ala Glu Asn Cys Glu 1585 1590 1595 1600 Phe Arg Glu Asn Glu Asp Ser Cys Val Thr Phe Met Arg Ala Ala Ser 1605 1610 1615 Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys Asp Thr Glu 1620 1625 1630 Gly Ile Pro Cys Leu Gly Ser Lys Val Lys Gly Ile Ile Glu Glu Ile 1635 1640 1645 Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu 1650 1655 1660 Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Leu 1665 1670 1675 1680 Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Ser Lys 1685 1690 1695 Val Arg Ser Asp Lys Ser Leu Lys Phe Thr Arg Met Gln Lys Ala Gly 1700 1705 1710 Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu Ala 1715 1720 1725 Glu Ala Val Ser Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro 1730 1735 1740 Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys Met 1745 1750 1755 1760 Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Thr Glu Asp 1765 1770 1775 Glu Glu Gln Leu Leu Gln Lys Val Met Asn Leu Trp Glu Lys Lys Gly 1780 1785 1790 Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Leu Arg 1795 1800 1805 Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe Gln Lys Cys Phe 1810 1815 1820 Leu Ile Phe Lys Leu Pro Arg Gln Arg Val Asp Ser Asp Gln Ser Ser 1825 1830 1835 1840 Trp Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Leu 1845 1850 1855 Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser 1860 1865 1870 Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys 1875 1880 1885 Met Ile Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Ile Phe 1890 1895 1900 Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp 1905 1910 1915 1920 Tyr Ile Glu Pro Trp Glu Arg Asn Ala Leu Gln Lys Arg Pro Ala Ala 1925 1930 1935 Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys His His His His His 1940 1945 1950 His <210> 30 <211> 5862 <212> DNA <213> Artificial Sequence <220> <223> SpyCas9-hTdT <400> 30 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgga caagaagtac agcatcggcc tggacatcgg taccaacagc 180 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaagtt caaggtgctg 240 ggcaacaccg accgccacag catcaagaag aacctgatcg gcgccctgct gttcgacagc 300 ggcgagaccg ccgaggccac ccgcctgaag cgcaccgccc gccgccgcta cacccgccgc 360 aagaaccgca tctgctacct gcaggagatc ttcagcaacg agatggccaa ggtggacgac 420 agcttcttcc accgcctgga ggagagcttc ctggtggagg aggacaagaa gcacgagcgc 480 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc 540 taccacctgc gcaagaagct ggtggacagc accgacaagg ccgacctgcg cctgatctac 600 ctggccctgg cccacatgat caagttccgc ggccacttcc tgatcgaggg cgacctgaac 660 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg 720 ttcgaggaga accccatcaa cgccagcggc gtggacgcca aggccatcct gagcgcccgc 780 ctgagcaaga gccgccgcct ggagaacctg atcgcccagc tgcccggcga gaagaagaac 840 ggcctgttcg gcaacctgat cgccctgagc ctgggcctga cccccaactt caagagcaac 900 ttcgacctgg ccgaggacgc caagctgcag ctgagcaagg acacctacga cgacgacctg 960 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgttcctggc cgccaagaac 1020 ctgagcgacg ccatcctgct gagcgacatc ctgcgcgtga acaccgagat caccaaggcc 1080 cccctgagcg ccagcatgat caagcgctac gacgagcacc accaggacct gaccctgctg 1140 aaggccctgg tgcgccagca gctgcccgag aagtacaagg agatcttctt cgaccagagc 1200 aagaacggct acgccggcta catcgacggc ggcgccagcc aggaggagtt ctacaagttc 1260 atcaagccca tcctggagaa gatggacggc accgaggagc tgctggtgaa gctgaaccgc 1320 gaggacctgc tgcgcaagca gcgcaccttc gacaacggca gcatccccca ccagatccac 1380 ctgggcgagc tgcacgccat cctgcgccgc caggaggact tctacccctt cctgaaggac 1440 aaccgcgaga agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccccctg 1500 gcccgcggca acagccgctt cgcctggatg acccgcaaga gcgaggagac catcaccccc 1560 tggaacttcg aggaggtggt ggacaagggc gccagcgccc agagcttcat cgagcgcatg 1620 accaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac 1680 gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga gggcatgcgc 1740 aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc 1800 aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttc 1860 gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg cacctaccac 1920 gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa cgaggacatc 1980 ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat cgaggagcgc 2040 ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa gcgccgccgc 2100 tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga caagcagagc 2160 ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa cttcatgcag 2220 ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca ggtgagcggc 2280 cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc catcaagaag 2340 ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg ccgccacaag 2400 cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa gggccagaag 2460 aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg cagccagatc 2520 ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta cctgtactac 2580 ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg cctgagcgac 2640 tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat cgacaacaag 2700 gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag cgaggaggtg 2760 gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat cacccagcgc 2820 aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga caaggccggc 2880 ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc ccagatcctg 2940 gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga ggtgaaggtg 3000 atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt ctacaaggtg 3060 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt ggtgggcacc 3120 gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga ctacaaggtg 3180 tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc caccgccaag 3240 tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct ggccaacggc 3300 gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat cgtgtgggac 3360 aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt gaacatcgtg 3420 aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc caagcgcaac 3480 agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg cggcttcgac 3540 agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg caagagcaag 3600 aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag cagcttcgag 3660 aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa ggacctgatc 3720 atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg catgctggcc 3780 agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta cgtgaacttc 3840 ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa cgagcagaag 3900 cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca gatcagcgag 3960 ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag cgcctacaac 4020 aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct gttcaccctg 4080 accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga ccgcaagcgc 4140 tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat caccggtctg 4200 tacgagaccc gcatcgacct gagccagctg ggcggcgacg cggccgcact cgacctgcag 4260 atggatccac cacgagcgtc ccacttgagc cctcggaaga agagaccccg gcagacgggt 4320 gccttgatgg cctcctctcc tcaagacatc aaatttcaag atttggtcgt cttcattttg 4380 gagaagaaaa tgggaaccac ccgcagagcg ttcctcatgg agctggcccg caggaaaggg 4440 ttcagggttg aaaatgagct cagtgattct gtcacccaca ttgtagcaga gaacaactcg 4500 ggttcggatg ttctggagtg gcttcaagca cagaaagtac aagtcagctc acaaccagag 4560 ctcctcgatg tctcctggct gatcgaatgc ataggagcag ggaaaccggt ggaaatgaca 4620 ggaaaacacc agcttgttgt gagaagagac tattcagata gcaccaaccc aggccccccg 4680 aagactccac caattgctgt acaaaagatc tcccagtatg cgtgtcagag aagaaccact 4740 ttaaacaact gtaaccagat attcacggat gcctttgata tactggctga aaactgtgag 4800 tttagagaaa atgaagactc ctgtgtgaca tttatgagag cagcttctgt attgaaatct 4860 ctgccattca caatcatcag tatgaaggac acagaaggaa ttccctgcct ggggtccaag 4920 gtgaagggta tcatagagga gattattgaa gatggagaaa gttctgaagt taaagctgtg 4980 ttaaatgatg aacgatatca atccttcaaa ctctttactt ctgtatttgg agtggggctg 5040 aagacttctg agaagtggtt caggatgggt ttcagaactc tgagtaaagt aaggtcggac 5100 aaaagcctga aatttacacg aatgcagaaa gcaggatttc tgtattatga agaccttgtc 5160 agctgtgtga ccagggcaga agcagaggcc gtcagtgtgc tggttaaaga ggctgtctgg 5220 gcatttcttc cggatgcttt cgtcaccatg acaggagggt tccggagggg taagaagatg 5280 gggcatgatg tagatttttt aattaccagc ccaggatcaa cagaggatga agagcaactt 5340 ttacagaaag tgatgaactt atgggaaaag aagggattac ttttatatta tgaccttgtg 5400 gagtcaacat ttgaaaagct caggttgcct agcaggaagg ttgatgcttt ggatcatttt 5460 caaaagtgct ttctgatttt caaattgcct cgtcaaagag tggacagtga ccagtccagc 5520 tggcaggaag gaaagacctg gaaggccatc cgtgtggatt tagttctgtg cccctacgag 5580 cgtcgtgcct ttgccctgtt gggatggact ggctcccggc agtttgagag agacctccgg 5640 cgctatgcca cacatgagcg gaagatgatt ctggataacc atgctttata tgacaagacc 5700 aagaggatat tcctcaaagc agaaagtgaa gaagaaattt ttgcgcatct gggattggat 5760 tatattgaac cgtgggaaag aaatgccctg cagaaaaggc cggcggccac gaaaaaggcc 5820 ggccaggcaa aaaagaaaaa gcaccaccac caccaccact ga 5862 <210> 31 <211> 2021 <212> PRT <213> Artificial Sequence <220> <223> SpyCas9-RecJ <400> 31 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Asp Lys 35 40 45 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 50 55 60 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 65 70 75 80 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 85 90 95 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 100 105 110 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 115 120 125 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 130 135 140 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 145 150 155 160 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 165 170 175 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 180 185 190 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 195 200 205 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 210 215 220 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 225 230 235 240 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 245 250 255 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 260 265 270 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 275 280 285 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 290 295 300 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 305 310 315 320 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 325 330 335 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 340 345 350 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 355 360 365 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 370 375 380 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 385 390 395 400 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 405 410 415 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 420 425 430 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 435 440 445 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 450 455 460 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 465 470 475 480 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 485 490 495 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 500 505 510 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 515 520 525 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 530 535 540 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 545 550 555 560 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 565 570 575 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 580 585 590 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 595 600 605 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 610 615 620 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 625 630 635 640 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 645 650 655 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 660 665 670 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 675 680 685 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 690 695 700 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 705 710 715 720 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 725 730 735 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 740 745 750 Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His 755 760 765 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 770 775 780 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 785 790 795 800 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 805 810 815 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 820 825 830 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 835 840 845 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 850 855 860 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 865 870 875 880 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 885 890 895 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 900 905 910 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 915 920 925 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 930 935 940 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 945 950 955 960 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 965 970 975 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 980 985 990 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 995 1000 1005 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1010 1015 1020 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1025 1030 1035 1040 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1045 1050 1055 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1060 1065 1070 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1075 1080 1085 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1090 1095 1100 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1105 1110 1115 1120 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1125 1130 1135 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1140 1145 1150 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1155 1160 1165 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1170 1175 1180 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1185 1190 1195 1200 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1205 1210 1215 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1220 1225 1230 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1235 1240 1245 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1250 1255 1260 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1265 1270 1275 1280 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1285 1290 1295 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1300 1305 1310 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1315 1320 1325 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1330 1335 1340 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1345 1350 1355 1360 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1365 1370 1375 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1380 1385 1390 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1395 1400 1405 Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp Leu Gln Val Lys Gln Gln 1410 1415 1420 Ile Gln Leu Arg Arg Arg Glu Val Asp Glu Thr Ala Asp Leu Pro Ala 1425 1430 1435 1440 Glu Leu Pro Pro Leu Leu Arg Arg Leu Tyr Ala Ser Arg Gly Val Arg 1445 1450 1455 Ser Ala Gln Glu Leu Glu Arg Ser Val Lys Gly Met Leu Pro Trp Gln 1460 1465 1470 Gln Leu Ser Gly Val Glu Lys Ala Val Glu Ile Leu Tyr Asn Ala Phe 1475 1480 1485 Arg Glu Gly Thr Arg Ile Ile Val Val Gly Asp Phe Asp Ala Asp Gly 1490 1495 1500 Ala Thr Ser Thr Ala Leu Ser Val Leu Ala Met Arg Ser Leu Gly Cys 1505 1510 1515 1520 Ser Asn Ile Asp Tyr Leu Val Pro Asn Arg Phe Glu Asp Gly Tyr Gly 1525 1530 1535 Leu Ser Pro Glu Val Val Asp Gln Ala His Ala Arg Gly Ala Gln Leu 1540 1545 1550 Ile Val Thr Val Asp Asn Gly Ile Ser Ser His Ala Gly Val Glu His 1555 1560 1565 Ala Arg Ser Leu Gly Ile Pro Val Ile Val Thr Asp His His Leu Pro 1570 1575 1580 Gly Glu Thr Leu Pro Ala Ala Glu Ala Ile Ile Asn Pro Asn Leu Arg 1585 1590 1595 1600 Asp Cys Asn Phe Pro Ser Lys Ser Leu Ala Gly Val Gly Val Ala Phe 1605 1610 1615 Tyr Leu Met Leu Ala Leu Arg Thr Phe Leu Arg Asp Gln Gly Trp Phe 1620 1625 1630 Asp Glu Arg Gly Ile Ala Ile Pro Asn Leu Ala Glu Leu Leu Asp Leu 1635 1640 1645 Val Ala Leu Gly Thr Val Ala Asp Val Val Pro Leu Asp Ala Asn Asn 1650 1655 1660 Arg Ile Leu Thr Trp Gln Gly Met Ser Arg Ile Arg Ala Gly Lys Cys 1665 1670 1675 1680 Arg Pro Gly Ile Lys Ala Leu Leu Glu Val Ala Asn Arg Asp Ala Gln 1685 1690 1695 Lys Leu Ala Ala Ser Asp Leu Gly Phe Ala Leu Gly Pro Arg Leu Asn 1700 1705 1710 Ala Ala Gly Arg Leu Asp Asp Met Ser Val Gly Val Ala Leu Leu Leu 1715 1720 1725 Cys Asp Asn Ile Gly Glu Ala Arg Val Leu Ala Asn Glu Leu Asp Ala 1730 1735 1740 Leu Asn Gln Thr Arg Lys Glu Ile Glu Gln Gly Met Gln Val Glu Ala 1745 1750 1755 1760 Leu Thr Leu Cys Glu Lys Leu Glu Arg Ser Arg Asp Thr Leu Pro Gly 1765 1770 1775 Gly Leu Ala Met Tyr His Pro Glu Trp His Gln Gly Val Val Gly Ile 1780 1785 1790 Leu Ala Ser Arg Ile Lys Glu Arg Phe His Arg Pro Val Ile Ala Phe 1795 1800 1805 Ala Pro Ala Gly Asp Gly Thr Leu Lys Gly Ser Gly Arg Ser Ile Gln 1810 1815 1820 Gly Leu His Met Arg Asp Ala Leu Glu Arg Leu Asp Thr Leu Tyr Pro 1825 1830 1835 1840 Gly Met Ile Leu Lys Phe Gly Gly His Ala Met Ala Ala Gly Leu Ser 1845 1850 1855 Leu Glu Glu Asp Lys Phe Glu Leu Phe Gln Gln Arg Phe Gly Glu Leu 1860 1865 1870 Val Thr Glu Trp Leu Asp Pro Ser Leu Leu Gln Gly Glu Val Val Ser 1875 1880 1885 Asp Gly Pro Leu Ser Pro Ala Glu Met Thr Met Glu Val Ala Gln Leu 1890 1895 1900 Leu Arg Asp Ala Gly Pro Trp Gly Gln Met Phe Pro Glu Pro Leu Phe 1905 1910 1915 1920 Asp Gly His Phe Arg Leu Leu Gln Gln Arg Leu Val Gly Glu Arg His 1925 1930 1935 Leu Lys Val Met Val Glu Pro Val Gly Gly Gly Pro Leu Leu Asp Gly 1940 1945 1950 Ile Ala Phe Asn Val Asp Thr Ala Leu Trp Pro Asp Asn Gly Val Arg 1955 1960 1965 Glu Val Gln Leu Ala Tyr Lys Leu Asp Ile Asn Glu Phe Arg Gly Asn 1970 1975 1980 Arg Ser Leu Gln Ile Ile Ile Asp Asn Ile Trp Pro Ile Leu Gln Lys 1985 1990 1995 2000 Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys His 2005 2010 2015 His His His His His 2020 <210> 32 <211> 6066 <212> DNA <213> Artificial Sequence <220> <223> SpyCas9-RecJ <400> 32 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgga caagaagtac agcatcggcc tggacatcgg taccaacagc 180 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaagtt caaggtgctg 240 ggcaacaccg accgccacag catcaagaag aacctgatcg gcgccctgct gttcgacagc 300 ggcgagaccg ccgaggccac ccgcctgaag cgcaccgccc gccgccgcta cacccgccgc 360 aagaaccgca tctgctacct gcaggagatc ttcagcaacg agatggccaa ggtggacgac 420 agcttcttcc accgcctgga ggagagcttc ctggtggagg aggacaagaa gcacgagcgc 480 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc 540 taccacctgc gcaagaagct ggtggacagc accgacaagg ccgacctgcg cctgatctac 600 ctggccctgg cccacatgat caagttccgc ggccacttcc tgatcgaggg cgacctgaac 660 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg 720 ttcgaggaga accccatcaa cgccagcggc gtggacgcca aggccatcct gagcgcccgc 780 ctgagcaaga gccgccgcct ggagaacctg atcgcccagc tgcccggcga gaagaagaac 840 ggcctgttcg gcaacctgat cgccctgagc ctgggcctga cccccaactt caagagcaac 900 ttcgacctgg ccgaggacgc caagctgcag ctgagcaagg acacctacga cgacgacctg 960 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgttcctggc cgccaagaac 1020 ctgagcgacg ccatcctgct gagcgacatc ctgcgcgtga acaccgagat caccaaggcc 1080 cccctgagcg ccagcatgat caagcgctac gacgagcacc accaggacct gaccctgctg 1140 aaggccctgg tgcgccagca gctgcccgag aagtacaagg agatcttctt cgaccagagc 1200 aagaacggct acgccggcta catcgacggc ggcgccagcc aggaggagtt ctacaagttc 1260 atcaagccca tcctggagaa gatggacggc accgaggagc tgctggtgaa gctgaaccgc 1320 gaggacctgc tgcgcaagca gcgcaccttc gacaacggca gcatccccca ccagatccac 1380 ctgggcgagc tgcacgccat cctgcgccgc caggaggact tctacccctt cctgaaggac 1440 aaccgcgaga agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccccctg 1500 gcccgcggca acagccgctt cgcctggatg acccgcaaga gcgaggagac catcaccccc 1560 tggaacttcg aggaggtggt ggacaagggc gccagcgccc agagcttcat cgagcgcatg 1620 accaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac 1680 gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga gggcatgcgc 1740 aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc 1800 aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttc 1860 gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg cacctaccac 1920 gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa cgaggacatc 1980 ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat cgaggagcgc 2040 ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa gcgccgccgc 2100 tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga caagcagagc 2160 ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa cttcatgcag 2220 ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca ggtgagcggc 2280 cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc catcaagaag 2340 ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg ccgccacaag 2400 cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa gggccagaag 2460 aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg cagccagatc 2520 ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta cctgtactac 2580 ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg cctgagcgac 2640 tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat cgacaacaag 2700 gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag cgaggaggtg 2760 gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat cacccagcgc 2820 aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga caaggccggc 2880 ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc ccagatcctg 2940 gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga ggtgaaggtg 3000 atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt ctacaaggtg 3060 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt ggtgggcacc 3120 gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga ctacaaggtg 3180 tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc caccgccaag 3240 tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct ggccaacggc 3300 gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat cgtgtgggac 3360 aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt gaacatcgtg 3420 aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc caagcgcaac 3480 agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg cggcttcgac 3540 agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg caagagcaag 3600 aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag cagcttcgag 3660 aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa ggacctgatc 3720 atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg catgctggcc 3780 agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta cgtgaacttc 3840 ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa cgagcagaag 3900 cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca gatcagcgag 3960 ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag cgcctacaac 4020 aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct gttcaccctg 4080 accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga ccgcaagcgc 4140 tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat caccggtctg 4200 tacgagaccc gcatcgacct gagccagctg ggcggcgacg cggccgcact cgacctgcag 4260 gtgaaacaac agatacaact tcgtcgccgt gaagtcgatg aaacggcaga cttgcccgct 4320 gaattgcctc ccttgctgcg ccgtttatac gccagccggg gcgtgcgcag tgcgcaagaa 4380 ctggaacgca gtgttaaagg tatgttgccc tggcagcaac tgagcggcgt cgaaaaggcc 4440 gttgagatcc tttacaacgc ttttcgcgaa ggaacgcgga ttattgtggt cggcgatttt 4500 gacgccgacg gcgcgaccag cacggctcta agcgtgctgg cgatgcgctc gcttggttgc 4560 agcaatatcg actatctggt accaaaccgt ttcgaagacg gttacggctt aagcccggaa 4620 gtagtcgatc aggcccatgc ccgtggcgcg cagttaattg tcacggtgga taacggtatt 4680 tcctcccatg cgggcgttga acacgctcgc tcgttgggca ttccggttat tgttaccgat 4740 caccatttgc cgggcgaaac attacccgca gcggaagcga tcattaaccc taacttgcgc 4800 gactgtaatt tcccgtcgaa atcactggca ggcgtgggtg tggcgtttta tctgatgctg 4860 gcgctgcgca cctttttgcg cgatcagggc tggtttgatg agcgtggcat cgcaattcct 4920 aacctggcag aactgctgga tctggtcgcg ctgggaacag tggcggacgt cgtgccgctg 4980 gacgctaata atcgcattct gacctggcag gggatgagtc gcatccgtgc cggaaagtgc 5040 cgtccaggga ttaaagcgct gctggaagtg gcaaaccgtg atgcacaaaa actcgccgcc 5100 agcgatttag gttttgcgct ggggccacgt ctcaatgctg ccggacgact ggacgatatg 5160 tccgtcggtg tggcgctctt gctgtgcgac aacatcggcg aagcgcgcgt gctggcaaat 5220 gaactcgatg cgctaaacca gacgcgaaaa gagatcgaac aaggaatgca agttgaagcc 5280 ctgaccctgt gcgagaaact ggagcgaagt cgcgacacgc tacccggcgg gctggcaatg 5340 tatcaccccg aatggcatca gggcgttgtc ggtattctgg cttcgcgcat caaagagcgt 5400 tttcaccgtc cggttatcgc ctttgcgcca gcaggtgatg gtacgctgaa aggttcaggt 5460 cgctccattc aggggctgca tatgcgtgat gcactggagc gattagacac actctaccct 5520 ggcatgatac tgaagtttgg cggtcatgcg atggcggcgg gtttgtcgct ggaagaggat 5580 aaattcgaac tctttcaaca acggtttggc gagctggtta ccgagtggct ggacccttcg 5640 ctattgcaag gcgaagtggt gtcagacggc ccgttaagcc cggccgaaat gaccatggaa 5700 gtggcgcagc tgctgcgcga tgctggcccg tgggggcaga tgttcccgga gccgctgttt 5760 gatggtcatt tccgtctgct gcaacagcgg ctggtgggcg aacgtcattt gaaagtcatg 5820 gtcgaaccgg tcggcggcgg tccgctgctg gatggtattg cttttaatgt cgataccgcc 5880 ctctggccgg ataacggcgt gcgcgaagtg caactggctt acaagctcga tatcaacgag 5940 tttcgcggca accgcagcct gcaaattatc atcgacaata tctggccaat tctgcagaaa 6000 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaagcacca ccaccaccac 6060 cactga 6066 <210> 33 <211> 2310 <212> PRT <213> Artificial Sequence <220> <223> SpyCas9-RecE <400> 33 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Asp Lys 35 40 45 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 50 55 60 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 65 70 75 80 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 85 90 95 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 100 105 110 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 115 120 125 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 130 135 140 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 145 150 155 160 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 165 170 175 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 180 185 190 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 195 200 205 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 210 215 220 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 225 230 235 240 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 245 250 255 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 260 265 270 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 275 280 285 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 290 295 300 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 305 310 315 320 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 325 330 335 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 340 345 350 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 355 360 365 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 370 375 380 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 385 390 395 400 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 405 410 415 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 420 425 430 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 435 440 445 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 450 455 460 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 465 470 475 480 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 485 490 495 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 500 505 510 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 515 520 525 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 530 535 540 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 545 550 555 560 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 565 570 575 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 580 585 590 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 595 600 605 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 610 615 620 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 625 630 635 640 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 645 650 655 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 660 665 670 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 675 680 685 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 690 695 700 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 705 710 715 720 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 725 730 735 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 740 745 750 Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His 755 760 765 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 770 775 780 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 785 790 795 800 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 805 810 815 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 820 825 830 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 835 840 845 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 850 855 860 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 865 870 875 880 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 885 890 895 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 900 905 910 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 915 920 925 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 930 935 940 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 945 950 955 960 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 965 970 975 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 980 985 990 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 995 1000 1005 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1010 1015 1020 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1025 1030 1035 1040 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1045 1050 1055 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1060 1065 1070 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1075 1080 1085 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1090 1095 1100 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1105 1110 1115 1120 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1125 1130 1135 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1140 1145 1150 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1155 1160 1165 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1170 1175 1180 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1185 1190 1195 1200 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1205 1210 1215 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1220 1225 1230 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1235 1240 1245 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1250 1255 1260 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1265 1270 1275 1280 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1285 1290 1295 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1300 1305 1310 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1315 1320 1325 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1330 1335 1340 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1345 1350 1355 1360 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1365 1370 1375 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1380 1385 1390 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1395 1400 1405 Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp Leu Gln Met Ser Thr Lys 1410 1415 1420 Pro Leu Phe Leu Leu Arg Lys Ala Lys Lys Ser Ser Gly Glu Pro Asp 1425 1430 1435 1440 Val Val Leu Trp Ala Ser Asn Asp Phe Glu Ser Thr Cys Ala Thr Leu 1445 1450 1455 Asp Tyr Leu Ile Val Lys Ser Gly Lys Lys Leu Ser Ser Tyr Phe Lys 1460 1465 1470 Ala Val Ala Thr Asn Phe Pro Val Val Asn Asp Leu Pro Ala Glu Gly 1475 1480 1485 Glu Ile Asp Phe Thr Trp Ser Glu Arg Tyr Gln Leu Ser Lys Asp Ser 1490 1495 1500 Met Thr Trp Glu Leu Lys Pro Gly Ala Ala Pro Asp Asn Ala His Tyr 1505 1510 1515 1520 Gln Gly Asn Thr Asn Val Asn Gly Glu Asp Met Thr Glu Ile Glu Glu 1525 1530 1535 Asn Met Leu Leu Pro Ile Ser Gly Gln Glu Leu Pro Ile Arg Trp Leu 1540 1545 1550 Ala Gln His Gly Ser Glu Lys Pro Val Thr His Val Ser Arg Asp Gly 1555 1560 1565 Leu Gln Ala Leu His Ile Ala Arg Ala Glu Glu Leu Pro Ala Val Thr 1570 1575 1580 Ala Leu Ala Val Ser His Lys Thr Ser Leu Leu Asp Pro Leu Glu Ile 1585 1590 1595 1600 Arg Glu Leu His Lys Leu Val Arg Asp Thr Asp Lys Val Phe Pro Asn 1605 1610 1615 Pro Gly Asn Ser Asn Leu Gly Leu Ile Thr Ala Phe Phe Glu Ala Tyr 1620 1625 1630 Leu Asn Ala Asp Tyr Thr Asp Arg Gly Leu Leu Thr Lys Glu Trp Met 1635 1640 1645 Lys Gly Asn Arg Val Ser His Ile Thr Arg Thr Ala Ser Gly Ala Asn 1650 1655 1660 Ala Gly Gly Gly Asn Leu Thr Asp Arg Gly Glu Gly Phe Val His Asp 1665 1670 1675 1680 Leu Thr Ser Leu Ala Arg Asp Val Ala Thr Gly Val Leu Ala Arg Ser 1685 1690 1695 Met Asp Leu Asp Ile Tyr Asn Leu His Pro Ala His Ala Lys Arg Ile 1700 1705 1710 Glu Glu Ile Ile Ala Glu Asn Lys Pro Pro Phe Ser Val Phe Arg Asp 1715 1720 1725 Lys Phe Ile Thr Met Pro Gly Gly Leu Asp Tyr Ser Arg Ala Ile Val 1730 1735 1740 Val Ala Ser Val Lys Glu Ala Pro Ile Gly Ile Glu Val Ile Pro Ala 1745 1750 1755 1760 His Val Thr Glu Tyr Leu Asn Lys Val Leu Thr Glu Thr Asp His Ala 1765 1770 1775 Asn Pro Asp Pro Glu Ile Val Asp Ile Ala Cys Gly Arg Ser Ser Ala 1780 1785 1790 Pro Met Pro Gln Arg Val Thr Glu Glu Gly Lys Gln Asp Asp Glu Glu 1795 1800 1805 Lys Pro Gln Pro Ser Gly Thr Thr Ala Val Glu Gln Gly Glu Ala Glu 1810 1815 1820 Thr Met Glu Pro Asp Ala Thr Glu His His Gln Asp Thr Gln Pro Leu 1825 1830 1835 1840 Asp Ala Gln Ser Gln Val Asn Ser Val Asp Ala Lys Tyr Gln Glu Leu 1845 1850 1855 Arg Ala Glu Leu His Glu Ala Arg Lys Asn Ile Pro Ser Lys Asn Pro 1860 1865 1870 Val Asp Asp Asp Lys Leu Leu Ala Ala Ser Arg Gly Glu Phe Val Asp 1875 1880 1885 Gly Ile Ser Asp Pro Asn Asp Pro Lys Trp Val Lys Gly Ile Gln Thr 1890 1895 1900 Arg Asp Cys Val Tyr Gln Asn Gln Pro Glu Thr Glu Lys Thr Ser Pro 1905 1910 1915 1920 Asp Met Asn Gln Pro Glu Pro Val Val Gln Gln Glu Pro Glu Ile Ala 1925 1930 1935 Cys Asn Ala Cys Gly Gln Thr Gly Gly Asp Asn Cys Pro Asp Cys Gly 1940 1945 1950 Ala Val Met Gly Asp Ala Thr Tyr Gln Glu Thr Phe Asp Glu Glu Ser 1955 1960 1965 Gln Val Glu Ala Lys Glu Asn Asp Pro Glu Glu Met Glu Gly Ala Glu 1970 1975 1980 His Pro His Asn Glu Asn Ala Gly Ser Asp Pro His Arg Asp Cys Ser 1985 1990 1995 2000 Asp Glu Thr Gly Glu Val Ala Asp Pro Val Ile Val Glu Asp Ile Glu 2005 2010 2015 Pro Gly Ile Tyr Tyr Gly Ile Ser Asn Glu Asn Tyr His Ala Gly Pro 2020 2025 2030 Gly Ile Ser Lys Ser Gln Leu Asp Asp Ile Ala Asp Thr Pro Ala Leu 2035 2040 2045 Tyr Leu Trp Arg Lys Asn Ala Pro Val Asp Thr Thr Lys Thr Lys Thr 2050 2055 2060 Leu Asp Leu Gly Thr Ala Phe His Cys Arg Val Leu Glu Pro Glu Glu 2065 2070 2075 2080 Phe Ser Asn Arg Phe Ile Val Ala Pro Glu Phe Asn Arg Arg Thr Asn 2085 2090 2095 Ala Gly Lys Glu Glu Glu Lys Ala Phe Leu Met Glu Cys Ala Ser Thr 2100 2105 2110 Gly Lys Thr Val Ile Thr Ala Glu Glu Gly Arg Lys Ile Glu Leu Met 2115 2120 2125 Tyr Gln Ser Val Met Ala Leu Pro Leu Gly Gln Trp Leu Val Glu Ser 2130 2135 2140 Ala Gly His Ala Glu Ser Ser Ile Tyr Trp Glu Asp Pro Glu Thr Gly 2145 2150 2155 2160 Ile Leu Cys Arg Cys Arg Pro Asp Lys Ile Ile Pro Glu Phe His Trp 2165 2170 2175 Ile Met Asp Val Lys Thr Thr Ala Asp Ile Gln Arg Phe Lys Thr Ala 2180 2185 2190 Tyr Tyr Asp Tyr Arg Tyr His Val Gln Asp Ala Phe Tyr Ser Asp Gly 2195 2200 2205 Tyr Glu Ala Gln Phe Gly Val Gln Pro Thr Phe Val Phe Leu Val Ala 2210 2215 2220 Ser Thr Thr Ile Glu Cys Gly Arg Tyr Pro Val Glu Ile Phe Met Met 2225 2230 2235 2240 Gly Glu Glu Ala Lys Leu Ala Gly Gln Gln Glu Tyr His Arg Asn Leu 2245 2250 2255 Arg Thr Leu Ser Asp Cys Leu Asn Thr Asp Glu Trp Pro Ala Ile Lys 2260 2265 2270 Thr Leu Ser Leu Pro Arg Trp Ala Lys Glu Tyr Ala Asn Asp Leu Gln 2275 2280 2285 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 2290 2295 2300 His His His His His His 2305 2310 <210> 34 <211> 6933 <212> DNA <213> Artificial Sequence <220> <223> SpyCas9-RecE <400> 34 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgga caagaagtac agcatcggcc tggacatcgg taccaacagc 180 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaagtt caaggtgctg 240 ggcaacaccg accgccacag catcaagaag aacctgatcg gcgccctgct gttcgacagc 300 ggcgagaccg ccgaggccac ccgcctgaag cgcaccgccc gccgccgcta cacccgccgc 360 aagaaccgca tctgctacct gcaggagatc ttcagcaacg agatggccaa ggtggacgac 420 agcttcttcc accgcctgga ggagagcttc ctggtggagg aggacaagaa gcacgagcgc 480 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc 540 taccacctgc gcaagaagct ggtggacagc accgacaagg ccgacctgcg cctgatctac 600 ctggccctgg cccacatgat caagttccgc ggccacttcc tgatcgaggg cgacctgaac 660 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg 720 ttcgaggaga accccatcaa cgccagcggc gtggacgcca aggccatcct gagcgcccgc 780 ctgagcaaga gccgccgcct ggagaacctg atcgcccagc tgcccggcga gaagaagaac 840 ggcctgttcg gcaacctgat cgccctgagc ctgggcctga cccccaactt caagagcaac 900 ttcgacctgg ccgaggacgc caagctgcag ctgagcaagg acacctacga cgacgacctg 960 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgttcctggc cgccaagaac 1020 ctgagcgacg ccatcctgct gagcgacatc ctgcgcgtga acaccgagat caccaaggcc 1080 cccctgagcg ccagcatgat caagcgctac gacgagcacc accaggacct gaccctgctg 1140 aaggccctgg tgcgccagca gctgcccgag aagtacaagg agatcttctt cgaccagagc 1200 aagaacggct acgccggcta catcgacggc ggcgccagcc aggaggagtt ctacaagttc 1260 atcaagccca tcctggagaa gatggacggc accgaggagc tgctggtgaa gctgaaccgc 1320 gaggacctgc tgcgcaagca gcgcaccttc gacaacggca gcatccccca ccagatccac 1380 ctgggcgagc tgcacgccat cctgcgccgc caggaggact tctacccctt cctgaaggac 1440 aaccgcgaga agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccccctg 1500 gcccgcggca acagccgctt cgcctggatg acccgcaaga gcgaggagac catcaccccc 1560 tggaacttcg aggaggtggt ggacaagggc gccagcgccc agagcttcat cgagcgcatg 1620 accaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac 1680 gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga gggcatgcgc 1740 aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc 1800 aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttc 1860 gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg cacctaccac 1920 gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa cgaggacatc 1980 ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat cgaggagcgc 2040 ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa gcgccgccgc 2100 tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga caagcagagc 2160 ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa cttcatgcag 2220 ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca ggtgagcggc 2280 cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc catcaagaag 2340 ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg ccgccacaag 2400 cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa gggccagaag 2460 aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg cagccagatc 2520 ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta cctgtactac 2580 ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg cctgagcgac 2640 tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat cgacaacaag 2700 gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag cgaggaggtg 2760 gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat cacccagcgc 2820 aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga caaggccggc 2880 ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc ccagatcctg 2940 gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga ggtgaaggtg 3000 atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt ctacaaggtg 3060 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt ggtgggcacc 3120 gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga ctacaaggtg 3180 tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc caccgccaag 3240 tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct ggccaacggc 3300 gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat cgtgtgggac 3360 aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt gaacatcgtg 3420 aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc caagcgcaac 3480 agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg cggcttcgac 3540 agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg caagagcaag 3600 aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag cagcttcgag 3660 aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa ggacctgatc 3720 atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg catgctggcc 3780 agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta cgtgaacttc 3840 ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa cgagcagaag 3900 cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca gatcagcgag 3960 ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag cgcctacaac 4020 aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct gttcaccctg 4080 accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga ccgcaagcgc 4140 tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat caccggtctg 4200 tacgagaccc gcatcgacct gagccagctg ggcggcgacg cggccgcact cgacctgcag 4260 atgagcacaa aaccactctt cctgttacgg aaagcgaaaa aatcatccgg tgaacctgac 4320 gtcgtcctgt gggcaagcaa cgattttgaa tcgacctgtg ccactctgga ctacctgatc 4380 gttaagtcag gtaaaaaact gagcagctat tttaaagctg ttgccacgaa ttttcctgtc 4440 gttaatgacc tgcccgctga aggtgagatc gattttacct ggagtgaacg ctatcaactc 4500 agcaaagact ccatgacatg ggaactaaaa ccgggagcag caccagacaa cgctcactat 4560 caaggcaata ccaacgtcaa cggcgaagac atgactgaga ttgaggagaa tatgctactc 4620 ccaatttctg gccaggaact gcccattcgt tggcttgctc aacacggcag cgaaaaaccg 4680 gtaacgcacg tttcacgcga cggactccag gcattacaca ttgctcgggc tgaagaacta 4740 ccggctgtta ctgccctggc tgtttcccac aaaaccagcc tgctcgaccc gctggaaatt 4800 cgcgaactcc acaaactggt tcgtgacact gacaaagttt tccctaatcc tggtaattca 4860 aacctgggac tgataactgc ttttttcgaa gcatacctga acgctgacta caccgatcga 4920 ggactgctga caaaagagtg gatgaagggt aatcgtgttt cacacatcac tcgcacggct 4980 tccggtgcta atgctggcgg cggaaacctc accgatcgcg gcgaaggttt cgtacacgat 5040 ctgacgtcac tggcgcgcga cgtagccact ggcgtactgg cccgttcaat ggatctggac 5100 atctataacc ttcatccggc acacgctaaa cgcattgagg aaattatcgc tgaaaataaa 5160 ccgccctttt ctgttttccg cgacaaattc atcaccatgc ctggcgggct ggattattcc 5220 cgcgccatcg tggttgcgtc cgtaaaagaa gcaccaattg ggatcgaggt catccccgcg 5280 cacgtcactg aatatctgaa caaagtactg actgaaaccg atcatgccaa ccctgatccg 5340 gaaatcgtgg atattgcctg cggtcgctcc tctgccccga tgccgcagcg agtaacagaa 5400 gaaggaaaac aggatgatga agaaaaaccg caaccatctg gaacaacggc agttgaacag 5460 ggagaggctg aaacaatgga accggacgca actgaacatc atcaggacac gcagccgctg 5520 gatgctcagt cacaggtaaa ttctgttgat gcgaaatatc aggaactgcg ggcagaactc 5580 catgaagccc ggaaaaacat tccatcaaaa aatcctgtcg atgacgataa attgcttgct 5640 gcatcacgtg gtgaatttgt tgacggaatt agcgacccga acgatccgaa atgggtaaag 5700 gggatccaga ctcgcgattg tgtgtaccag aaccagccag aaacggaaaa aaccagccca 5760 gatatgaatc aacctgagcc agtagtgcaa caggaaccgg aaatagcctg caatgcctgc 5820 ggccagactg gcggggataa ctgccctgac tgtggtgcgg tgatgggcga cgcaacatac 5880 caggaaacat tcgatgaaga gagtcaggtt gaagctaagg aaaatgatcc ggaggaaatg 5940 gaaggcgctg aacatccgca caatgagaat gctggcagcg atccgcatcg cgattgcagt 6000 gatgaaactg gcgaagtcgc agatcccgta atcgtagaag acatagagcc aggtatttat 6060 tacggaattt cgaatgagaa ttaccacgcg ggtcccggta tcagtaagtc tcagctcgat 6120 gacattgctg atactccggc actatatttg tggcgtaaaa atgcccccgt ggacaccaca 6180 aagacaaaaa cgctcgattt aggaactgct ttccactgcc gggtacttga accggaagaa 6240 ttcagtaacc gctttatcgt agcacctgaa tttaaccgcc gtacaaacgc cggaaaagaa 6300 gaagagaaag cgtttctgat ggaatgcgca agcacaggaa aaacggttat cactgcggaa 6360 gaaggccgga aaattgaact catgtatcaa agcgttatgg ctttgccgct ggggcaatgg 6420 cttgttgaaa gcgccggaca cgctgaatca tcaatttact gggaagatcc tgaaacagga 6480 attttgtgtc ggtgccgtcc ggacaaaatt atccctgaat ttcactggat catggacgtg 6540 aaaactacgg cggatattca acgattcaaa accgcttatt acgactaccg ctatcacgtt 6600 caggatgcat tctacagtga cggttatgaa gcacagtttg gagtgcagcc aactttcgtt 6660 tttctggttg ccagcacaac tattgaatgc ggacgttatc cggttgaaat tttcatgatg 6720 ggcgaagaag caaaactggc aggtcaacag gaatatcacc gcaatctgcg aaccctgtct 6780 gactgcctga ataccgatga atggccagct attaagacat tatcactgcc ccgctgggct 6840 aaggaatatg caaatgacct gcagaaaagg ccggcggcca cgaaaaaggc cggccaggca 6900 aaaaagaaaa agcaccacca ccaccaccac tga 6933 <210> 35 <211> 1670 <212> PRT <213> Artificial Sequence <220> <223> SpyCas9-lambda <400> 35 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Asp Lys 35 40 45 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 50 55 60 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 65 70 75 80 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 85 90 95 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 100 105 110 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 115 120 125 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 130 135 140 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 145 150 155 160 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 165 170 175 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 180 185 190 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 195 200 205 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 210 215 220 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 225 230 235 240 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 245 250 255 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 260 265 270 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 275 280 285 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 290 295 300 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 305 310 315 320 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 325 330 335 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 340 345 350 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 355 360 365 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 370 375 380 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 385 390 395 400 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 405 410 415 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 420 425 430 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 435 440 445 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 450 455 460 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 465 470 475 480 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 485 490 495 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 500 505 510 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 515 520 525 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 530 535 540 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 545 550 555 560 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 565 570 575 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 580 585 590 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 595 600 605 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 610 615 620 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 625 630 635 640 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 645 650 655 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 660 665 670 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 675 680 685 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 690 695 700 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 705 710 715 720 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 725 730 735 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 740 745 750 Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His 755 760 765 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 770 775 780 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 785 790 795 800 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 805 810 815 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 820 825 830 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 835 840 845 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 850 855 860 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 865 870 875 880 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 885 890 895 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 900 905 910 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 915 920 925 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 930 935 940 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 945 950 955 960 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 965 970 975 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 980 985 990 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 995 1000 1005 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1010 1015 1020 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1025 1030 1035 1040 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1045 1050 1055 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1060 1065 1070 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1075 1080 1085 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1090 1095 1100 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1105 1110 1115 1120 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1125 1130 1135 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1140 1145 1150 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1155 1160 1165 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1170 1175 1180 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1185 1190 1195 1200 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1205 1210 1215 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1220 1225 1230 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1235 1240 1245 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1250 1255 1260 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1265 1270 1275 1280 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1285 1290 1295 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1300 1305 1310 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1315 1320 1325 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1330 1335 1340 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1345 1350 1355 1360 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1365 1370 1375 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1380 1385 1390 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1395 1400 1405 Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp Leu Glu Met Thr Pro Asp 1410 1415 1420 Ile Ile Leu Gln Arg Thr Gly Ile Asp Val Arg Ala Val Glu Gln Gly 1425 1430 1435 1440 Asp Asp Ala Trp His Lys Leu Arg Leu Gly Val Ile Thr Ala Ser Glu 1445 1450 1455 Val His Asn Val Ile Ala Lys Pro Arg Ser Gly Lys Lys Trp Pro Asp 1460 1465 1470 Met Lys Met Ser Tyr Phe His Thr Leu Leu Ala Glu Val Cys Thr Gly 1475 1480 1485 Val Ala Pro Glu Val Asn Ala Lys Ala Leu Ala Trp Gly Lys Gln Tyr 1490 1495 1500 Glu Asn Asp Ala Arg Thr Leu Phe Glu Phe Thr Ser Gly Val Asn Val 1505 1510 1515 1520 Thr Glu Ser Pro Ile Ile Tyr Arg Asp Glu Ser Met Arg Thr Ala Cys 1525 1530 1535 Ser Pro Asp Gly Leu Cys Ser Asp Gly Asn Gly Leu Glu Leu Lys Cys 1540 1545 1550 Pro Phe Thr Ser Arg Asp Phe Met Lys Phe Arg Leu Gly Gly Phe Glu 1555 1560 1565 Ala Ile Lys Ser Ala Tyr Met Ala Gln Val Gln Tyr Ser Met Trp Val 1570 1575 1580 Thr Arg Lys Asn Ala Trp Tyr Phe Ala Asn Tyr Asp Pro Arg Met Lys 1585 1590 1595 1600 Arg Glu Gly Leu His Tyr Val Val Ile Glu Arg Asp Glu Lys Tyr Met 1605 1610 1615 Ala Ser Phe Asp Glu Ile Val Pro Glu Phe Ile Glu Lys Met Asp Glu 1620 1625 1630 Ala Leu Ala Glu Ile Gly Phe Val Phe Gly Glu Gln Trp Arg Leu Glu 1635 1640 1645 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1650 1655 1660 His His His His His His 1665 1670 <210> 36 <211> 5013 <212> DNA <213> Artificial Sequence <220> <223> SpyCas9-lambda <400> 36 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgga caagaagtac agcatcggcc tggacatcgg taccaacagc 180 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaagtt caaggtgctg 240 ggcaacaccg accgccacag catcaagaag aacctgatcg gcgccctgct gttcgacagc 300 ggcgagaccg ccgaggccac ccgcctgaag cgcaccgccc gccgccgcta cacccgccgc 360 aagaaccgca tctgctacct gcaggagatc ttcagcaacg agatggccaa ggtggacgac 420 agcttcttcc accgcctgga ggagagcttc ctggtggagg aggacaagaa gcacgagcgc 480 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc 540 taccacctgc gcaagaagct ggtggacagc accgacaagg ccgacctgcg cctgatctac 600 ctggccctgg cccacatgat caagttccgc ggccacttcc tgatcgaggg cgacctgaac 660 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg 720 ttcgaggaga accccatcaa cgccagcggc gtggacgcca aggccatcct gagcgcccgc 780 ctgagcaaga gccgccgcct ggagaacctg atcgcccagc tgcccggcga gaagaagaac 840 ggcctgttcg gcaacctgat cgccctgagc ctgggcctga cccccaactt caagagcaac 900 ttcgacctgg ccgaggacgc caagctgcag ctgagcaagg acacctacga cgacgacctg 960 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgttcctggc cgccaagaac 1020 ctgagcgacg ccatcctgct gagcgacatc ctgcgcgtga acaccgagat caccaaggcc 1080 cccctgagcg ccagcatgat caagcgctac gacgagcacc accaggacct gaccctgctg 1140 aaggccctgg tgcgccagca gctgcccgag aagtacaagg agatcttctt cgaccagagc 1200 aagaacggct acgccggcta catcgacggc ggcgccagcc aggaggagtt ctacaagttc 1260 atcaagccca tcctggagaa gatggacggc accgaggagc tgctggtgaa gctgaaccgc 1320 gaggacctgc tgcgcaagca gcgcaccttc gacaacggca gcatccccca ccagatccac 1380 ctgggcgagc tgcacgccat cctgcgccgc caggaggact tctacccctt cctgaaggac 1440 aaccgcgaga agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccccctg 1500 gcccgcggca acagccgctt cgcctggatg acccgcaaga gcgaggagac catcaccccc 1560 tggaacttcg aggaggtggt ggacaagggc gccagcgccc agagcttcat cgagcgcatg 1620 accaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac 1680 gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga gggcatgcgc 1740 aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc 1800 aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttc 1860 gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg cacctaccac 1920 gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa cgaggacatc 1980 ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat cgaggagcgc 2040 ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa gcgccgccgc 2100 tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga caagcagagc 2160 ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa cttcatgcag 2220 ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca ggtgagcggc 2280 cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc catcaagaag 2340 ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg ccgccacaag 2400 cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa gggccagaag 2460 aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg cagccagatc 2520 ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta cctgtactac 2580 ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg cctgagcgac 2640 tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat cgacaacaag 2700 gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag cgaggaggtg 2760 gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat cacccagcgc 2820 aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga caaggccggc 2880 ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc ccagatcctg 2940 gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga ggtgaaggtg 3000 atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt ctacaaggtg 3060 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt ggtgggcacc 3120 gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga ctacaaggtg 3180 tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc caccgccaag 3240 tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct ggccaacggc 3300 gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat cgtgtgggac 3360 aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt gaacatcgtg 3420 aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc caagcgcaac 3480 agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg cggcttcgac 3540 agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg caagagcaag 3600 aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag cagcttcgag 3660 aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa ggacctgatc 3720 atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg catgctggcc 3780 agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta cgtgaacttc 3840 ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa cgagcagaag 3900 cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca gatcagcgag 3960 ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag cgcctacaac 4020 aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct gttcaccctg 4080 accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga ccgcaagcgc 4140 tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat caccggtctg 4200 tacgagaccc gcatcgacct gagccagctg ggcggcgacg cggccgcact cgacctcgag 4260 atgacaccgg acattatcct gcagcgtacc gggatcgatg tgagagctgt cgaacagggg 4320 gatgatgcgt ggcacaaatt acggctcggc gtcatcaccg cttcagaagt tcacaacgtg 4380 atagcaaaac cccgctccgg aaagaagtgg cctgacatga aaatgtccta cttccacacc 4440 ctgcttgctg aggtttgcac cggtgtggct ccggaagtta acgctaaagc actggcctgg 4500 ggaaaacagt acgagaacga cgccagaacc ctgtttgaat tcacttccgg cgtgaatgtt 4560 actgaatccc cgatcatcta tcgcgacgaa agtatgcgta ccgcctgctc tcccgatggt 4620 ttatgcagtg acggcaacgg ccttgaactg aaatgcccgt ttacctcccg ggatttcatg 4680 aagttccggc tcggtggttt cgaggccata aagtcagctt acatggccca ggtgcagtac 4740 agcatgtggg tgacgcgaaa aaatgcctgg tactttgcca actatgaccc gcgtatgaag 4800 cgtgaaggcc tgcattatgt cgtgattgag cgggatgaaa agtacatggc gagttttgac 4860 gagatcgtgc cggagttcat cgaaaaaatg gacgaggcac tggctgaaat tggttttgta 4920 tttggggagc aatggcgact cgagaaaagg ccggcggcca cgaaaaaggc cggccaggca 4980 aaaaagaaaa agcaccacca ccaccaccac tga 5013 <210> 37 <211> 1799 <212> PRT <213> Artificial Sequence <220> <223> SpyCas9-mungbean <400> 37 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Asp Lys 35 40 45 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 50 55 60 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 65 70 75 80 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 85 90 95 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 100 105 110 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 115 120 125 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 130 135 140 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 145 150 155 160 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 165 170 175 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 180 185 190 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 195 200 205 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 210 215 220 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 225 230 235 240 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 245 250 255 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 260 265 270 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 275 280 285 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 290 295 300 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 305 310 315 320 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 325 330 335 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 340 345 350 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 355 360 365 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 370 375 380 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 385 390 395 400 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 405 410 415 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 420 425 430 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 435 440 445 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 450 455 460 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 465 470 475 480 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 485 490 495 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 500 505 510 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 515 520 525 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 530 535 540 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 545 550 555 560 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 565 570 575 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 580 585 590 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 595 600 605 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 610 615 620 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 625 630 635 640 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 645 650 655 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 660 665 670 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 675 680 685 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 690 695 700 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 705 710 715 720 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 725 730 735 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 740 745 750 Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His 755 760 765 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 770 775 780 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 785 790 795 800 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 805 810 815 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 820 825 830 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 835 840 845 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 850 855 860 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 865 870 875 880 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 885 890 895 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 900 905 910 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 915 920 925 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 930 935 940 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 945 950 955 960 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 965 970 975 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 980 985 990 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 995 1000 1005 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1010 1015 1020 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1025 1030 1035 1040 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1045 1050 1055 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1060 1065 1070 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1075 1080 1085 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1090 1095 1100 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1105 1110 1115 1120 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1125 1130 1135 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1140 1145 1150 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1155 1160 1165 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1170 1175 1180 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1185 1190 1195 1200 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1205 1210 1215 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1220 1225 1230 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1235 1240 1245 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1250 1255 1260 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1265 1270 1275 1280 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1285 1290 1295 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1300 1305 1310 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1315 1320 1325 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1330 1335 1340 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1345 1350 1355 1360 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1365 1370 1375 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1380 1385 1390 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1395 1400 1405 Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp Leu Gln Met Gln Thr Leu 1410 1415 1420 Gln Met Ser Leu Leu Thr Gln Pro Tyr Val Gln Pro Arg Phe Pro Cys 1425 1430 1435 1440 Lys Arg Tyr Pro Thr Phe Ser Ala Ser Cys Arg Thr Gln Lys Thr Ala 1445 1450 1455 Ile Thr Lys Thr Glu Lys Val Phe Phe Ser Glu Ser Phe Asp Gln Thr 1460 1465 1470 Arg Cys Thr Gln Pro Leu Ser Glu Lys Lys Lys Arg Val Phe Phe Leu 1475 1480 1485 Asp Val Asn Pro Leu Cys Tyr Glu Gly Ser Lys Pro Ser Leu Arg Ser 1490 1495 1500 Phe Gly Arg Trp Leu Ser Leu Phe Leu His Gln Val Ser Leu Thr Asp 1505 1510 1515 1520 Pro Val Ile Ala Val Ile Asp Gly Glu Gly Gly Ser Glu His Arg Arg 1525 1530 1535 Lys Leu Leu Pro Ser Tyr Lys Ala His Arg Lys Lys Phe Met Arg His 1540 1545 1550 Met Ser Ser Gly His Val Gly Arg Ser His Gln Val Ile Asn Asp Val 1555 1560 1565 Leu Gly Lys Cys Asn Val Pro Val Ile Lys Val Ala Gly His Glu Ala 1570 1575 1580 Asp Asp Val Val Ala Thr Leu Ala Gly Gln Val Val Asn Lys Gly Phe 1585 1590 1595 1600 Arg Val Val Ile Gly Ser Pro Asp Lys Asp Phe Lys Gln Leu Ile Ser 1605 1610 1615 Glu Asp Val Gln Ile Val Met Pro Leu Pro Glu Leu Gln Arg Trp Ser 1620 1625 1630 Phe Tyr Thr Leu Arg His Tyr Arg Asp Gln Tyr Asn Cys Asp Pro Glu 1635 1640 1645 Ser Asp Leu Ser Phe Arg Cys Ile Val Gly Asp Glu Val Asp Gly Val 1650 1655 1660 Pro Gly Ile Gln His Leu Val Pro Ser Phe Gly Arg Lys Thr Ala Met 1665 1670 1675 1680 Lys Leu Ile Lys Lys His Gly Ser Leu Glu Thr Leu Leu Asn Ala Ala 1685 1690 1695 Ala Ile Arg Thr Val Gly Arg Pro Tyr Ala Gln Asp Ala Leu Lys Asn 1700 1705 1710 His Ala Asp Tyr Leu Arg Arg Asn Tyr Glu Val Leu Ala Leu Lys Arg 1715 1720 1725 Asp Val Asn Ile Gln Leu Tyr Asp Glu Trp Leu Val Lys Arg Asp Asn 1730 1735 1740 His Asn Asp Lys Thr Ala Leu Ser Ser Phe Phe Lys Tyr Leu Gly Glu 1745 1750 1755 1760 Ser Lys Glu Leu Ser Tyr Asn Gly Arg Pro Ile Ser Tyr Asn Gly Leu 1765 1770 1775 Gln Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys 1780 1785 1790 Lys His His His His His His 1795 <210> 38 <211> 5400 <212> DNA <213> Artificial Sequence <220> <223> SpyCas9-mungbean <400> 38 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgga caagaagtac agcatcggcc tggacatcgg taccaacagc 180 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaagtt caaggtgctg 240 ggcaacaccg accgccacag catcaagaag aacctgatcg gcgccctgct gttcgacagc 300 ggcgagaccg ccgaggccac ccgcctgaag cgcaccgccc gccgccgcta cacccgccgc 360 aagaaccgca tctgctacct gcaggagatc ttcagcaacg agatggccaa ggtggacgac 420 agcttcttcc accgcctgga ggagagcttc ctggtggagg aggacaagaa gcacgagcgc 480 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc 540 taccacctgc gcaagaagct ggtggacagc accgacaagg ccgacctgcg cctgatctac 600 ctggccctgg cccacatgat caagttccgc ggccacttcc tgatcgaggg cgacctgaac 660 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg 720 ttcgaggaga accccatcaa cgccagcggc gtggacgcca aggccatcct gagcgcccgc 780 ctgagcaaga gccgccgcct ggagaacctg atcgcccagc tgcccggcga gaagaagaac 840 ggcctgttcg gcaacctgat cgccctgagc ctgggcctga cccccaactt caagagcaac 900 ttcgacctgg ccgaggacgc caagctgcag ctgagcaagg acacctacga cgacgacctg 960 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgttcctggc cgccaagaac 1020 ctgagcgacg ccatcctgct gagcgacatc ctgcgcgtga acaccgagat caccaaggcc 1080 cccctgagcg ccagcatgat caagcgctac gacgagcacc accaggacct gaccctgctg 1140 aaggccctgg tgcgccagca gctgcccgag aagtacaagg agatcttctt cgaccagagc 1200 aagaacggct acgccggcta catcgacggc ggcgccagcc aggaggagtt ctacaagttc 1260 atcaagccca tcctggagaa gatggacggc accgaggagc tgctggtgaa gctgaaccgc 1320 gaggacctgc tgcgcaagca gcgcaccttc gacaacggca gcatccccca ccagatccac 1380 ctgggcgagc tgcacgccat cctgcgccgc caggaggact tctacccctt cctgaaggac 1440 aaccgcgaga agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccccctg 1500 gcccgcggca acagccgctt cgcctggatg acccgcaaga gcgaggagac catcaccccc 1560 tggaacttcg aggaggtggt ggacaagggc gccagcgccc agagcttcat cgagcgcatg 1620 accaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac 1680 gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga gggcatgcgc 1740 aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc 1800 aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttc 1860 gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg cacctaccac 1920 gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa cgaggacatc 1980 ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat cgaggagcgc 2040 ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa gcgccgccgc 2100 tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga caagcagagc 2160 ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa cttcatgcag 2220 ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca ggtgagcggc 2280 cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc catcaagaag 2340 ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg ccgccacaag 2400 cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa gggccagaag 2460 aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg cagccagatc 2520 ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta cctgtactac 2580 ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg cctgagcgac 2640 tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat cgacaacaag 2700 gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag cgaggaggtg 2760 gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat cacccagcgc 2820 aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga caaggccggc 2880 ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc ccagatcctg 2940 gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga ggtgaaggtg 3000 atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt ctacaaggtg 3060 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt ggtgggcacc 3120 gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga ctacaaggtg 3180 tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc caccgccaag 3240 tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct ggccaacggc 3300 gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat cgtgtgggac 3360 aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt gaacatcgtg 3420 aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc caagcgcaac 3480 agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg cggcttcgac 3540 agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg caagagcaag 3600 aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag cagcttcgag 3660 aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa ggacctgatc 3720 atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg catgctggcc 3780 agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta cgtgaacttc 3840 ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa cgagcagaag 3900 cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca gatcagcgag 3960 ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag cgcctacaac 4020 aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct gttcaccctg 4080 accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga ccgcaagcgc 4140 tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat caccggtctg 4200 tacgagaccc gcatcgacct gagccagctg ggcggcgacg cggccgcact cgacctgcag 4260 atgcaaacgt tacagatgag tctgttgaca caaccttacg ttcagcctcg tttcccttgc 4320 aagcgttacc cgaccttctc cgcatcctgc agaactcaaa agacagcgat cacgaaaaca 4380 gagaaggtgt ttttcagtga gtcatttgat caaacacgtt gcacgcagcc tctctcggaa 4440 aagaagaaga gggtgttctt tttggacgtt aacccgctct gttacgaagg aagcaagccc 4500 agcttgcgct ccttcgggcg gtggctctct ctgtttctcc atcaagtcag cctcactgac 4560 cccgtcattg ctgttattga tggagaagga ggcagcgagc atcgcagaaa gttgctacct 4620 tcatataaag cacataggaa aaagttcatg agacacatgt caagtggcca tgttgggagg 4680 tctcatcaag ttataaatga tgttcttgga aaatgcaacg tgccagttat aaaggttgct 4740 ggtcatgaag ctgatgatgt tgtagctact ctagctggac aagttgtcaa taaagggttt 4800 cgagtggtca ttggctcccc tgataaggat tttaagcagc ttatatctga agatgtgcaa 4860 atagttatgc ctttgccaga gttacaaagg tggtccttct acactctgag gcactacagg 4920 gatcagtata attgtgatcc agaatctgat ctgagcttta gatgcattgt aggtgatgaa 4980 gtagacggcg ttcctggtat ccagcatttg gtccctagtt ttggtcggaa gactgctatg 5040 aaacttatta aaaaacatgg ttccttggaa actttattaa atgcggctgc aataaggact 5100 gtaggcagac catatgcaca ggatgccctc aaaaaccatg ctgattacct tcggagaaac 5160 tatgaagttc ttgccttgaa aagggatgta aatatccaac tttatgatga gtggttggtt 5220 aagagagaca atcacaatga taaaactgca ctatcttcct tcttcaaata tttgggagaa 5280 agtaaggagc tcagttacaa tggcagacct atctcttaca atggtctgca gaaaaggccg 5340 gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagc accaccacca ccaccactga 5400 5400 <210> 39 <211> 1720 <212> PRT <213> Artificial Sequence <220> <223> SpyCas9-T5 <400> 39 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Asp Lys 35 40 45 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 50 55 60 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 65 70 75 80 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 85 90 95 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 100 105 110 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 115 120 125 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 130 135 140 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 145 150 155 160 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 165 170 175 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 180 185 190 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 195 200 205 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 210 215 220 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 225 230 235 240 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 245 250 255 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 260 265 270 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 275 280 285 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 290 295 300 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 305 310 315 320 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 325 330 335 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 340 345 350 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 355 360 365 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 370 375 380 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 385 390 395 400 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 405 410 415 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 420 425 430 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 435 440 445 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 450 455 460 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 465 470 475 480 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 485 490 495 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 500 505 510 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 515 520 525 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 530 535 540 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 545 550 555 560 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 565 570 575 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 580 585 590 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 595 600 605 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 610 615 620 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 625 630 635 640 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 645 650 655 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 660 665 670 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 675 680 685 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 690 695 700 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 705 710 715 720 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 725 730 735 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 740 745 750 Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His 755 760 765 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 770 775 780 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 785 790 795 800 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 805 810 815 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 820 825 830 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 835 840 845 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 850 855 860 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 865 870 875 880 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 885 890 895 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 900 905 910 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 915 920 925 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 930 935 940 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 945 950 955 960 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 965 970 975 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 980 985 990 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 995 1000 1005 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1010 1015 1020 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1025 1030 1035 1040 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1045 1050 1055 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1060 1065 1070 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1075 1080 1085 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1090 1095 1100 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1105 1110 1115 1120 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1125 1130 1135 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1140 1145 1150 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1155 1160 1165 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1170 1175 1180 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1185 1190 1195 1200 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1205 1210 1215 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1220 1225 1230 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1235 1240 1245 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1250 1255 1260 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1265 1270 1275 1280 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1285 1290 1295 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1300 1305 1310 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1315 1320 1325 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1330 1335 1340 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1345 1350 1355 1360 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1365 1370 1375 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1380 1385 1390 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1395 1400 1405 Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp Leu Gln Met Ala Ser Arg 1410 1415 1420 Arg Asn Leu Met Ile Val Asp Gly Thr Asn Leu Gly Phe Arg Phe Lys 1425 1430 1435 1440 His Asn Asn Ser Lys Lys Pro Phe Ala Ser Ser Tyr Val Ser Thr Ile 1445 1450 1455 Gln Ser Leu Ala Lys Ser Tyr Ser Ala Arg Thr Thr Ile Val Leu Gly 1460 1465 1470 Asp Lys Gly Lys Ser Val Phe Arg Leu Glu His Leu Pro Glu Tyr Lys 1475 1480 1485 Gly Asn Arg Asp Glu Lys Tyr Ala Gln Arg Thr Glu Glu Glu Lys Ala 1490 1495 1500 Leu Asp Glu Gln Phe Phe Glu Tyr Leu Lys Asp Ala Phe Glu Leu Cys 1505 1510 1515 1520 Lys Thr Thr Phe Pro Thr Phe Thr Ile Arg Gly Val Glu Ala Asp Asp 1525 1530 1535 Met Ala Ala Tyr Ile Val Lys Leu Ile Gly His Leu Tyr Asp His Val 1540 1545 1550 Trp Leu Ile Ser Thr Asp Gly Asp Trp Asp Thr Leu Leu Thr Asp Lys 1555 1560 1565 Val Ser Arg Phe Ser Phe Thr Thr Arg Arg Glu Tyr His Leu Arg Asp 1570 1575 1580 Met Tyr Glu His His Asn Val Asp Asp Val Glu Gln Phe Ile Ser Leu 1585 1590 1595 1600 Lys Ala Ile Met Gly Asp Leu Gly Asp Asn Ile Arg Gly Val Glu Gly 1605 1610 1615 Ile Gly Ala Lys Arg Gly Tyr Asn Ile Ile Arg Glu Phe Gly Asn Val 1620 1625 1630 Leu Asp Ile Ile Asp Gln Leu Pro Leu Pro Gly Lys Gln Lys Tyr Ile 1635 1640 1645 Gln Asn Leu Asn Ala Ser Glu Glu Leu Leu Phe Arg Asn Leu Ile Leu 1650 1655 1660 Val Asp Leu Pro Thr Tyr Cys Val Asp Ala Ile Ala Ala Val Gly Gln 1665 1670 1675 1680 Asp Val Leu Asp Lys Phe Thr Lys Asp Ile Leu Glu Ile Ala Glu Gln 1685 1690 1695 Leu Gln Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys 1700 1705 1710 Lys Lys His His His His His His 1715 1720 <210> 40 <211> 5163 <212> DNA <213> Artificial Sequence <220> <223> SpyCas9-T5 <400> 40 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgga caagaagtac agcatcggcc tggacatcgg taccaacagc 180 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaagtt caaggtgctg 240 ggcaacaccg accgccacag catcaagaag aacctgatcg gcgccctgct gttcgacagc 300 ggcgagaccg ccgaggccac ccgcctgaag cgcaccgccc gccgccgcta cacccgccgc 360 aagaaccgca tctgctacct gcaggagatc ttcagcaacg agatggccaa ggtggacgac 420 agcttcttcc accgcctgga ggagagcttc ctggtggagg aggacaagaa gcacgagcgc 480 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc 540 taccacctgc gcaagaagct ggtggacagc accgacaagg ccgacctgcg cctgatctac 600 ctggccctgg cccacatgat caagttccgc ggccacttcc tgatcgaggg cgacctgaac 660 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg 720 ttcgaggaga accccatcaa cgccagcggc gtggacgcca aggccatcct gagcgcccgc 780 ctgagcaaga gccgccgcct ggagaacctg atcgcccagc tgcccggcga gaagaagaac 840 ggcctgttcg gcaacctgat cgccctgagc ctgggcctga cccccaactt caagagcaac 900 ttcgacctgg ccgaggacgc caagctgcag ctgagcaagg acacctacga cgacgacctg 960 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgttcctggc cgccaagaac 1020 ctgagcgacg ccatcctgct gagcgacatc ctgcgcgtga acaccgagat caccaaggcc 1080 cccctgagcg ccagcatgat caagcgctac gacgagcacc accaggacct gaccctgctg 1140 aaggccctgg tgcgccagca gctgcccgag aagtacaagg agatcttctt cgaccagagc 1200 aagaacggct acgccggcta catcgacggc ggcgccagcc aggaggagtt ctacaagttc 1260 atcaagccca tcctggagaa gatggacggc accgaggagc tgctggtgaa gctgaaccgc 1320 gaggacctgc tgcgcaagca gcgcaccttc gacaacggca gcatccccca ccagatccac 1380 ctgggcgagc tgcacgccat cctgcgccgc caggaggact tctacccctt cctgaaggac 1440 aaccgcgaga agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccccctg 1500 gcccgcggca acagccgctt cgcctggatg acccgcaaga gcgaggagac catcaccccc 1560 tggaacttcg aggaggtggt ggacaagggc gccagcgccc agagcttcat cgagcgcatg 1620 accaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac 1680 gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga gggcatgcgc 1740 aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc 1800 aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttc 1860 gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg cacctaccac 1920 gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa cgaggacatc 1980 ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat cgaggagcgc 2040 ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa gcgccgccgc 2100 tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga caagcagagc 2160 ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa cttcatgcag 2220 ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca ggtgagcggc 2280 cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc catcaagaag 2340 ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg ccgccacaag 2400 cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa gggccagaag 2460 aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg cagccagatc 2520 ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta cctgtactac 2580 ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg cctgagcgac 2640 tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat cgacaacaag 2700 gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag cgaggaggtg 2760 gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat cacccagcgc 2820 aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga caaggccggc 2880 ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc ccagatcctg 2940 gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga ggtgaaggtg 3000 atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt ctacaaggtg 3060 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt ggtgggcacc 3120 gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga ctacaaggtg 3180 tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc caccgccaag 3240 tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct ggccaacggc 3300 gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat cgtgtgggac 3360 aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt gaacatcgtg 3420 aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc caagcgcaac 3480 agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg cggcttcgac 3540 agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg caagagcaag 3600 aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag cagcttcgag 3660 aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa ggacctgatc 3720 atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg catgctggcc 3780 agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta cgtgaacttc 3840 ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa cgagcagaag 3900 cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca gatcagcgag 3960 ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag cgcctacaac 4020 aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct gttcaccctg 4080 accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga ccgcaagcgc 4140 tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat caccggtctg 4200 tacgagaccc gcatcgacct gagccagctg ggcggcgacg cggccgcact cgacctgcag 4260 atggcttccc gtcgtaatct aatgattgtc gatggaacta acttaggctt tcgcttcaaa 4320 cataacaata gtaaaaaacc atttgcctca agttatgttt caactattca atctctggca 4380 aaatcctact ctgccagaac tacgattgtt ctaggtgata agggaaaatc tgtatttcgt 4440 ctagaacatc taccagagta taaaggtaat cgtgatgaaa agtacgcaca acgtacggaa 4500 gaggagaaag cgctagatga gcagttcttt gagtatttga aggatgcttt cgagttgtgt 4560 aaaactacat tcccaacttt taccattcgt ggtgtagaag cagacgatat ggcagcttat 4620 attgttaagc tcatcgggca tctttatgat cacgtttggc taatatctac agatggtgac 4680 tgggatactt tattaacgga taaagtttct cgtttttctt tcacaacacg tcgtgagtat 4740 catcttcgtg atatgtatga acatcataat gttgatgatg ttgagcagtt tatctccctg 4800 aaagcaatta tgggagatct aggagataat attcgtggtg ttgaaggaat aggagcaaaa 4860 cgcggatata atattattcg tgagtttggt aacgtactgg atattattga tcagcttcca 4920 ctgcctggaa agcagaaata tatacagaac ctgaatgcat cggaagaact gcttttccga 4980 aacttgattc tggttgattt acctacctac tgtgtggatg ctattgctgc tgtaggtcaa 5040 gatgtgttag ataagtttac aaaagatatt ttggagattg cagaacaact gcagaaaagg 5100 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agcaccacca ccaccaccac 5160 tga 5163 <210> 41 <211> 2021 <212> PRT <213> Artificial Sequence <220> <223> RecJ-SpyCas9 <400> 41 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Val Lys Gln Gln Ile Gln Leu Arg Arg Arg Glu Val Asp Glu 35 40 45 Thr Ala Asp Leu Pro Ala Glu Leu Pro Pro Leu Leu Arg Arg Leu Tyr 50 55 60 Ala Ser Arg Gly Val Arg Ser Ala Gln Glu Leu Glu Arg Ser Val Lys 65 70 75 80 Gly Met Leu Pro Trp Gln Gln Leu Ser Gly Val Glu Lys Ala Val Glu 85 90 95 Ile Leu Tyr Asn Ala Phe Arg Glu Gly Thr Arg Ile Ile Val Val Gly 100 105 110 Asp Phe Asp Ala Asp Gly Ala Thr Ser Thr Ala Leu Ser Val Leu Ala 115 120 125 Met Arg Ser Leu Gly Cys Ser Asn Ile Asp Tyr Leu Val Pro Asn Arg 130 135 140 Phe Glu Asp Gly Tyr Gly Leu Ser Pro Glu Val Val Asp Gln Ala His 145 150 155 160 Ala Arg Gly Ala Gln Leu Ile Val Thr Val Asp Asn Gly Ile Ser Ser 165 170 175 His Ala Gly Val Glu His Ala Arg Ser Leu Gly Ile Pro Val Ile Val 180 185 190 Thr Asp His His Leu Pro Gly Glu Thr Leu Pro Ala Ala Glu Ala Ile 195 200 205 Ile Asn Pro Asn Leu Arg Asp Cys Asn Phe Pro Ser Lys Ser Leu Ala 210 215 220 Gly Val Gly Val Ala Phe Tyr Leu Met Leu Ala Leu Arg Thr Phe Leu 225 230 235 240 Arg Asp Gln Gly Trp Phe Asp Glu Arg Gly Ile Ala Ile Pro Asn Leu 245 250 255 Ala Glu Leu Leu Asp Leu Val Ala Leu Gly Thr Val Ala Asp Val Val 260 265 270 Pro Leu Asp Ala Asn Asn Arg Ile Leu Thr Trp Gln Gly Met Ser Arg 275 280 285 Ile Arg Ala Gly Lys Cys Arg Pro Gly Ile Lys Ala Leu Leu Glu Val 290 295 300 Ala Asn Arg Asp Ala Gln Lys Leu Ala Ala Ser Asp Leu Gly Phe Ala 305 310 315 320 Leu Gly Pro Arg Leu Asn Ala Ala Gly Arg Leu Asp Asp Met Ser Val 325 330 335 Gly Val Ala Leu Leu Leu Cys Asp Asn Ile Gly Glu Ala Arg Val Leu 340 345 350 Ala Asn Glu Leu Asp Ala Leu Asn Gln Thr Arg Lys Glu Ile Glu Gln 355 360 365 Gly Met Gln Val Glu Ala Leu Thr Leu Cys Glu Lys Leu Glu Arg Ser 370 375 380 Arg Asp Thr Leu Pro Gly Gly Leu Ala Met Tyr His Pro Glu Trp His 385 390 395 400 Gln Gly Val Val Gly Ile Leu Ala Ser Arg Ile Lys Glu Arg Phe His 405 410 415 Arg Pro Val Ile Ala Phe Ala Pro Ala Gly Asp Gly Thr Leu Lys Gly 420 425 430 Ser Gly Arg Ser Ile Gln Gly Leu His Met Arg Asp Ala Leu Glu Arg 435 440 445 Leu Asp Thr Leu Tyr Pro Gly Met Ile Leu Lys Phe Gly Gly His Ala 450 455 460 Met Ala Ala Gly Leu Ser Leu Glu Glu Asp Lys Phe Glu Leu Phe Gln 465 470 475 480 Gln Arg Phe Gly Glu Leu Val Thr Glu Trp Leu Asp Pro Ser Leu Leu 485 490 495 Gln Gly Glu Val Val Ser Asp Gly Pro Leu Ser Pro Ala Glu Met Thr 500 505 510 Met Glu Val Ala Gln Leu Leu Arg Asp Ala Gly Pro Trp Gly Gln Met 515 520 525 Phe Pro Glu Pro Leu Phe Asp Gly His Phe Arg Leu Leu Gln Gln Arg 530 535 540 Leu Val Gly Glu Arg His Leu Lys Val Met Val Glu Pro Val Gly Gly 545 550 555 560 Gly Pro Leu Leu Asp Gly Ile Ala Phe Asn Val Asp Thr Ala Leu Trp 565 570 575 Pro Asp Asn Gly Val Arg Glu Val Gln Leu Ala Tyr Lys Leu Asp Ile 580 585 590 Asn Glu Phe Arg Gly Asn Arg Ser Leu Gln Ile Ile Ile Asp Asn Ile 595 600 605 Trp Pro Ile Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg 610 615 620 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 625 630 635 640 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 645 650 655 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 660 665 670 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 675 680 685 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 690 695 700 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 705 710 715 720 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 725 730 735 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 740 745 750 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 755 760 765 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 770 775 780 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 785 790 795 800 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 805 810 815 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 820 825 830 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 835 840 845 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 850 855 860 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 865 870 875 880 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 885 890 895 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 900 905 910 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 915 920 925 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 930 935 940 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 945 950 955 960 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 965 970 975 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 980 985 990 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 995 1000 1005 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 1010 1015 1020 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 1025 1030 1035 1040 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 1045 1050 1055 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 1060 1065 1070 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 1075 1080 1085 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 1090 1095 1100 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 1105 1110 1115 1120 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 1125 1130 1135 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 1140 1145 1150 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 1155 1160 1165 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 1170 1175 1180 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 1185 1190 1195 1200 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 1205 1210 1215 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 1220 1225 1230 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 1235 1240 1245 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 1250 1255 1260 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 1265 1270 1275 1280 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 1285 1290 1295 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 1300 1305 1310 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 1315 1320 1325 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 1330 1335 1340 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 1345 1350 1355 1360 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 1365 1370 1375 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 1380 1385 1390 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 1395 1400 1405 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 1410 1415 1420 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 1425 1430 1435 1440 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 1445 1450 1455 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 1460 1465 1470 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 1475 1480 1485 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 1490 1495 1500 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 1505 1510 1515 1520 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 1525 1530 1535 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 1540 1545 1550 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 1555 1560 1565 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 1570 1575 1580 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 1585 1590 1595 1600 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 1605 1610 1615 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 1620 1625 1630 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys 1635 1640 1645 Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser 1650 1655 1660 Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu 1665 1670 1675 1680 Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile 1685 1690 1695 Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser 1700 1705 1710 Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly 1715 1720 1725 Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile 1730 1735 1740 Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 1745 1750 1755 1760 Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly 1765 1770 1775 Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 1780 1785 1790 Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 1795 1800 1805 Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys 1810 1815 1820 Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser 1825 1830 1835 1840 Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr 1845 1850 1855 Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1860 1865 1870 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His 1875 1880 1885 Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val 1890 1895 1900 Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys 1905 1910 1915 1920 His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu 1925 1930 1935 Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp 1940 1945 1950 Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 1955 1960 1965 Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile 1970 1975 1980 Asp Leu Ser Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp Leu Glu Lys 1985 1990 1995 2000 Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys His 2005 2010 2015 His His His His His 2020 <210> 42 <211> 6066 <212> DNA <213> Artificial Sequence <220> <223> RecJ-SpyCas9 <400> 42 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgtgaaaca acagatacaa 120 cttcgtcgcc gtgaagtcga tgaaacggca gacttgcccg ctgaattgcc tcccttgctg 180 cgccgtttat acgccagccg gggcgtgcgc agtgcgcaag aactggaacg cagtgttaaa 240 ggtatgttgc cctggcagca actgagcggc gtcgaaaagg ccgttgagat cctttacaac 300 gcttttcgcg aaggaacgcg gattattgtg gtcggcgatt ttgacgccga cggcgcgacc 360 agcacggctc taagcgtgct ggcgatgcgc tcgcttggtt gcagcaatat cgactatctg 420 gtaccaaacc gtttcgaaga cggttacggc ttaagcccgg aagtagtcga tcaggcccat 480 gcccgtggcg cgcagttaat tgtcacggtg gataacggta tttcctccca tgcgggcgtt 540 gaacacgctc gctcgttggg cattccggtt attgttaccg atcaccattt gccgggcgaa 600 acattacccg cagcggaagc gatcattaac cctaacttgc gcgactgtaa tttcccgtcg 660 aaatcactgg caggcgtggg tgtggcgttt tatctgatgc tggcgctgcg cacctttttg 720 cgcgatcagg gctggtttga tgagcgtggc atcgcaattc ctaacctggc agaactgctg 780 gatctggtcg cgctgggaac agtggcggac gtcgtgccgc tggacgctaa taatcgcatt 840 ctgacctggc aggggatgag tcgcatccgt gccggaaagt gccgtccagg gattaaagcg 900 ctgctggaag tggcaaaccg tgatgcacaa aaactcgccg ccagcgattt aggttttgcg 960 ctggggccac gtctcaatgc tgccggacga ctggacgata tgtccgtcgg tgtggcgctc 1020 ttgctgtgcg acaacatcgg cgaagcgcgc gtgctggcaa atgaactcga tgcgctaaac 1080 cagacgcgaa aagagatcga acaaggaatg caagttgaag ccctgaccct gtgcgagaaa 1140 ctggagcgaa gtcgcgacac gctacccggc gggctggcaa tgtatcaccc cgaatggcat 1200 cagggcgttg tcggtattct ggcttcgcgc atcaaagagc gttttcaccg tccggttatc 1260 gcctttgcgc cagcaggtga tggtacgctg aaaggttcag gtcgctccat tcaggggctg 1320 catatgcgtg atgcactgga gcgattagac acactctacc ctggcatgat actgaagttt 1380 ggcggtcatg cgatggcggc gggtttgtcg ctggaagagg ataaattcga actctttcaa 1440 caacggtttg gcgagctggt taccgagtgg ctggaccctt cgctattgca aggcgaagtg 1500 gtgtcagacg gcccgttaag cccggccgaa atgaccatgg aagtggcgca gctgctgcgc 1560 gatgctggcc cgtgggggca gatgttcccg gagccgctgt ttgatggtca tttccgtctg 1620 ctgcaacagc ggctggtggg cgaacgtcat ttgaaagtca tggtcgaacc ggtcggcggc 1680 ggtccgctgc tggatggtat tgcttttaat gtcgataccg ccctctggcc ggataacggc 1740 gtgcgcgaag tgcaactggc ttacaagctc gatatcaacg agtttcgcgg caaccgcagc 1800 ctgcaaatta tcatcgacaa tatctggcca attggatccg aattcgagct ccgtcgacaa 1860 gcttgcggcc gcatggacaa gaagtacagc atcggcctgg acatcggtac caacagcgtg 1920 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaagttcaa ggtgctgggc 1980 aacaccgacc gccacagcat caagaagaac ctgatcggcg ccctgctgtt cgacagcggc 2040 gagaccgccg aggccacccg cctgaagcgc accgcccgcc gccgctacac ccgccgcaag 2100 aaccgcatct gctacctgca ggagatcttc agcaacgaga tggccaaggt ggacgacagc 2160 ttcttccacc gcctggagga gagcttcctg gtggaggagg acaagaagca cgagcgccac 2220 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac 2280 cacctgcgca agaagctggt ggacagcacc gacaaggccg acctgcgcct gatctacctg 2340 gccctggccc acatgatcaa gttccgcggc cacttcctga tcgagggcga cctgaacccc 2400 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc 2460 gaggagaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgag cgcccgcctg 2520 agcaagagcc gccgcctgga gaacctgatc gcccagctgc ccggcgagaa gaagaacggc 2580 ctgttcggca acctgatcgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc 2640 gacctggccg aggacgccaa gctgcagctg agcaaggaca cctacgacga cgacctggac 2700 aacctgctgg cccagatcgg cgaccagtac gccgacctgt tcctggccgc caagaacctg 2760 agcgacgcca tcctgctgag cgacatcctg cgcgtgaaca ccgagatcac caaggccccc 2820 ctgagcgcca gcatgatcaa gcgctacgac gagcaccacc aggacctgac cctgctgaag 2880 gccctggtgc gccagcagct gcccgagaag tacaaggaga tcttcttcga ccagagcaag 2940 aacggctacg ccggctacat cgacggcggc gccagccagg aggagttcta caagttcatc 3000 aagcccatcc tggagaagat ggacggcacc gaggagctgc tggtgaagct gaaccgcgag 3060 gacctgctgc gcaagcagcg caccttcgac aacggcagca tcccccacca gatccacctg 3120 ggcgagctgc acgccatcct gcgccgccag gaggacttct accccttcct gaaggacaac 3180 cgcgagaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccccctggcc 3240 cgcggcaaca gccgcttcgc ctggatgacc cgcaagagcg aggagaccat caccccctgg 3300 aacttcgagg aggtggtgga caagggcgcc agcgcccaga gcttcatcga gcgcatgacc 3360 aacttcgaca agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag 3420 tacttcaccg tgtacaacga gctgaccaag gtgaagtacg tgaccgaggg catgcgcaag 3480 cccgccttcc tgagcggcga gcagaagaag gccatcgtgg acctgctgtt caagaccaac 3540 cgcaaggtga ccgtgaagca gctgaaggag gactacttca agaagatcga gtgcttcgac 3600 agcgtggaga tcagcggcgt ggaggaccgc ttcaacgcca gcctgggcac ctaccacgac 3660 ctgctgaaga tcatcaagga caaggacttc ctggacaacg aggagaacga ggacatcctg 3720 gaggacatcg tgctgaccct gaccctgttc gaggaccgcg agatgatcga ggagcgcctg 3780 aagacctacg cccacctgtt cgacgacaag gtgatgaagc agctgaagcg ccgccgctac 3840 accggctggg gccgcctgag ccgcaagctt atcaacggca tccgcgacaa gcagagcggc 3900 aagaccatcc tggacttcct gaagagcgac ggcttcgcca accgcaactt catgcagctg 3960 atccacgacg acagcctgac cttcaaggag gacatccaga aggcccaggt gagcggccag 4020 ggcgacagcc tgcacgagca catcgccaac ctggccggca gccccgccat caagaagggc 4080 atcctgcaga ccgtgaaggt ggtggacgag ctggtgaagg tgatgggccg ccacaagccc 4140 gagaacatcg tgatcgagat ggcccgcgag aaccagacca cccagaaggg ccagaagaac 4200 agccgcgagc gcatgaagcg catcgaggag ggcatcaagg agctgggcag ccagatcctg 4260 aaggagcacc ccgtggagaa cacccagctg cagaacgaga agctgtacct gtactacctg 4320 cagaacggcc gcgacatgta cgtggaccag gagctggaca tcaaccgcct gagcgactac 4380 gacgtggacc acatcgtgcc ccagagcttc ctgaaggacg acagcatcga caacaaggtg 4440 ctgacccgca gcgacaagaa ccgcggcaag agcgacaacg tgcccagcga ggaggtggtg 4500 aagaagatga agaactactg gcgccagctg ctgaacgcca agctgatcac ccagcgcaag 4560 ttcgacaacc tgaccaaggc cgagcgcggc ggcctgagcg agctggacaa ggccggcttc 4620 atcaagcgcc agctggtgga gacccgccag atcaccaagc acgtggccca gatcctggac 4680 agccgcatga acaccaagta cgacgagaac gacaagctga tccgcgaggt gaaggtgatc 4740 accctgaaga gcaagctggt gagcgacttc cgcaaggact tccagttcta caaggtgcgc 4800 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtggt gggcaccgcc 4860 ctgatcaaga agtaccccaa gctggagagc gagttcgtgt acggcgacta caaggtgtac 4920 gacgtgcgca agatgatcgc caagagcgag caggagatcg gcaaggccac cgccaagtac 4980 ttcttctaca gcaacatcat gaacttcttc aagaccgaga tcaccctggc caacggcgag 5040 atccgcaagc gccccctgat cgagaccaac ggcgagaccg gcgagatcgt gtgggacaag 5100 ggccgcgact tcgccaccgt gcgcaaggtg ctgagcatgc cccaggtgaa catcgtgaag 5160 aagaccgagg tgcagaccgg cggcttcagc aaggagagca tcctgcccaa gcgcaacagc 5220 gacaagctga tcgcccgcaa gaaggactgg gaccccaaga agtacggcgg cttcgacagc 5280 cccaccgtgg cctacagcgt gctggtggtg gccaaggtgg agaagggcaa gagcaagaag 5340 ctgaagagcg tgaaggagct gctgggcatc accatcatgg agcgcagcag cttcgagaag 5400 aaccccatcg acttcctgga ggccaagggc tacaaggagg tgaagaagga cctgatcatc 5460 aagctgccca agtacagcct gttcgagctg gagaacggcc gcaagcgcat gctggccagc 5520 gccggcgagc tgcagaaggg caacgagctg gccctgccca gcaagtacgt gaacttcctg 5580 tacctggcca gccactacga gaagctgaag ggcagccccg aggacaacga gcagaagcag 5640 ctgttcgtgg agcagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc 5700 agcaagcgcg tgatcctggc cgacgccaac ctggacaagg tgctgagcgc ctacaacaag 5760 caccgcgaca agcccatccg cgagcaggcc gagaacatca tccacctgtt caccctgacc 5820 aacctgggcg cccccgccgc cttcaagtac ttcgacacca ccatcgaccg caagcgctac 5880 accagcacca aggaggtgct ggacgccacc ctgatccacc agagcatcac cggtctgtac 5940 gagacccgca tcgacctgag ccagctgggc ggcgacgcgg ccgcactcga cctcgagaaa 6000 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaagcacca ccaccaccac 6060 cactga 6066 <210> 43 <211> 2310 <212> PRT <213> Artificial Sequence <220> <223> RecE-SpyCas9 <400> 43 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Ser Thr Lys Pro Leu Phe Leu Leu Arg Lys Ala Lys Lys 35 40 45 Ser Ser Gly Glu Pro Asp Val Val Leu Trp Ala Ser Asn Asp Phe Glu 50 55 60 Ser Thr Cys Ala Thr Leu Asp Tyr Leu Ile Val Lys Ser Gly Lys Lys 65 70 75 80 Leu Ser Ser Tyr Phe Lys Ala Val Ala Thr Asn Phe Pro Val Val Asn 85 90 95 Asp Leu Pro Ala Glu Gly Glu Ile Asp Phe Thr Trp Ser Glu Arg Tyr 100 105 110 Gln Leu Ser Lys Asp Ser Met Thr Trp Glu Leu Lys Pro Gly Ala Ala 115 120 125 Pro Asp Asn Ala His Tyr Gln Gly Asn Thr Asn Val Asn Gly Glu Asp 130 135 140 Met Thr Glu Ile Glu Glu Asn Met Leu Leu Pro Ile Ser Gly Gln Glu 145 150 155 160 Leu Pro Ile Arg Trp Leu Ala Gln His Gly Ser Glu Lys Pro Val Thr 165 170 175 His Val Ser Arg Asp Gly Leu Gln Ala Leu His Ile Ala Arg Ala Glu 180 185 190 Glu Leu Pro Ala Val Thr Ala Leu Ala Val Ser His Lys Thr Ser Leu 195 200 205 Leu Asp Pro Leu Glu Ile Arg Glu Leu His Lys Leu Val Arg Asp Thr 210 215 220 Asp Lys Val Phe Pro Asn Pro Gly Asn Ser Asn Leu Gly Leu Ile Thr 225 230 235 240 Ala Phe Phe Glu Ala Tyr Leu Asn Ala Asp Tyr Thr Asp Arg Gly Leu 245 250 255 Leu Thr Lys Glu Trp Met Lys Gly Asn Arg Val Ser His Ile Thr Arg 260 265 270 Thr Ala Ser Gly Ala Asn Ala Gly Gly Gly Asn Leu Thr Asp Arg Gly 275 280 285 Glu Gly Phe Val His Asp Leu Thr Ser Leu Ala Arg Asp Val Ala Thr 290 295 300 Gly Val Leu Ala Arg Ser Met Asp Leu Asp Ile Tyr Asn Leu His Pro 305 310 315 320 Ala His Ala Lys Arg Ile Glu Glu Ile Ile Ala Glu Asn Lys Pro Pro 325 330 335 Phe Ser Val Phe Arg Asp Lys Phe Ile Thr Met Pro Gly Gly Leu Asp 340 345 350 Tyr Ser Arg Ala Ile Val Val Ala Ser Val Lys Glu Ala Pro Ile Gly 355 360 365 Ile Glu Val Ile Pro Ala His Val Thr Glu Tyr Leu Asn Lys Val Leu 370 375 380 Thr Glu Thr Asp His Ala Asn Pro Asp Pro Glu Ile Val Asp Ile Ala 385 390 395 400 Cys Gly Arg Ser Ser Ala Pro Met Pro Gln Arg Val Thr Glu Glu Gly 405 410 415 Lys Gln Asp Asp Glu Glu Lys Pro Gln Pro Ser Gly Thr Thr Ala Val 420 425 430 Glu Gln Gly Glu Ala Glu Thr Met Glu Pro Asp Ala Thr Glu His His 435 440 445 Gln Asp Thr Gln Pro Leu Asp Ala Gln Ser Gln Val Asn Ser Val Asp 450 455 460 Ala Lys Tyr Gln Glu Leu Arg Ala Glu Leu His Glu Ala Arg Lys Asn 465 470 475 480 Ile Pro Ser Lys Asn Pro Val Asp Asp Asp Lys Leu Leu Ala Ala Ser 485 490 495 Arg Gly Glu Phe Val Asp Gly Ile Ser Asp Pro Asn Asp Pro Lys Trp 500 505 510 Val Lys Gly Ile Gln Thr Arg Asp Cys Val Tyr Gln Asn Gln Pro Glu 515 520 525 Thr Glu Lys Thr Ser Pro Asp Met Asn Gln Pro Glu Pro Val Val Gln 530 535 540 Gln Glu Pro Glu Ile Ala Cys Asn Ala Cys Gly Gln Thr Gly Gly Asp 545 550 555 560 Asn Cys Pro Asp Cys Gly Ala Val Met Gly Asp Ala Thr Tyr Gln Glu 565 570 575 Thr Phe Asp Glu Glu Ser Gln Val Glu Ala Lys Glu Asn Asp Pro Glu 580 585 590 Glu Met Glu Gly Ala Glu His Pro His Asn Glu Asn Ala Gly Ser Asp 595 600 605 Pro His Arg Asp Cys Ser Asp Glu Thr Gly Glu Val Ala Asp Pro Val 610 615 620 Ile Val Glu Asp Ile Glu Pro Gly Ile Tyr Tyr Gly Ile Ser Asn Glu 625 630 635 640 Asn Tyr His Ala Gly Pro Gly Ile Ser Lys Ser Gln Leu Asp Asp Ile 645 650 655 Ala Asp Thr Pro Ala Leu Tyr Leu Trp Arg Lys Asn Ala Pro Val Asp 660 665 670 Thr Thr Lys Thr Lys Thr Leu Asp Leu Gly Thr Ala Phe His Cys Arg 675 680 685 Val Leu Glu Pro Glu Glu Phe Ser Asn Arg Phe Ile Val Ala Pro Glu 690 695 700 Phe Asn Arg Arg Thr Asn Ala Gly Lys Glu Glu Glu Lys Ala Phe Leu 705 710 715 720 Met Glu Cys Ala Ser Thr Gly Lys Thr Val Ile Thr Ala Glu Glu Gly 725 730 735 Arg Lys Ile Glu Leu Met Tyr Gln Ser Val Met Ala Leu Pro Leu Gly 740 745 750 Gln Trp Leu Val Glu Ser Ala Gly His Ala Glu Ser Ser Ile Tyr Trp 755 760 765 Glu Asp Pro Glu Thr Gly Ile Leu Cys Arg Cys Arg Pro Asp Lys Ile 770 775 780 Ile Pro Glu Phe His Trp Ile Met Asp Val Lys Thr Thr Ala Asp Ile 785 790 795 800 Gln Arg Phe Lys Thr Ala Tyr Tyr Asp Tyr Arg Tyr His Val Gln Asp 805 810 815 Ala Phe Tyr Ser Asp Gly Tyr Glu Ala Gln Phe Gly Val Gln Pro Thr 820 825 830 Phe Val Phe Leu Val Ala Ser Thr Thr Ile Glu Cys Gly Arg Tyr Pro 835 840 845 Val Glu Ile Phe Met Met Gly Glu Glu Ala Lys Leu Ala Gly Gln Gln 850 855 860 Glu Tyr His Arg Asn Leu Arg Thr Leu Ser Asp Cys Leu Asn Thr Asp 865 870 875 880 Glu Trp Pro Ala Ile Lys Thr Leu Ser Leu Pro Arg Trp Ala Lys Glu 885 890 895 Tyr Ala Asn Asp Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly 900 905 910 Arg Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser 915 920 925 Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys 930 935 940 Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu 945 950 955 960 Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg 965 970 975 Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile 980 985 990 Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp 995 1000 1005 Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys 1010 1015 1020 Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala 1025 1030 1035 1040 Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val 1045 1050 1055 Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala 1060 1065 1070 His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn 1075 1080 1085 Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr 1090 1095 1100 Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp 1105 1110 1115 1120 Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu 1125 1130 1135 Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly 1140 1145 1150 Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn 1155 1160 1165 Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr 1170 1175 1180 Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala 1185 1190 1195 1200 Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser 1205 1210 1215 Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala 1220 1225 1230 Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu 1235 1240 1245 Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe 1250 1255 1260 Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala 1265 1270 1275 1280 Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met 1285 1290 1295 Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu 1300 1305 1310 Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His 1315 1320 1325 Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro 1330 1335 1340 Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg 1345 1350 1355 1360 Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala 1365 1370 1375 Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu 1380 1385 1390 Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met 1395 1400 1405 Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His 1410 1415 1420 Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val 1425 1430 1435 1440 Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu 1445 1450 1455 Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val 1460 1465 1470 Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe 1475 1480 1485 Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu 1490 1495 1500 Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu 1505 1510 1515 1520 Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu 1525 1530 1535 Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr 1540 1545 1550 Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg 1555 1560 1565 Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg 1570 1575 1580 Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly 1585 1590 1595 1600 Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr 1605 1610 1615 Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser 1620 1625 1630 Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys 1635 1640 1645 Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met 1650 1655 1660 Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn 1665 1670 1675 1680 Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg 1685 1690 1695 Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His 1700 1705 1710 Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr 1715 1720 1725 Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn 1730 1735 1740 Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu 1745 1750 1755 1760 Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn 1765 1770 1775 Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met 1780 1785 1790 Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg 1795 1800 1805 Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu 1810 1815 1820 Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile 1825 1830 1835 1840 Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr 1845 1850 1855 Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys 1860 1865 1870 Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val 1875 1880 1885 Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala 1890 1895 1900 Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu 1905 1910 1915 1920 Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1925 1930 1935 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr 1940 1945 1950 Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly 1955 1960 1965 Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu 1970 1975 1980 Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu 1985 1990 1995 2000 Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly 2005 2010 2015 Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu 2020 2025 2030 Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp 2035 2040 2045 Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys 2050 2055 2060 Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr 2065 2070 2075 2080 Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu 2085 2090 2095 Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro 2100 2105 2110 Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala 2115 2120 2125 Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys 2130 2135 2140 Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly 2145 2150 2155 2160 Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 2165 2170 2175 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg 2180 2185 2190 Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn 2195 2200 2205 Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His 2210 2215 2220 Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe 2225 2230 2235 2240 Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu 2245 2250 2255 Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg 2260 2265 2270 Ile Asp Leu Ser Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp Leu Glu 2275 2280 2285 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 2290 2295 2300 His His His His His His 2305 2310 <210> 44 <211> 6933 <212> DNA <213> Artificial Sequence <220> <223> RecE-SpyCas9 <400> 44 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgagcac aaaaccactc 120 ttcctgttac ggaaagcgaa aaaatcatcc ggtgaacctg acgtcgtcct gtgggcaagc 180 aacgattttg aatcgacctg tgccactctg gactacctga tcgttaagtc aggtaaaaaa 240 ctgagcagct attttaaagc tgttgccacg aattttcctg tcgttaatga cctgcccgct 300 gaaggtgaga tcgattttac ctggagtgaa cgctatcaac tcagcaaaga ctccatgaca 360 tgggaactaa aaccgggagc agcaccagac aacgctcact atcaaggcaa taccaacgtc 420 aacggcgaag acatgactga gattgaggag aatatgctac tcccaatttc tggccaggaa 480 ctgcccattc gttggcttgc tcaacacggc agcgaaaaac cggtaacgca cgtttcacgc 540 gacggactcc aggcattaca cattgctcgg gctgaagaac taccggctgt tactgccctg 600 gctgtttccc acaaaaccag cctgctcgac ccgctggaaa ttcgcgaact ccacaaactg 660 gttcgtgaca ctgacaaagt tttccctaat cctggtaatt caaacctggg actgataact 720 gcttttttcg aagcatacct gaacgctgac tacaccgatc gaggactgct gacaaaagag 780 tggatgaagg gtaatcgtgt ttcacacatc actcgcacgg cttccggtgc taatgctggc 840 ggcggaaacc tcaccgatcg cggcgaaggt ttcgtacacg atctgacgtc actggcgcgc 900 gacgtagcca ctggcgtact ggcccgttca atggatctgg acatctataa ccttcatccg 960 gcacacgcta aacgcattga ggaaattatc gctgaaaata aaccgccctt ttctgttttc 1020 cgcgacaaat tcatcaccat gcctggcggg ctggattatt cccgcgccat cgtggttgcg 1080 tccgtaaaag aagcaccaat tgggatcgag gtcatccccg cgcacgtcac tgaatatctg 1140 aacaaagtac tgactgaaac cgatcatgcc aaccctgatc cggaaatcgt ggatattgcc 1200 tgcggtcgct cctctgcccc gatgccgcag cgagtaacag aagaaggaaa acaggatgat 1260 gaagaaaaac cgcaaccatc tggaacaacg gcagttgaac agggagaggc tgaaacaatg 1320 gaaccggacg caactgaaca tcatcaggac acgcagccgc tggatgctca gtcacaggta 1380 aattctgttg atgcgaaata tcaggaactg cgggcagaac tccatgaagc ccggaaaaac 1440 attccatcaa aaaatcctgt cgatgacgat aaattgcttg ctgcatcacg tggtgaattt 1500 gttgacggaa ttagcgaccc gaacgatccg aaatgggtaa aggggatcca gactcgcgat 1560 tgtgtgtacc agaaccagcc agaaacggaa aaaaccagcc cagatatgaa tcaacctgag 1620 ccagtagtgc aacaggaacc ggaaatagcc tgcaatgcct gcggccagac tggcggggat 1680 aactgccctg actgtggtgc ggtgatgggc gacgcaacat accaggaaac attcgatgaa 1740 gagagtcagg ttgaagctaa ggaaaatgat ccggaggaaa tggaaggcgc tgaacatccg 1800 cacaatgaga atgctggcag cgatccgcat cgcgattgca gtgatgaaac tggcgaagtc 1860 gcagatcccg taatcgtaga agacatagag ccaggtattt attacggaat ttcgaatgag 1920 aattaccacg cgggtcccgg tatcagtaag tctcagctcg atgacattgc tgatactccg 1980 gcactatatt tgtggcgtaa aaatgccccc gtggacacca caaagacaaa aacgctcgat 2040 ttaggaactg ctttccactg ccgggtactt gaaccggaag aattcagtaa ccgctttatc 2100 gtagcacctg aatttaaccg ccgtacaaac gccggaaaag aagaagagaa agcgtttctg 2160 atggaatgcg caagcacagg aaaaacggtt atcactgcgg aagaaggccg gaaaattgaa 2220 ctcatgtatc aaagcgttat ggctttgccg ctggggcaat ggcttgttga aagcgccgga 2280 cacgctgaat catcaattta ctgggaagat cctgaaacag gaattttgtg tcggtgccgt 2340 ccggacaaaa ttatccctga atttcactgg atcatggacg tgaaaactac ggcggatatt 2400 caacgattca aaaccgctta ttacgactac cgctatcacg ttcaggatgc attctacagt 2460 gacggttatg aagcacagtt tggagtgcag ccaactttcg tttttctggt tgccagcaca 2520 actattgaat gcggacgtta tccggttgaa attttcatga tgggcgaaga agcaaaactg 2580 gcaggtcaac aggaatatca ccgcaatctg cgaaccctgt ctgactgcct gaataccgat 2640 gaatggccag ctattaagac attatcactg ccccgctggg ctaaggaata tgcaaatgac 2700 ggatccgaat tcgagctccg tcgacaagct tgcggccgca tggacaagaa gtacagcatc 2760 ggcctggaca tcggtaccaa cagcgtgggc tgggccgtga tcaccgacga gtacaaggtg 2820 cccagcaaga agttcaaggt gctgggcaac accgaccgcc acagcatcaa gaagaacctg 2880 atcggcgccc tgctgttcga cagcggcgag accgccgagg ccacccgcct gaagcgcacc 2940 gcccgccgcc gctacacccg ccgcaagaac cgcatctgct acctgcagga gatcttcagc 3000 aacgagatgg ccaaggtgga cgacagcttc ttccaccgcc tggaggagag cttcctggtg 3060 gaggaggaca agaagcacga gcgccacccc atcttcggca acatcgtgga cgaggtggcc 3120 taccacgaga agtaccccac catctaccac ctgcgcaaga agctggtgga cagcaccgac 3180 aaggccgacc tgcgcctgat ctacctggcc ctggcccaca tgatcaagtt ccgcggccac 3240 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag 3300 ctggtgcaga cctacaacca gctgttcgag gagaacccca tcaacgccag cggcgtggac 3360 gccaaggcca tcctgagcgc ccgcctgagc aagagccgcc gcctggagaa cctgatcgcc 3420 cagctgcccg gcgagaagaa gaacggcctg ttcggcaacc tgatcgccct gagcctgggc 3480 ctgaccccca acttcaagag caacttcgac ctggccgagg acgccaagct gcagctgagc 3540 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc 3600 gacctgttcc tggccgccaa gaacctgagc gacgccatcc tgctgagcga catcctgcgc 3660 gtgaacaccg agatcaccaa ggcccccctg agcgccagca tgatcaagcg ctacgacgag 3720 caccaccagg acctgaccct gctgaaggcc ctggtgcgcc agcagctgcc cgagaagtac 3780 aaggagatct tcttcgacca gagcaagaac ggctacgccg gctacatcga cggcggcgcc 3840 agccaggagg agttctacaa gttcatcaag cccatcctgg agaagatgga cggcaccgag 3900 gagctgctgg tgaagctgaa ccgcgaggac ctgctgcgca agcagcgcac cttcgacaac 3960 ggcagcatcc cccaccagat ccacctgggc gagctgcacg ccatcctgcg ccgccaggag 4020 gacttctacc ccttcctgaa ggacaaccgc gagaagatcg agaagatcct gaccttccgc 4080 atcccctact acgtgggccc cctggcccgc ggcaacagcc gcttcgcctg gatgacccgc 4140 aagagcgagg agaccatcac cccctggaac ttcgaggagg tggtggacaa gggcgccagc 4200 gcccagagct tcatcgagcg catgaccaac ttcgacaaga acctgcccaa cgagaaggtg 4260 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt acaacgagct gaccaaggtg 4320 aagtacgtga ccgagggcat gcgcaagccc gccttcctga gcggcgagca gaagaaggcc 4380 atcgtggacc tgctgttcaa gaccaaccgc aaggtgaccg tgaagcagct gaaggaggac 4440 tacttcaaga agatcgagtg cttcgacagc gtggagatca gcggcgtgga ggaccgcttc 4500 aacgccagcc tgggcaccta ccacgacctg ctgaagatca tcaaggacaa ggacttcctg 4560 gacaacgagg agaacgagga catcctggag gacatcgtgc tgaccctgac cctgttcgag 4620 gaccgcgaga tgatcgagga gcgcctgaag acctacgccc acctgttcga cgacaaggtg 4680 atgaagcagc tgaagcgccg ccgctacacc ggctggggcc gcctgagccg caagcttatc 4740 aacggcatcc gcgacaagca gagcggcaag accatcctgg acttcctgaa gagcgacggc 4800 ttcgccaacc gcaacttcat gcagctgatc cacgacgaca gcctgacctt caaggaggac 4860 atccagaagg cccaggtgag cggccagggc gacagcctgc acgagcacat cgccaacctg 4920 gccggcagcc ccgccatcaa gaagggcatc ctgcagaccg tgaaggtggt ggacgagctg 4980 gtgaaggtga tgggccgcca caagcccgag aacatcgtga tcgagatggc ccgcgagaac 5040 cagaccaccc agaagggcca gaagaacagc cgcgagcgca tgaagcgcat cgaggagggc 5100 atcaaggagc tgggcagcca gatcctgaag gagcaccccg tggagaacac ccagctgcag 5160 aacgagaagc tgtacctgta ctacctgcag aacggccgcg acatgtacgt ggaccaggag 5220 ctggacatca accgcctgag cgactacgac gtggaccaca tcgtgcccca gagcttcctg 5280 aaggacgaca gcatcgacaa caaggtgctg acccgcagcg acaagaaccg cggcaagagc 5340 gacaacgtgc ccagcgagga ggtggtgaag aagatgaaga actactggcg ccagctgctg 5400 aacgccaagc tgatcaccca gcgcaagttc gacaacctga ccaaggccga gcgcggcggc 5460 ctgagcgagc tggacaaggc cggcttcatc aagcgccagc tggtggagac ccgccagatc 5520 accaagcacg tggcccagat cctggacagc cgcatgaaca ccaagtacga cgagaacgac 5580 aagctgatcc gcgaggtgaa ggtgatcacc ctgaagagca agctggtgag cgacttccgc 5640 aaggacttcc agttctacaa ggtgcgcgag atcaacaact accaccacgc ccacgacgcc 5700 tacctgaacg ccgtggtggg caccgccctg atcaagaagt accccaagct ggagagcgag 5760 ttcgtgtacg gcgactacaa ggtgtacgac gtgcgcaaga tgatcgccaa gagcgagcag 5820 gagatcggca aggccaccgc caagtacttc ttctacagca acatcatgaa cttcttcaag 5880 accgagatca ccctggccaa cggcgagatc cgcaagcgcc ccctgatcga gaccaacggc 5940 gagaccggcg agatcgtgtg ggacaagggc cgcgacttcg ccaccgtgcg caaggtgctg 6000 agcatgcccc aggtgaacat cgtgaagaag accgaggtgc agaccggcgg cttcagcaag 6060 gagagcatcc tgcccaagcg caacagcgac aagctgatcg cccgcaagaa ggactgggac 6120 cccaagaagt acggcggctt cgacagcccc accgtggcct acagcgtgct ggtggtggcc 6180 aaggtggaga agggcaagag caagaagctg aagagcgtga aggagctgct gggcatcacc 6240 atcatggagc gcagcagctt cgagaagaac cccatcgact tcctggaggc caagggctac 6300 aaggaggtga agaaggacct gatcatcaag ctgcccaagt acagcctgtt cgagctggag 6360 aacggccgca agcgcatgct ggccagcgcc ggcgagctgc agaagggcaa cgagctggcc 6420 ctgcccagca agtacgtgaa cttcctgtac ctggccagcc actacgagaa gctgaagggc 6480 agccccgagg acaacgagca gaagcagctg ttcgtggagc agcacaagca ctacctggac 6540 gagatcatcg agcagatcag cgagttcagc aagcgcgtga tcctggccga cgccaacctg 6600 gacaaggtgc tgagcgccta caacaagcac cgcgacaagc ccatccgcga gcaggccgag 6660 aacatcatcc acctgttcac cctgaccaac ctgggcgccc ccgccgcctt caagtacttc 6720 gacaccacca tcgaccgcaa gcgctacacc agcaccaagg aggtgctgga cgccaccctg 6780 atccaccaga gcatcaccgg tctgtacgag acccgcatcg acctgagcca gctgggcggc 6840 gacgcggccg cactcgacct cgagaaaagg ccggcggcca cgaaaaaggc cggccaggca 6900 aaaaagaaaa agcaccacca ccaccaccac tga 6933 <210> 45 <211> 1682 <212> PRT <213> Artificial Sequence <220> <223> GFP-SpyCas9 <400> 45 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile 35 40 45 Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser 50 55 60 Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe 65 70 75 80 Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr 85 90 95 Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met 100 105 110 Lys Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln 115 120 125 Glu Arg Thr Ile Ser Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala 130 135 140 Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys 145 150 155 160 Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu 165 170 175 Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys 180 185 190 Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly 195 200 205 Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp 210 215 220 Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala 225 230 235 240 Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu 245 250 255 Phe Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 260 265 270 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Asp Lys 275 280 285 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 290 295 300 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 305 310 315 320 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 325 330 335 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 340 345 350 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 355 360 365 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 370 375 380 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 385 390 395 400 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 405 410 415 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 420 425 430 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 435 440 445 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 450 455 460 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 465 470 475 480 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 485 490 495 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 500 505 510 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 515 520 525 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 530 535 540 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 545 550 555 560 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 565 570 575 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 580 585 590 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 595 600 605 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 610 615 620 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 625 630 635 640 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 645 650 655 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 660 665 670 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 675 680 685 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 690 695 700 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 705 710 715 720 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 725 730 735 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 740 745 750 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 755 760 765 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 770 775 780 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 785 790 795 800 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 805 810 815 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 820 825 830 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 835 840 845 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 850 855 860 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 865 870 875 880 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 885 890 895 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 900 905 910 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 915 920 925 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 930 935 940 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 945 950 955 960 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 965 970 975 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 980 985 990 Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His 995 1000 1005 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 1010 1015 1020 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 1025 1030 1035 1040 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 1045 1050 1055 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 1060 1065 1070 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 1075 1080 1085 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 1090 1095 1100 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 1105 1110 1115 1120 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 1125 1130 1135 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 1140 1145 1150 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 1155 1160 1165 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 1170 1175 1180 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 1185 1190 1195 1200 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 1205 1210 1215 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 1220 1225 1230 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 1235 1240 1245 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1250 1255 1260 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1265 1270 1275 1280 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1285 1290 1295 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1300 1305 1310 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1315 1320 1325 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1330 1335 1340 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1345 1350 1355 1360 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1365 1370 1375 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1380 1385 1390 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1395 1400 1405 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1410 1415 1420 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1425 1430 1435 1440 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1445 1450 1455 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1460 1465 1470 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1475 1480 1485 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1490 1495 1500 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1505 1510 1515 1520 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1525 1530 1535 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1540 1545 1550 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1555 1560 1565 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1570 1575 1580 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1585 1590 1595 1600 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1605 1610 1615 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1620 1625 1630 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1635 1640 1645 Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp Leu Glu Lys Arg Pro Ala 1650 1655 1660 Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys His His His His 1665 1670 1675 1680 His His <210> 46 <211> 5049 <212> DNA <213> Artificial Sequence <220> <223> GFP-SpyCas9 <400> 46 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgagtaa aggagaagaa 120 cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa 180 ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt 240 atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac tttctcttat 300 ggtgttcaat gcttttcaag atacccagat catatgaagc ggcacgactt cttcaagagc 360 gccatgcctg agggatacgt gcaggagagg accatctctt tcaaggacga cgggaactac 420 aagacacgtg ctgaagtcaa gtttgaggga gacaccctcg tcaacaggat cgagcttaag 480 ggaatcgatt tcaaggagga cggaaacatc ctcggccaca agttggaata caactacaac 540 tcccacaacg tatacatcac ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa 600 attagacaca acattgaaga tggaagcgtt caactagcag accattatca acaaaatact 660 ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc 720 ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct 780 gctgggatta cacatggcat ggatgaacta tacaaaggat ccgaattcga gctccgtcga 840 caagcttgcg gccgcatgga caagaagtac agcatcggcc tggacatcgg taccaacagc 900 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaagtt caaggtgctg 960 ggcaacaccg accgccacag catcaagaag aacctgatcg gcgccctgct gttcgacagc 1020 ggcgagaccg ccgaggccac ccgcctgaag cgcaccgccc gccgccgcta cacccgccgc 1080 aagaaccgca tctgctacct gcaggagatc ttcagcaacg agatggccaa ggtggacgac 1140 agcttcttcc accgcctgga ggagagcttc ctggtggagg aggacaagaa gcacgagcgc 1200 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc 1260 taccacctgc gcaagaagct ggtggacagc accgacaagg ccgacctgcg cctgatctac 1320 ctggccctgg cccacatgat caagttccgc ggccacttcc tgatcgaggg cgacctgaac 1380 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg 1440 ttcgaggaga accccatcaa cgccagcggc gtggacgcca aggccatcct gagcgcccgc 1500 ctgagcaaga gccgccgcct ggagaacctg atcgcccagc tgcccggcga gaagaagaac 1560 ggcctgttcg gcaacctgat cgccctgagc ctgggcctga cccccaactt caagagcaac 1620 ttcgacctgg ccgaggacgc caagctgcag ctgagcaagg acacctacga cgacgacctg 1680 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgttcctggc cgccaagaac 1740 ctgagcgacg ccatcctgct gagcgacatc ctgcgcgtga acaccgagat caccaaggcc 1800 cccctgagcg ccagcatgat caagcgctac gacgagcacc accaggacct gaccctgctg 1860 aaggccctgg tgcgccagca gctgcccgag aagtacaagg agatcttctt cgaccagagc 1920 aagaacggct acgccggcta catcgacggc ggcgccagcc aggaggagtt ctacaagttc 1980 atcaagccca tcctggagaa gatggacggc accgaggagc tgctggtgaa gctgaaccgc 2040 gaggacctgc tgcgcaagca gcgcaccttc gacaacggca gcatccccca ccagatccac 2100 ctgggcgagc tgcacgccat cctgcgccgc caggaggact tctacccctt cctgaaggac 2160 aaccgcgaga agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccccctg 2220 gcccgcggca acagccgctt cgcctggatg acccgcaaga gcgaggagac catcaccccc 2280 tggaacttcg aggaggtggt ggacaagggc gccagcgccc agagcttcat cgagcgcatg 2340 accaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac 2400 gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga gggcatgcgc 2460 aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc 2520 aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttc 2580 gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg cacctaccac 2640 gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa cgaggacatc 2700 ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat cgaggagcgc 2760 ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa gcgccgccgc 2820 tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga caagcagagc 2880 ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa cttcatgcag 2940 ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca ggtgagcggc 3000 cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc catcaagaag 3060 ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg ccgccacaag 3120 cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa gggccagaag 3180 aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg cagccagatc 3240 ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta cctgtactac 3300 ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg cctgagcgac 3360 tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat cgacaacaag 3420 gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag cgaggaggtg 3480 gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat cacccagcgc 3540 aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga caaggccggc 3600 ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc ccagatcctg 3660 gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga ggtgaaggtg 3720 atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt ctacaaggtg 3780 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt ggtgggcacc 3840 gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga ctacaaggtg 3900 tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc caccgccaag 3960 tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct ggccaacggc 4020 gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat cgtgtgggac 4080 aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt gaacatcgtg 4140 aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc caagcgcaac 4200 agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg cggcttcgac 4260 agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg caagagcaag 4320 aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag cagcttcgag 4380 aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa ggacctgatc 4440 atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg catgctggcc 4500 agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta cgtgaacttc 4560 ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa cgagcagaag 4620 cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca gatcagcgag 4680 ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag cgcctacaac 4740 aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct gttcaccctg 4800 accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga ccgcaagcgc 4860 tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat caccggtctg 4920 tacgagaccc gcatcgacct gagccagctg ggcggcgacg cggccgcact cgacctcgag 4980 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagca ccaccaccac 5040 caccactga 5049 <210> 47 <211> 1560 <212> PRT <213> Artificial Sequence <220> <223> SSB-SpyCas9 <400> 47 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Ala Ser Arg Gly Val Asn Lys Val Ile Leu Val Gly Asn 35 40 45 Leu Gly Gln Asp Pro Glu Val Arg Tyr Met Pro Asn Gly Gly Ala Val 50 55 60 Ala Asn Ile Thr Leu Ala Thr Ser Glu Ser Trp Arg Asp Lys Ala Thr 65 70 75 80 Gly Glu Met Lys Glu Gln Thr Glu Trp His Arg Val Val Leu Phe Gly 85 90 95 Lys Leu Ala Glu Val Ala Ser Glu Tyr Leu Arg Lys Gly Ser Gln Val 100 105 110 Tyr Ile Glu Gly Gln Leu Arg Thr Arg Lys Trp Thr Asp Gln Ser Gly 115 120 125 Gln Asp Arg Tyr Thr Thr Glu Val Val Val Asn Val Gly Gly Thr Met 130 135 140 Gln Met Leu Gly Gly Arg Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala 145 150 155 160 Cys Gly Arg Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr 165 170 175 Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser 180 185 190 Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys 195 200 205 Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala 210 215 220 Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn 225 230 235 240 Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val 245 250 255 Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu 260 265 270 Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu 275 280 285 Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys 290 295 300 Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala 305 310 315 320 Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp 325 330 335 Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val 340 345 350 Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly 355 360 365 Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg 370 375 380 Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu 385 390 395 400 Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys 405 410 415 Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp 420 425 430 Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln 435 440 445 Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu 450 455 460 Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu 465 470 475 480 Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr 485 490 495 Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu 500 505 510 Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly 515 520 525 Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu 530 535 540 Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp 545 550 555 560 Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln 565 570 575 Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe 580 585 590 Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr 595 600 605 Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg 610 615 620 Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn 625 630 635 640 Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu 645 650 655 Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro 660 665 670 Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr 675 680 685 Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser 690 695 700 Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg 705 710 715 720 Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu 725 730 735 Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala 740 745 750 Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp 755 760 765 Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu 770 775 780 Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys 785 790 795 800 Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg 805 810 815 Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly 820 825 830 Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser 835 840 845 Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser 850 855 860 Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly 865 870 875 880 Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile 885 890 895 Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys 900 905 910 Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg 915 920 925 Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met 930 935 940 Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys 945 950 955 960 Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu 965 970 975 Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp 980 985 990 Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser 995 1000 1005 Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp 1010 1015 1020 Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys 1025 1030 1035 1040 Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr 1045 1050 1055 Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser 1060 1065 1070 Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg 1075 1080 1085 Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr 1090 1095 1100 Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr 1105 1110 1115 1120 Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr 1125 1130 1135 Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu 1140 1145 1150 Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu 1155 1160 1165 Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met 1170 1175 1180 Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe 1185 1190 1195 1200 Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1205 1210 1215 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr 1220 1225 1230 Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys 1235 1240 1245 Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln 1250 1255 1260 Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp 1265 1270 1275 1280 Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly 1285 1290 1295 Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val 1300 1305 1310 Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly 1315 1320 1325 Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe 1330 1335 1340 Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys 1345 1350 1355 1360 Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met 1365 1370 1375 Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro 1380 1385 1390 Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu 1395 1400 1405 Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln 1410 1415 1420 His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser 1425 1430 1435 1440 Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1445 1450 1455 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile 1460 1465 1470 Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys 1475 1480 1485 Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu 1490 1495 1500 Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu 1505 1510 1515 1520 Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp 1525 1530 1535 Leu Glu Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys 1540 1545 1550 Lys Lys His His His His His His 1555 1560 <210> 48 <211> 4683 <212> DNA <213> Artificial Sequence <220> <223> SSB-SpyCas9 <400> 48 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggccag cagaggcgta 120 aacaaggtta ttctcgttgg taatctgggt caggacccgg aagtacgcta catgccaaat 180 ggtggcgcag ttgccaacat tacgctggct acttccgaat cctggcgtga taaagcgacc 240 ggcgagatga aagaacagac tgaatggcac cgcgttgtgc tgttcggcaa actggcagaa 300 gtggcgagcg aatatctgcg taaaggttct caggtttata tcgaaggtca gctgcgtacc 360 cgtaaatgga ccgatcaatc cggtcaggat cgctacacca cagaagtcgt ggtgaacgtt 420 ggcggcacca tgcagatgct gggtggtcgt ggatccgaat tcgagctccg tcgacaagct 480 tgcggccgca tggacaagaa gtacagcatc ggcctggaca tcggtaccaa cagcgtgggc 540 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga agttcaaggt gctgggcaac 600 accgaccgcc acagcatcaa gaagaacctg atcggcgccc tgctgttcga cagcggcgag 660 accgccgagg ccacccgcct gaagcgcacc gcccgccgcc gctacacccg ccgcaagaac 720 cgcatctgct acctgcagga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc 780 ttccaccgcc tggaggagag cttcctggtg gaggaggaca agaagcacga gcgccacccc 840 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac 900 ctgcgcaaga agctggtgga cagcaccgac aaggccgacc tgcgcctgat ctacctggcc 960 ctggcccaca tgatcaagtt ccgcggccac ttcctgatcg agggcgacct gaaccccgac 1020 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag 1080 gagaacccca tcaacgccag cggcgtggac gccaaggcca tcctgagcgc ccgcctgagc 1140 aagagccgcc gcctggagaa cctgatcgcc cagctgcccg gcgagaagaa gaacggcctg 1200 ttcggcaacc tgatcgccct gagcctgggc ctgaccccca acttcaagag caacttcgac 1260 ctggccgagg acgccaagct gcagctgagc aaggacacct acgacgacga cctggacaac 1320 ctgctggccc agatcggcga ccagtacgcc gacctgttcc tggccgccaa gaacctgagc 1380 gacgccatcc tgctgagcga catcctgcgc gtgaacaccg agatcaccaa ggcccccctg 1440 agcgccagca tgatcaagcg ctacgacgag caccaccagg acctgaccct gctgaaggcc 1500 ctggtgcgcc agcagctgcc cgagaagtac aaggagatct tcttcgacca gagcaagaac 1560 ggctacgccg gctacatcga cggcggcgcc agccaggagg agttctacaa gttcatcaag 1620 cccatcctgg agaagatgga cggcaccgag gagctgctgg tgaagctgaa ccgcgaggac 1680 ctgctgcgca agcagcgcac cttcgacaac ggcagcatcc cccaccagat ccacctgggc 1740 gagctgcacg ccatcctgcg ccgccaggag gacttctacc ccttcctgaa ggacaaccgc 1800 gagaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc cctggcccgc 1860 ggcaacagcc gcttcgcctg gatgacccgc aagagcgagg agaccatcac cccctggaac 1920 ttcgaggagg tggtggacaa gggcgccagc gcccagagct tcatcgagcg catgaccaac 1980 ttcgacaaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac 2040 ttcaccgtgt acaacgagct gaccaaggtg aagtacgtga ccgagggcat gcgcaagccc 2100 gccttcctga gcggcgagca gaagaaggcc atcgtggacc tgctgttcaa gaccaaccgc 2160 aaggtgaccg tgaagcagct gaaggaggac tacttcaaga agatcgagtg cttcgacagc 2220 gtggagatca gcggcgtgga ggaccgcttc aacgccagcc tgggcaccta ccacgacctg 2280 ctgaagatca tcaaggacaa ggacttcctg gacaacgagg agaacgagga catcctggag 2340 gacatcgtgc tgaccctgac cctgttcgag gaccgcgaga tgatcgagga gcgcctgaag 2400 acctacgccc acctgttcga cgacaaggtg atgaagcagc tgaagcgccg ccgctacacc 2460 ggctggggcc gcctgagccg caagcttatc aacggcatcc gcgacaagca gagcggcaag 2520 accatcctgg acttcctgaa gagcgacggc ttcgccaacc gcaacttcat gcagctgatc 2580 cacgacgaca gcctgacctt caaggaggac atccagaagg cccaggtgag cggccagggc 2640 gacagcctgc acgagcacat cgccaacctg gccggcagcc ccgccatcaa gaagggcatc 2700 ctgcagaccg tgaaggtggt ggacgagctg gtgaaggtga tgggccgcca caagcccgag 2760 aacatcgtga tcgagatggc ccgcgagaac cagaccaccc agaagggcca gaagaacagc 2820 cgcgagcgca tgaagcgcat cgaggagggc atcaaggagc tgggcagcca gatcctgaag 2880 gagcaccccg tggagaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag 2940 aacggccgcg acatgtacgt ggaccaggag ctggacatca accgcctgag cgactacgac 3000 gtggaccaca tcgtgcccca gagcttcctg aaggacgaca gcatcgacaa caaggtgctg 3060 acccgcagcg acaagaaccg cggcaagagc gacaacgtgc ccagcgagga ggtggtgaag 3120 aagatgaaga actactggcg ccagctgctg aacgccaagc tgatcaccca gcgcaagttc 3180 gacaacctga ccaaggccga gcgcggcggc ctgagcgagc tggacaaggc cggcttcatc 3240 aagcgccagc tggtggagac ccgccagatc accaagcacg tggcccagat cctggacagc 3300 cgcatgaaca ccaagtacga cgagaacgac aagctgatcc gcgaggtgaa ggtgatcacc 3360 ctgaagagca agctggtgag cgacttccgc aaggacttcc agttctacaa ggtgcgcgag 3420 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtggtggg caccgccctg 3480 atcaagaagt accccaagct ggagagcgag ttcgtgtacg gcgactacaa ggtgtacgac 3540 gtgcgcaaga tgatcgccaa gagcgagcag gagatcggca aggccaccgc caagtacttc 3600 ttctacagca acatcatgaa cttcttcaag accgagatca ccctggccaa cggcgagatc 3660 cgcaagcgcc ccctgatcga gaccaacggc gagaccggcg agatcgtgtg ggacaagggc 3720 cgcgacttcg ccaccgtgcg caaggtgctg agcatgcccc aggtgaacat cgtgaagaag 3780 accgaggtgc agaccggcgg cttcagcaag gagagcatcc tgcccaagcg caacagcgac 3840 aagctgatcg cccgcaagaa ggactgggac cccaagaagt acggcggctt cgacagcccc 3900 accgtggcct acagcgtgct ggtggtggcc aaggtggaga agggcaagag caagaagctg 3960 aagagcgtga aggagctgct gggcatcacc atcatggagc gcagcagctt cgagaagaac 4020 cccatcgact tcctggaggc caagggctac aaggaggtga agaaggacct gatcatcaag 4080 ctgcccaagt acagcctgtt cgagctggag aacggccgca agcgcatgct ggccagcgcc 4140 ggcgagctgc agaagggcaa cgagctggcc ctgcccagca agtacgtgaa cttcctgtac 4200 ctggccagcc actacgagaa gctgaagggc agccccgagg acaacgagca gaagcagctg 4260 ttcgtggagc agcacaagca ctacctggac gagatcatcg agcagatcag cgagttcagc 4320 aagcgcgtga tcctggccga cgccaacctg gacaaggtgc tgagcgccta caacaagcac 4380 cgcgacaagc ccatccgcga gcaggccgag aacatcatcc acctgttcac cctgaccaac 4440 ctgggcgccc ccgccgcctt caagtacttc gacaccacca tcgaccgcaa gcgctacacc 4500 agcaccaagg aggtgctgga cgccaccctg atccaccaga gcatcaccgg tctgtacgag 4560 acccgcatcg acctgagcca gctgggcggc gacgcggccg cactcgacct cgagaaaagg 4620 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agcaccacca ccaccaccac 4680 tga 4683 <210> 49 <211> 2139 <212> PRT <213> Artificial Sequence <220> <223> SSB-SpyCas9-RecJ <400> 49 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Ala Ser Arg Gly Val Asn Lys Val Ile Leu Val Gly Asn 35 40 45 Leu Gly Gln Asp Pro Glu Val Arg Tyr Met Pro Asn Gly Gly Ala Val 50 55 60 Ala Asn Ile Thr Leu Ala Thr Ser Glu Ser Trp Arg Asp Lys Ala Thr 65 70 75 80 Gly Glu Met Lys Glu Gln Thr Glu Trp His Arg Val Val Leu Phe Gly 85 90 95 Lys Leu Ala Glu Val Ala Ser Glu Tyr Leu Arg Lys Gly Ser Gln Val 100 105 110 Tyr Ile Glu Gly Gln Leu Arg Thr Arg Lys Trp Thr Asp Gln Ser Gly 115 120 125 Gln Asp Arg Tyr Thr Thr Glu Val Val Val Asn Val Gly Gly Thr Met 130 135 140 Gln Met Leu Gly Gly Arg Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala 145 150 155 160 Cys Gly Arg Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr 165 170 175 Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser 180 185 190 Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys 195 200 205 Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala 210 215 220 Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn 225 230 235 240 Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val 245 250 255 Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu 260 265 270 Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu 275 280 285 Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys 290 295 300 Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala 305 310 315 320 Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp 325 330 335 Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val 340 345 350 Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly 355 360 365 Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg 370 375 380 Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu 385 390 395 400 Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys 405 410 415 Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp 420 425 430 Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln 435 440 445 Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu 450 455 460 Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu 465 470 475 480 Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr 485 490 495 Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu 500 505 510 Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly 515 520 525 Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu 530 535 540 Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp 545 550 555 560 Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln 565 570 575 Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe 580 585 590 Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr 595 600 605 Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg 610 615 620 Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn 625 630 635 640 Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu 645 650 655 Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro 660 665 670 Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr 675 680 685 Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser 690 695 700 Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg 705 710 715 720 Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu 725 730 735 Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala 740 745 750 Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp 755 760 765 Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu 770 775 780 Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys 785 790 795 800 Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg 805 810 815 Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly 820 825 830 Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser 835 840 845 Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser 850 855 860 Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly 865 870 875 880 Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile 885 890 895 Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys 900 905 910 Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg 915 920 925 Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met 930 935 940 Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys 945 950 955 960 Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu 965 970 975 Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp 980 985 990 Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser 995 1000 1005 Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp 1010 1015 1020 Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys 1025 1030 1035 1040 Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr 1045 1050 1055 Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser 1060 1065 1070 Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg 1075 1080 1085 Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr 1090 1095 1100 Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr 1105 1110 1115 1120 Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr 1125 1130 1135 Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu 1140 1145 1150 Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu 1155 1160 1165 Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met 1170 1175 1180 Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe 1185 1190 1195 1200 Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1205 1210 1215 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr 1220 1225 1230 Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys 1235 1240 1245 Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln 1250 1255 1260 Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp 1265 1270 1275 1280 Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly 1285 1290 1295 Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val 1300 1305 1310 Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly 1315 1320 1325 Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe 1330 1335 1340 Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys 1345 1350 1355 1360 Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met 1365 1370 1375 Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro 1380 1385 1390 Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu 1395 1400 1405 Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln 1410 1415 1420 His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser 1425 1430 1435 1440 Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1445 1450 1455 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile 1460 1465 1470 Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys 1475 1480 1485 Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu 1490 1495 1500 Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu 1505 1510 1515 1520 Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ala Ala Ala Leu Asp 1525 1530 1535 Leu Gln Val Lys Gln Gln Ile Gln Leu Arg Arg Arg Glu Val Asp Glu 1540 1545 1550 Thr Ala Asp Leu Pro Ala Glu Leu Pro Pro Leu Leu Arg Arg Leu Tyr 1555 1560 1565 Ala Ser Arg Gly Val Arg Ser Ala Gln Glu Leu Glu Arg Ser Val Lys 1570 1575 1580 Gly Met Leu Pro Trp Gln Gln Leu Ser Gly Val Glu Lys Ala Val Glu 1585 1590 1595 1600 Ile Leu Tyr Asn Ala Phe Arg Glu Gly Thr Arg Ile Ile Val Val Gly 1605 1610 1615 Asp Phe Asp Ala Asp Gly Ala Thr Ser Thr Ala Leu Ser Val Leu Ala 1620 1625 1630 Met Arg Ser Leu Gly Cys Ser Asn Ile Asp Tyr Leu Val Pro Asn Arg 1635 1640 1645 Phe Glu Asp Gly Tyr Gly Leu Ser Pro Glu Val Val Asp Gln Ala His 1650 1655 1660 Ala Arg Gly Ala Gln Leu Ile Val Thr Val Asp Asn Gly Ile Ser Ser 1665 1670 1675 1680 His Ala Gly Val Glu His Ala Arg Ser Leu Gly Ile Pro Val Ile Val 1685 1690 1695 Thr Asp His His Leu Pro Gly Asp Thr Leu Pro Ala Ala Glu Ala Ile 1700 1705 1710 Ile Asn Pro Asn Leu Arg Asp Cys Asn Phe Pro Ser Lys Ser Leu Ala 1715 1720 1725 Gly Val Gly Val Ala Phe Tyr Leu Met Leu Ala Leu Arg Thr Phe Leu 1730 1735 1740 Arg Asp Gln Gly Trp Phe Asp Glu Arg Asn Ile Ala Ile Pro Asn Leu 1745 1750 1755 1760 Ala Glu Leu Leu Asp Leu Val Ala Leu Gly Thr Val Ala Asp Val Val 1765 1770 1775 Pro Leu Asp Ala Asn Asn Arg Ile Leu Thr Trp Gln Gly Met Ser Arg 1780 1785 1790 Ile Arg Ala Gly Lys Cys Arg Pro Gly Ile Lys Ala Leu Leu Glu Val 1795 1800 1805 Ala Asn Arg Asp Ala Gln Lys Leu Ala Ala Ser Asp Leu Gly Phe Ala 1810 1815 1820 Leu Gly Pro Arg Leu Asn Ala Ala Gly Arg Leu Asp Asp Met Ser Val 1825 1830 1835 1840 Gly Val Ala Leu Leu Leu Cys Asp Asn Ile Gly Glu Ala Arg Val Leu 1845 1850 1855 Ala Asn Glu Leu Asp Ala Leu Asn Gln Thr Arg Lys Glu Ile Glu Gln 1860 1865 1870 Gly Met Gln Ile Glu Ala Leu Thr Leu Cys Glu Lys Leu Glu Arg Ser 1875 1880 1885 Arg Asp Thr Leu Pro Gly Gly Leu Ala Met Tyr His Pro Glu Trp His 1890 1895 1900 Gln Gly Val Val Gly Ile Leu Ala Ser Arg Ile Lys Glu Arg Phe His 1905 1910 1915 1920 Arg Pro Val Ile Ala Phe Ala Pro Ala Gly Asp Gly Thr Leu Lys Gly 1925 1930 1935 Ser Gly Arg Ser Ile Gln Gly Leu His Met Arg Asp Ala Leu Glu Arg 1940 1945 1950 Leu Asp Thr Leu Tyr Pro Gly Met Met Leu Lys Phe Gly Gly His Ala 1955 1960 1965 Met Ala Ala Gly Leu Ser Leu Glu Glu Asp Lys Phe Lys Leu Phe Gln 1970 1975 1980 Gln Arg Phe Gly Glu Leu Val Thr Glu Trp Leu Asp Pro Ser Leu Leu 1985 1990 1995 2000 Gln Gly Glu Val Val Ser Asp Gly Pro Leu Ser Pro Ala Glu Met Thr 2005 2010 2015 Met Glu Val Ala Gln Leu Leu Arg Asp Ala Gly Pro Trp Gly Gln Met 2020 2025 2030 Phe Pro Glu Pro Leu Phe Asp Gly His Phe Arg Leu Leu Gln Gln Arg 2035 2040 2045 Leu Val Gly Glu Arg His Leu Lys Val Met Val Glu Pro Val Gly Gly 2050 2055 2060 Gly Pro Leu Leu Asp Gly Ile Ala Phe Asn Val Asp Thr Ala Leu Trp 2065 2070 2075 2080 Pro Asp Asn Gly Val Arg Glu Val Gln Leu Ala Tyr Lys Leu Asp Ile 2085 2090 2095 Asn Glu Phe Arg Gly Asn Arg Ser Leu Gln Ile Ile Ile Asp Asn Ile 2100 2105 2110 Trp Pro Ile Leu Gln Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln 2115 2120 2125 Ala Lys Lys Lys Lys His His His His His His 2130 2135 <210> 50 <211> 6420 <212> DNA <213> Artificial Sequence <220> <223> SSB-SpyCas9-RecJ <400> 50 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggccag cagaggcgta 120 aacaaggtta ttctcgttgg taatctgggt caggacccgg aagtacgcta catgccaaat 180 ggtggcgcag ttgccaacat tacgctggct acttccgaat cctggcgtga taaagcgacc 240 ggcgagatga aagaacagac tgaatggcac cgcgttgtgc tgttcggcaa actggcagaa 300 gtggcgagcg aatatctgcg taaaggttct caggtttata tcgaaggtca gctgcgtacc 360 cgtaaatgga ccgatcaatc cggtcaggat cgctacacca cagaagtcgt ggtgaacgtt 420 ggcggcacca tgcagatgct gggtggtcgt ggatccgaat tcgagctccg tcgacaagct 480 tgcggccgca tggacaagaa gtacagcatc ggcctggaca tcggtaccaa cagcgtgggc 540 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga agttcaaggt gctgggcaac 600 accgaccgcc acagcatcaa gaagaacctg atcggcgccc tgctgttcga cagcggcgag 660 accgccgagg ccacccgcct gaagcgcacc gcccgccgcc gctacacccg ccgcaagaac 720 cgcatctgct acctgcagga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc 780 ttccaccgcc tggaggagag cttcctggtg gaggaggaca agaagcacga gcgccacccc 840 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac 900 ctgcgcaaga agctggtgga cagcaccgac aaggccgacc tgcgcctgat ctacctggcc 960 ctggcccaca tgatcaagtt ccgcggccac ttcctgatcg agggcgacct gaaccccgac 1020 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag 1080 gagaacccca tcaacgccag cggcgtggac gccaaggcca tcctgagcgc ccgcctgagc 1140 aagagccgcc gcctggagaa cctgatcgcc cagctgcccg gcgagaagaa gaacggcctg 1200 ttcggcaacc tgatcgccct gagcctgggc ctgaccccca acttcaagag caacttcgac 1260 ctggccgagg acgccaagct gcagctgagc aaggacacct acgacgacga cctggacaac 1320 ctgctggccc agatcggcga ccagtacgcc gacctgttcc tggccgccaa gaacctgagc 1380 gacgccatcc tgctgagcga catcctgcgc gtgaacaccg agatcaccaa ggcccccctg 1440 agcgccagca tgatcaagcg ctacgacgag caccaccagg acctgaccct gctgaaggcc 1500 ctggtgcgcc agcagctgcc cgagaagtac aaggagatct tcttcgacca gagcaagaac 1560 ggctacgccg gctacatcga cggcggcgcc agccaggagg agttctacaa gttcatcaag 1620 cccatcctgg agaagatgga cggcaccgag gagctgctgg tgaagctgaa ccgcgaggac 1680 ctgctgcgca agcagcgcac cttcgacaac ggcagcatcc cccaccagat ccacctgggc 1740 gagctgcacg ccatcctgcg ccgccaggag gacttctacc ccttcctgaa ggacaaccgc 1800 gagaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc cctggcccgc 1860 ggcaacagcc gcttcgcctg gatgacccgc aagagcgagg agaccatcac cccctggaac 1920 ttcgaggagg tggtggacaa gggcgccagc gcccagagct tcatcgagcg catgaccaac 1980 ttcgacaaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac 2040 ttcaccgtgt acaacgagct gaccaaggtg aagtacgtga ccgagggcat gcgcaagccc 2100 gccttcctga gcggcgagca gaagaaggcc atcgtggacc tgctgttcaa gaccaaccgc 2160 aaggtgaccg tgaagcagct gaaggaggac tacttcaaga agatcgagtg cttcgacagc 2220 gtggagatca gcggcgtgga ggaccgcttc aacgccagcc tgggcaccta ccacgacctg 2280 ctgaagatca tcaaggacaa ggacttcctg gacaacgagg agaacgagga catcctggag 2340 gacatcgtgc tgaccctgac cctgttcgag gaccgcgaga tgatcgagga gcgcctgaag 2400 acctacgccc acctgttcga cgacaaggtg atgaagcagc tgaagcgccg ccgctacacc 2460 ggctggggcc gcctgagccg caagcttatc aacggcatcc gcgacaagca gagcggcaag 2520 accatcctgg acttcctgaa gagcgacggc ttcgccaacc gcaacttcat gcagctgatc 2580 cacgacgaca gcctgacctt caaggaggac atccagaagg cccaggtgag cggccagggc 2640 gacagcctgc acgagcacat cgccaacctg gccggcagcc ccgccatcaa gaagggcatc 2700 ctgcagaccg tgaaggtggt ggacgagctg gtgaaggtga tgggccgcca caagcccgag 2760 aacatcgtga tcgagatggc ccgcgagaac cagaccaccc agaagggcca gaagaacagc 2820 cgcgagcgca tgaagcgcat cgaggagggc atcaaggagc tgggcagcca gatcctgaag 2880 gagcaccccg tggagaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag 2940 aacggccgcg acatgtacgt ggaccaggag ctggacatca accgcctgag cgactacgac 3000 gtggaccaca tcgtgcccca gagcttcctg aaggacgaca gcatcgacaa caaggtgctg 3060 acccgcagcg acaagaaccg cggcaagagc gacaacgtgc ccagcgagga ggtggtgaag 3120 aagatgaaga actactggcg ccagctgctg aacgccaagc tgatcaccca gcgcaagttc 3180 gacaacctga ccaaggccga gcgcggcggc ctgagcgagc tggacaaggc cggcttcatc 3240 aagcgccagc tggtggagac ccgccagatc accaagcacg tggcccagat cctggacagc 3300 cgcatgaaca ccaagtacga cgagaacgac aagctgatcc gcgaggtgaa ggtgatcacc 3360 ctgaagagca agctggtgag cgacttccgc aaggacttcc agttctacaa ggtgcgcgag 3420 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtggtggg caccgccctg 3480 atcaagaagt accccaagct ggagagcgag ttcgtgtacg gcgactacaa ggtgtacgac 3540 gtgcgcaaga tgatcgccaa gagcgagcag gagatcggca aggccaccgc caagtacttc 3600 ttctacagca acatcatgaa cttcttcaag accgagatca ccctggccaa cggcgagatc 3660 cgcaagcgcc ccctgatcga gaccaacggc gagaccggcg agatcgtgtg ggacaagggc 3720 cgcgacttcg ccaccgtgcg caaggtgctg agcatgcccc aggtgaacat cgtgaagaag 3780 accgaggtgc agaccggcgg cttcagcaag gagagcatcc tgcccaagcg caacagcgac 3840 aagctgatcg cccgcaagaa ggactgggac cccaagaagt acggcggctt cgacagcccc 3900 accgtggcct acagcgtgct ggtggtggcc aaggtggaga agggcaagag caagaagctg 3960 aagagcgtga aggagctgct gggcatcacc atcatggagc gcagcagctt cgagaagaac 4020 cccatcgact tcctggaggc caagggctac aaggaggtga agaaggacct gatcatcaag 4080 ctgcccaagt acagcctgtt cgagctggag aacggccgca agcgcatgct ggccagcgcc 4140 ggcgagctgc agaagggcaa cgagctggcc ctgcccagca agtacgtgaa cttcctgtac 4200 ctggccagcc actacgagaa gctgaagggc agccccgagg acaacgagca gaagcagctg 4260 ttcgtggagc agcacaagca ctacctggac gagatcatcg agcagatcag cgagttcagc 4320 aagcgcgtga tcctggccga cgccaacctg gacaaggtgc tgagcgccta caacaagcac 4380 cgcgacaagc ccatccgcga gcaggccgag aacatcatcc acctgttcac cctgaccaac 4440 ctgggcgccc ccgccgcctt caagtacttc gacaccacca tcgaccgcaa gcgctacacc 4500 agcaccaagg aggtgctgga cgccaccctg atccaccaga gcatcaccgg tctgtacgag 4560 acccgcatcg acctgagcca gctgggcggc gacgcggccg cactcgacct gcaggtgaaa 4620 caacagatac aacttcgtcg ccgtgaagtc gatgaaacgg cagacttgcc cgctgaattg 4680 cctcccttgc tgcgccgttt atacgccagc cggggagtac gcagtgcgca agaactggaa 4740 cgcagtgtta aaggtatgct gccctggcag caactgagcg gcgtcgaaaa ggccgttgag 4800 atcctttaca acgcttttcg cgaaggaacg cggattattg tggtcggtga tttcgacgcc 4860 gacggcgcga ccagcacggc tctaagcgtg ctggcgatgc gctcgcttgg ttgcagcaat 4920 atcgactacc tggtaccaaa ccgtttcgaa gacggttacg gcttaagccc ggaagtggtc 4980 gatcaggccc atgcccgtgg cgcgcagtta attgtcacgg tggataacgg tatttcctcc 5040 catgcggggg ttgagcacgc tcgctcgttg ggcatcccgg ttattgttac cgatcaccat 5100 ttgccaggcg acacattacc cgcagcggaa gcgatcatta accctaactt gcgcgactgt 5160 aatttcccgt cgaaatcact ggcaggcgtg ggtgtggcgt tttatctgat gctggcgctg 5220 cgcacctttt tgcgcgatca gggctggttt gatgagcgta acatcgcaat tcctaacctg 5280 gcagaactgc tggatctggt cgcgctgggg acagtggcgg acgtcgtgcc gctggacgct 5340 aataatcgca ttctgacctg gcaggggatg agtcgcatcc gagccggaaa gtgccgtccg 5400 gggattaaag cgctgcttga agtggcaaac cgtgatgcac aaaaactcgc cgccagcgat 5460 ttaggttttg cgctggggcc acgtctcaat gctgccggac gactggacga tatgtccgtc 5520 ggtgtggcgc tgttgttgtg cgacaacatc ggcgaagcgc gcgtgctggc aaatgaactc 5580 gatgcgctaa accagacgcg aaaagagatc gaacaaggaa tgcaaattga agccctgacc 5640 ctgtgcgaga aactggagcg cagccgtgac acgctacccg gcgggctggc aatgtatcac 5700 cccgaatggc atcagggcgt tgtcggtatt ctggcttcgc gcatcaaaga gcgttttcac 5760 cgtccggtta tcgcgtttgc gccagcaggt gacggtacgc tgaaaggttc cggtcgctcc 5820 attcaggggc tgcatatgcg tgatgcgctg gagcgattag acacactcta ccctggcatg 5880 atgctgaagt ttggcggtca tgcgatggcg gcgggtttgt cgctggaaga ggataaattc 5940 aaactctttc aacaacggtt tggcgaactg gttactgagt ggctggaccc ttcgctattg 6000 caaggcgaag tggtatcaga cggtccgtta agcccggccg aaatgaccat ggaagtggcg 6060 cagctgctgc gcgatgctgg cccgtggggg cagatgttcc cggagccgct gtttgacggt 6120 catttccgtc tgctgcaaca gcggctggtg ggcgaacgtc atttgaaggt gatggtcgaa 6180 ccggtcggcg gcggtccact gctggatggt attgctttta atgtcgatac cgccctctgg 6240 ccggataacg gcgtgcgcga agtgcaactg gcttataagc tcgatatcaa cgagtttcgc 6300 ggcaaccgca gcctgcaaat tatcatcgac aatatctggc caattctgca gaaaaggccg 6360 gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagc accaccacca ccaccactga 6420 6420 <210> 51 <211> 1533 <212> PRT <213> Artificial Sequence <220> <223> DSB-SpyCas9 <400> 51 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Ala Lys Lys Glu Met Val Glu Phe Asp Glu Ala Ile His 35 40 45 Gly Glu Asp Leu Ala Lys Phe Ile Lys Glu Ala Ser Asp His Lys Leu 50 55 60 Lys Ile Ser Gly Tyr Asn Glu Leu Ile Lys Asp Ile Arg Ile Arg Ala 65 70 75 80 Lys Asp Glu Leu Gly Val Asp Gly Lys Met Phe Asn Arg Leu Leu Ala 85 90 95 Leu Tyr His Lys Asp Asn Arg Asp Val Phe Glu Ala Glu Thr Glu Glu 100 105 110 Val Val Glu Leu Tyr Asp Thr Val Phe Ser Lys Gly Ser Glu Phe Glu 115 120 125 Leu Arg Arg Gln Ala Cys Gly Arg Met Asp Lys Lys Tyr Ser Ile Gly 130 135 140 Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu 145 150 155 160 Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg 165 170 175 His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly 180 185 190 Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr 195 200 205 Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn 210 215 220 Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser 225 230 235 240 Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly 245 250 255 Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr 260 265 270 His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg 275 280 285 Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe 290 295 300 Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu 305 310 315 320 Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro 325 330 335 Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu 340 345 350 Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu 355 360 365 Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu 370 375 380 Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu 385 390 395 400 Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala 405 410 415 Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu 420 425 430 Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile 435 440 445 Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His 450 455 460 His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro 465 470 475 480 Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala 485 490 495 Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile 500 505 510 Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys 515 520 525 Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly 530 535 540 Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg 545 550 555 560 Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile 565 570 575 Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala 580 585 590 Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr 595 600 605 Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala 610 615 620 Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn 625 630 635 640 Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val 645 650 655 Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys 660 665 670 Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu 675 680 685 Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr 690 695 700 Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu 705 710 715 720 Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile 725 730 735 Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu 740 745 750 Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile 755 760 765 Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met 770 775 780 Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg 785 790 795 800 Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu 805 810 815 Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu 820 825 830 Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln 835 840 845 Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala 850 855 860 Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val 865 870 875 880 Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val 885 890 895 Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn 900 905 910 Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly 915 920 925 Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn 930 935 940 Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val 945 950 955 960 Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His 965 970 975 Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val 980 985 990 Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser 995 1000 1005 Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn 1010 1015 1020 Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu 1025 1030 1035 1040 Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln 1045 1050 1055 Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp 1060 1065 1070 Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu 1075 1080 1085 Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys 1090 1095 1100 Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala 1105 1110 1115 1120 His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys 1125 1130 1135 Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr 1140 1145 1150 Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala 1155 1160 1165 Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr 1170 1175 1180 Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu 1185 1190 1195 1200 Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe 1205 1210 1215 Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys 1220 1225 1230 Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro 1235 1240 1245 Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1250 1255 1260 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu 1265 1270 1275 1280 Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val 1285 1290 1295 Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys 1300 1305 1310 Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys 1315 1320 1325 Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn 1330 1335 1340 Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn 1345 1350 1355 1360 Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser 1365 1370 1375 His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln 1380 1385 1390 Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln 1395 1400 1405 Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp 1410 1415 1420 Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu 1425 1430 1435 1440 Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala 1445 1450 1455 Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr 1460 1465 1470 Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile 1475 1480 1485 Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1490 1495 1500 Ala Ala Ala Leu Asp Leu Glu Lys Arg Pro Ala Ala Thr Lys Lys Ala 1505 1510 1515 1520 Gly Gln Ala Lys Lys Lys Lys His His His His His His 1525 1530 <210> 52 <211> 4602 <212> DNA <213> Artificial Sequence <220> <223> DSB-SpyCas9 <400> 52 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggctaa aaaagaaatg 120 gttgaatttg atgaagctat ccatggcgaa gacttggcta aatttattaa agaagcatct 180 gatcataaac tgaaaatttc cggttataat gaactgatta aagatattcg aattcgtgct 240 aaagatgaac ttggcgttga tggtaagatg tttaatcgtc tattagcttt gtatcataaa 300 gataaccgtg atgtgtttga agctgaaact gaagaggtag ttgaacttta tgacacagtt 360 ttctctaaag gatccgaatt cgagctccgt cgacaagctt gcggccgcat ggacaagaag 420 tacagcatcg gcctggacat cggtaccaac agcgtgggct gggccgtgat caccgacgag 480 tacaaggtgc ccagcaagaa gttcaaggtg ctgggcaaca ccgaccgcca cagcatcaag 540 aagaacctga tcggcgccct gctgttcgac agcggcgaga ccgccgaggc cacccgcctg 600 aagcgcaccg cccgccgccg ctacacccgc cgcaagaacc gcatctgcta cctgcaggag 660 atcttcagca acgagatggc caaggtggac gacagcttct tccaccgcct ggaggagagc 720 ttcctggtgg aggaggacaa gaagcacgag cgccacccca tcttcggcaa catcgtggac 780 gaggtggcct accacgagaa gtaccccacc atctaccacc tgcgcaagaa gctggtggac 840 agcaccgaca aggccgacct gcgcctgatc tacctggccc tggcccacat gatcaagttc 900 cgcggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg 960 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg agaaccccat caacgccagc 1020 ggcgtggacg ccaaggccat cctgagcgcc cgcctgagca agagccgccg cctggagaac 1080 ctgatcgccc agctgcccgg cgagaagaag aacggcctgt tcggcaacct gatcgccctg 1140 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga cgccaagctg 1200 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac 1260 cagtacgccg acctgttcct ggccgccaag aacctgagcg acgccatcct gctgagcgac 1320 atcctgcgcg tgaacaccga gatcaccaag gcccccctga gcgccagcat gatcaagcgc 1380 tacgacgagc accaccagga cctgaccctg ctgaaggccc tggtgcgcca gcagctgccc 1440 gagaagtaca aggagatctt cttcgaccag agcaagaacg gctacgccgg ctacatcgac 1500 ggcggcgcca gccaggagga gttctacaag ttcatcaagc ccatcctgga gaagatggac 1560 ggcaccgagg agctgctggt gaagctgaac cgcgaggacc tgctgcgcaa gcagcgcacc 1620 ttcgacaacg gcagcatccc ccaccagatc cacctgggcg agctgcacgc catcctgcgc 1680 cgccaggagg acttctaccc cttcctgaag gacaaccgcg agaagatcga gaagatcctg 1740 accttccgca tcccctacta cgtgggcccc ctggcccgcg gcaacagccg cttcgcctgg 1800 atgacccgca agagcgagga gaccatcacc ccctggaact tcgaggaggt ggtggacaag 1860 ggcgccagcg cccagagctt catcgagcgc atgaccaact tcgacaagaa cctgcccaac 1920 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta caacgagctg 1980 accaaggtga agtacgtgac cgagggcatg cgcaagcccg ccttcctgag cggcgagcag 2040 aagaaggcca tcgtggacct gctgttcaag accaaccgca aggtgaccgt gaagcagctg 2100 aaggaggact acttcaagaa gatcgagtgc ttcgacagcg tggagatcag cggcgtggag 2160 gaccgcttca acgccagcct gggcacctac cacgacctgc tgaagatcat caaggacaag 2220 gacttcctgg acaacgagga gaacgaggac atcctggagg acatcgtgct gaccctgacc 2280 ctgttcgagg accgcgagat gatcgaggag cgcctgaaga cctacgccca cctgttcgac 2340 gacaaggtga tgaagcagct gaagcgccgc cgctacaccg gctggggccg cctgagccgc 2400 aagcttatca acggcatccg cgacaagcag agcggcaaga ccatcctgga cttcctgaag 2460 agcgacggct tcgccaaccg caacttcatg cagctgatcc acgacgacag cctgaccttc 2520 aaggaggaca tccagaaggc ccaggtgagc ggccagggcg acagcctgca cgagcacatc 2580 gccaacctgg ccggcagccc cgccatcaag aagggcatcc tgcagaccgt gaaggtggtg 2640 gacgagctgg tgaaggtgat gggccgccac aagcccgaga acatcgtgat cgagatggcc 2700 cgcgagaacc agaccaccca gaagggccag aagaacagcc gcgagcgcat gaagcgcatc 2760 gaggagggca tcaaggagct gggcagccag atcctgaagg agcaccccgt ggagaacacc 2820 cagctgcaga acgagaagct gtacctgtac tacctgcaga acggccgcga catgtacgtg 2880 gaccaggagc tggacatcaa ccgcctgagc gactacgacg tggaccacat cgtgccccag 2940 agcttcctga aggacgacag catcgacaac aaggtgctga cccgcagcga caagaaccgc 3000 ggcaagagcg acaacgtgcc cagcgaggag gtggtgaaga agatgaagaa ctactggcgc 3060 cagctgctga acgccaagct gatcacccag cgcaagttcg acaacctgac caaggccgag 3120 cgcggcggcc tgagcgagct ggacaaggcc ggcttcatca agcgccagct ggtggagacc 3180 cgccagatca ccaagcacgt ggcccagatc ctggacagcc gcatgaacac caagtacgac 3240 gagaacgaca agctgatccg cgaggtgaag gtgatcaccc tgaagagcaa gctggtgagc 3300 gacttccgca aggacttcca gttctacaag gtgcgcgaga tcaacaacta ccaccacgcc 3360 cacgacgcct acctgaacgc cgtggtgggc accgccctga tcaagaagta ccccaagctg 3420 gagagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcgcaagat gatcgccaag 3480 agcgagcagg agatcggcaa ggccaccgcc aagtacttct tctacagcaa catcatgaac 3540 ttcttcaaga ccgagatcac cctggccaac ggcgagatcc gcaagcgccc cctgatcgag 3600 accaacggcg agaccggcga gatcgtgtgg gacaagggcc gcgacttcgc caccgtgcgc 3660 aaggtgctga gcatgcccca ggtgaacatc gtgaagaaga ccgaggtgca gaccggcggc 3720 ttcagcaagg agagcatcct gcccaagcgc aacagcgaca agctgatcgc ccgcaagaag 3780 gactgggacc ccaagaagta cggcggcttc gacagcccca ccgtggccta cagcgtgctg 3840 gtggtggcca aggtggagaa gggcaagagc aagaagctga agagcgtgaa ggagctgctg 3900 ggcatcacca tcatggagcg cagcagcttc gagaagaacc ccatcgactt cctggaggcc 3960 aagggctaca aggaggtgaa gaaggacctg atcatcaagc tgcccaagta cagcctgttc 4020 gagctggaga acggccgcaa gcgcatgctg gccagcgccg gcgagctgca gaagggcaac 4080 gagctggccc tgcccagcaa gtacgtgaac ttcctgtacc tggccagcca ctacgagaag 4140 ctgaagggca gccccgagga caacgagcag aagcagctgt tcgtggagca gcacaagcac 4200 tacctggacg agatcatcga gcagatcagc gagttcagca agcgcgtgat cctggccgac 4260 gccaacctgg acaaggtgct gagcgcctac aacaagcacc gcgacaagcc catccgcgag 4320 caggccgaga acatcatcca cctgttcacc ctgaccaacc tgggcgcccc cgccgccttc 4380 aagtacttcg acaccaccat cgaccgcaag cgctacacca gcaccaagga ggtgctggac 4440 gccaccctga tccaccagag catcaccggt ctgtacgaga cccgcatcga cctgagccag 4500 ctgggcggcg acgcggccgc actcgacctc gagaaaaggc cggcggccac gaaaaaggcc 4560 ggccaggcaa aaaagaaaaa gcaccaccac caccaccact ga 4602 <210> 53 <211> 2112 <212> PRT <213> Artificial Sequence <220> <223> DSB-SpyCas9-RecJ <400> 53 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Ala Lys Lys Glu Met Val Glu Phe Asp Glu Ala Ile His 35 40 45 Gly Glu Asp Leu Ala Lys Phe Ile Lys Glu Ala Ser Asp His Lys Leu 50 55 60 Lys Ile Ser Gly Tyr Asn Glu Leu Ile Lys Asp Ile Arg Ile Arg Ala 65 70 75 80 Lys Asp Glu Leu Gly Val Asp Gly Lys Met Phe Asn Arg Leu Leu Ala 85 90 95 Leu Tyr His Lys Asp Asn Arg Asp Val Phe Glu Ala Glu Thr Glu Glu 100 105 110 Val Val Glu Leu Tyr Asp Thr Val Phe Ser Lys Gly Ser Glu Phe Glu 115 120 125 Leu Arg Arg Gln Ala Cys Gly Arg Met Asp Lys Lys Tyr Ser Ile Gly 130 135 140 Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu 145 150 155 160 Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg 165 170 175 His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly 180 185 190 Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr 195 200 205 Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn 210 215 220 Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser 225 230 235 240 Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly 245 250 255 Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr 260 265 270 His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg 275 280 285 Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe 290 295 300 Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu 305 310 315 320 Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro 325 330 335 Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu 340 345 350 Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu 355 360 365 Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu 370 375 380 Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu 385 390 395 400 Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala 405 410 415 Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu 420 425 430 Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile 435 440 445 Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His 450 455 460 His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro 465 470 475 480 Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala 485 490 495 Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile 500 505 510 Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys 515 520 525 Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly 530 535 540 Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg 545 550 555 560 Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile 565 570 575 Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala 580 585 590 Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr 595 600 605 Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala 610 615 620 Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn 625 630 635 640 Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val 645 650 655 Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys 660 665 670 Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu 675 680 685 Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr 690 695 700 Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu 705 710 715 720 Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile 725 730 735 Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu 740 745 750 Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile 755 760 765 Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met 770 775 780 Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg 785 790 795 800 Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu 805 810 815 Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu 820 825 830 Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln 835 840 845 Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala 850 855 860 Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val 865 870 875 880 Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val 885 890 895 Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn 900 905 910 Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly 915 920 925 Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn 930 935 940 Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val 945 950 955 960 Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His 965 970 975 Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val 980 985 990 Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser 995 1000 1005 Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn 1010 1015 1020 Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu 1025 1030 1035 1040 Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln 1045 1050 1055 Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp 1060 1065 1070 Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu 1075 1080 1085 Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys 1090 1095 1100 Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala 1105 1110 1115 1120 His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys 1125 1130 1135 Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr 1140 1145 1150 Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala 1155 1160 1165 Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr 1170 1175 1180 Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu 1185 1190 1195 1200 Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe 1205 1210 1215 Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys 1220 1225 1230 Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro 1235 1240 1245 Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1250 1255 1260 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu 1265 1270 1275 1280 Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val 1285 1290 1295 Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys 1300 1305 1310 Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys 1315 1320 1325 Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn 1330 1335 1340 Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn 1345 1350 1355 1360 Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser 1365 1370 1375 His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln 1380 1385 1390 Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln 1395 1400 1405 Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp 1410 1415 1420 Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu 1425 1430 1435 1440 Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala 1445 1450 1455 Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr 1460 1465 1470 Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile 1475 1480 1485 Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1490 1495 1500 Ala Ala Ala Leu Asp Leu Gln Val Lys Gln Gln Ile Gln Leu Arg Arg 1505 1510 1515 1520 Arg Glu Val Asp Glu Thr Ala Asp Leu Pro Ala Glu Leu Pro Pro Leu 1525 1530 1535 Leu Arg Arg Leu Tyr Ala Ser Arg Gly Val Arg Ser Ala Gln Glu Leu 1540 1545 1550 Glu Arg Ser Val Lys Gly Met Leu Pro Trp Gln Gln Leu Ser Gly Val 1555 1560 1565 Glu Lys Ala Val Glu Ile Leu Tyr Asn Ala Phe Arg Glu Gly Thr Arg 1570 1575 1580 Ile Ile Val Val Gly Asp Phe Asp Ala Asp Gly Ala Thr Ser Thr Ala 1585 1590 1595 1600 Leu Ser Val Leu Ala Met Arg Ser Leu Gly Cys Ser Asn Ile Asp Tyr 1605 1610 1615 Leu Val Pro Asn Arg Phe Glu Asp Gly Tyr Gly Leu Ser Pro Glu Val 1620 1625 1630 Val Asp Gln Ala His Ala Arg Gly Ala Gln Leu Ile Val Thr Val Asp 1635 1640 1645 Asn Gly Ile Ser Ser His Ala Gly Val Glu His Ala Arg Ser Leu Gly 1650 1655 1660 Ile Pro Val Ile Val Thr Asp His His Leu Pro Gly Asp Thr Leu Pro 1665 1670 1675 1680 Ala Ala Glu Ala Ile Ile Asn Pro Asn Leu Arg Asp Cys Asn Phe Pro 1685 1690 1695 Ser Lys Ser Leu Ala Gly Val Gly Val Ala Phe Tyr Leu Met Leu Ala 1700 1705 1710 Leu Arg Thr Phe Leu Arg Asp Gln Gly Trp Phe Asp Glu Arg Asn Ile 1715 1720 1725 Ala Ile Pro Asn Leu Ala Glu Leu Leu Asp Leu Val Ala Leu Gly Thr 1730 1735 1740 Val Ala Asp Val Val Pro Leu Asp Ala Asn Asn Arg Ile Leu Thr Trp 1745 1750 1755 1760 Gln Gly Met Ser Arg Ile Arg Ala Gly Lys Cys Arg Pro Gly Ile Lys 1765 1770 1775 Ala Leu Leu Glu Val Ala Asn Arg Asp Ala Gln Lys Leu Ala Ala Ser 1780 1785 1790 Asp Leu Gly Phe Ala Leu Gly Pro Arg Leu Asn Ala Ala Gly Arg Leu 1795 1800 1805 Asp Asp Met Ser Val Gly Val Ala Leu Leu Leu Cys Asp Asn Ile Gly 1810 1815 1820 Glu Ala Arg Val Leu Ala Asn Glu Leu Asp Ala Leu Asn Gln Thr Arg 1825 1830 1835 1840 Lys Glu Ile Glu Gln Gly Met Gln Ile Glu Ala Leu Thr Leu Cys Glu 1845 1850 1855 Lys Leu Glu Arg Ser Arg Asp Thr Leu Pro Gly Gly Leu Ala Met Tyr 1860 1865 1870 His Pro Glu Trp His Gln Gly Val Val Gly Ile Leu Ala Ser Arg Ile 1875 1880 1885 Lys Glu Arg Phe His Arg Pro Val Ile Ala Phe Ala Pro Ala Gly Asp 1890 1895 1900 Gly Thr Leu Lys Gly Ser Gly Arg Ser Ile Gln Gly Leu His Met Arg 1905 1910 1915 1920 Asp Ala Leu Glu Arg Leu Asp Thr Leu Tyr Pro Gly Met Met Leu Lys 1925 1930 1935 Phe Gly Gly His Ala Met Ala Ala Gly Leu Ser Leu Glu Glu Asp Lys 1940 1945 1950 Phe Lys Leu Phe Gln Gln Arg Phe Gly Glu Leu Val Thr Glu Trp Leu 1955 1960 1965 Asp Pro Ser Leu Leu Gln Gly Glu Val Val Ser Asp Gly Pro Leu Ser 1970 1975 1980 Pro Ala Glu Met Thr Met Glu Val Ala Gln Leu Leu Arg Asp Ala Gly 1985 1990 1995 2000 Pro Trp Gly Gln Met Phe Pro Glu Pro Leu Phe Asp Gly His Phe Arg 2005 2010 2015 Leu Leu Gln Gln Arg Leu Val Gly Glu Arg His Leu Lys Val Met Val 2020 2025 2030 Glu Pro Val Gly Gly Gly Pro Leu Leu Asp Gly Ile Ala Phe Asn Val 2035 2040 2045 Asp Thr Ala Leu Trp Pro Asp Asn Gly Val Arg Glu Val Gln Leu Ala 2050 2055 2060 Tyr Lys Leu Asp Ile Asn Glu Phe Arg Gly Asn Arg Ser Leu Gln Ile 2065 2070 2075 2080 Ile Ile Asp Asn Ile Trp Pro Ile Leu Gln Lys Arg Pro Ala Ala Thr 2085 2090 2095 Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys His His His His His His 2100 2105 2110 <210> 54 <211> 6339 <212> DNA <213> Artificial Sequence <220> <223> DSB-SpyCas9-RecJ <400> 54 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggctaa aaaagaaatg 120 gttgaatttg atgaagctat ccatggcgaa gacttggcta aatttattaa agaagcatct 180 gatcataaac tgaaaatttc cggttataat gaactgatta aagatattcg aattcgtgct 240 aaagatgaac ttggcgttga tggtaagatg tttaatcgtc tattagcttt gtatcataaa 300 gataaccgtg atgtgtttga agctgaaact gaagaggtag ttgaacttta tgacacagtt 360 ttctctaaag gatccgaatt cgagctccgt cgacaagctt gcggccgcat ggacaagaag 420 tacagcatcg gcctggacat cggtaccaac agcgtgggct gggccgtgat caccgacgag 480 tacaaggtgc ccagcaagaa gttcaaggtg ctgggcaaca ccgaccgcca cagcatcaag 540 aagaacctga tcggcgccct gctgttcgac agcggcgaga ccgccgaggc cacccgcctg 600 aagcgcaccg cccgccgccg ctacacccgc cgcaagaacc gcatctgcta cctgcaggag 660 atcttcagca acgagatggc caaggtggac gacagcttct tccaccgcct ggaggagagc 720 ttcctggtgg aggaggacaa gaagcacgag cgccacccca tcttcggcaa catcgtggac 780 gaggtggcct accacgagaa gtaccccacc atctaccacc tgcgcaagaa gctggtggac 840 agcaccgaca aggccgacct gcgcctgatc tacctggccc tggcccacat gatcaagttc 900 cgcggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg 960 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg agaaccccat caacgccagc 1020 ggcgtggacg ccaaggccat cctgagcgcc cgcctgagca agagccgccg cctggagaac 1080 ctgatcgccc agctgcccgg cgagaagaag aacggcctgt tcggcaacct gatcgccctg 1140 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga cgccaagctg 1200 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac 1260 cagtacgccg acctgttcct ggccgccaag aacctgagcg acgccatcct gctgagcgac 1320 atcctgcgcg tgaacaccga gatcaccaag gcccccctga gcgccagcat gatcaagcgc 1380 tacgacgagc accaccagga cctgaccctg ctgaaggccc tggtgcgcca gcagctgccc 1440 gagaagtaca aggagatctt cttcgaccag agcaagaacg gctacgccgg ctacatcgac 1500 ggcggcgcca gccaggagga gttctacaag ttcatcaagc ccatcctgga gaagatggac 1560 ggcaccgagg agctgctggt gaagctgaac cgcgaggacc tgctgcgcaa gcagcgcacc 1620 ttcgacaacg gcagcatccc ccaccagatc cacctgggcg agctgcacgc catcctgcgc 1680 cgccaggagg acttctaccc cttcctgaag gacaaccgcg agaagatcga gaagatcctg 1740 accttccgca tcccctacta cgtgggcccc ctggcccgcg gcaacagccg cttcgcctgg 1800 atgacccgca agagcgagga gaccatcacc ccctggaact tcgaggaggt ggtggacaag 1860 ggcgccagcg cccagagctt catcgagcgc atgaccaact tcgacaagaa cctgcccaac 1920 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta caacgagctg 1980 accaaggtga agtacgtgac cgagggcatg cgcaagcccg ccttcctgag cggcgagcag 2040 aagaaggcca tcgtggacct gctgttcaag accaaccgca aggtgaccgt gaagcagctg 2100 aaggaggact acttcaagaa gatcgagtgc ttcgacagcg tggagatcag cggcgtggag 2160 gaccgcttca acgccagcct gggcacctac cacgacctgc tgaagatcat caaggacaag 2220 gacttcctgg acaacgagga gaacgaggac atcctggagg acatcgtgct gaccctgacc 2280 ctgttcgagg accgcgagat gatcgaggag cgcctgaaga cctacgccca cctgttcgac 2340 gacaaggtga tgaagcagct gaagcgccgc cgctacaccg gctggggccg cctgagccgc 2400 aagcttatca acggcatccg cgacaagcag agcggcaaga ccatcctgga cttcctgaag 2460 agcgacggct tcgccaaccg caacttcatg cagctgatcc acgacgacag cctgaccttc 2520 aaggaggaca tccagaaggc ccaggtgagc ggccagggcg acagcctgca cgagcacatc 2580 gccaacctgg ccggcagccc cgccatcaag aagggcatcc tgcagaccgt gaaggtggtg 2640 gacgagctgg tgaaggtgat gggccgccac aagcccgaga acatcgtgat cgagatggcc 2700 cgcgagaacc agaccaccca gaagggccag aagaacagcc gcgagcgcat gaagcgcatc 2760 gaggagggca tcaaggagct gggcagccag atcctgaagg agcaccccgt ggagaacacc 2820 cagctgcaga acgagaagct gtacctgtac tacctgcaga acggccgcga catgtacgtg 2880 gaccaggagc tggacatcaa ccgcctgagc gactacgacg tggaccacat cgtgccccag 2940 agcttcctga aggacgacag catcgacaac aaggtgctga cccgcagcga caagaaccgc 3000 ggcaagagcg acaacgtgcc cagcgaggag gtggtgaaga agatgaagaa ctactggcgc 3060 cagctgctga acgccaagct gatcacccag cgcaagttcg acaacctgac caaggccgag 3120 cgcggcggcc tgagcgagct ggacaaggcc ggcttcatca agcgccagct ggtggagacc 3180 cgccagatca ccaagcacgt ggcccagatc ctggacagcc gcatgaacac caagtacgac 3240 gagaacgaca agctgatccg cgaggtgaag gtgatcaccc tgaagagcaa gctggtgagc 3300 gacttccgca aggacttcca gttctacaag gtgcgcgaga tcaacaacta ccaccacgcc 3360 cacgacgcct acctgaacgc cgtggtgggc accgccctga tcaagaagta ccccaagctg 3420 gagagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcgcaagat gatcgccaag 3480 agcgagcagg agatcggcaa ggccaccgcc aagtacttct tctacagcaa catcatgaac 3540 ttcttcaaga ccgagatcac cctggccaac ggcgagatcc gcaagcgccc cctgatcgag 3600 accaacggcg agaccggcga gatcgtgtgg gacaagggcc gcgacttcgc caccgtgcgc 3660 aaggtgctga gcatgcccca ggtgaacatc gtgaagaaga ccgaggtgca gaccggcggc 3720 ttcagcaagg agagcatcct gcccaagcgc aacagcgaca agctgatcgc ccgcaagaag 3780 gactgggacc ccaagaagta cggcggcttc gacagcccca ccgtggccta cagcgtgctg 3840 gtggtggcca aggtggagaa gggcaagagc aagaagctga agagcgtgaa ggagctgctg 3900 ggcatcacca tcatggagcg cagcagcttc gagaagaacc ccatcgactt cctggaggcc 3960 aagggctaca aggaggtgaa gaaggacctg atcatcaagc tgcccaagta cagcctgttc 4020 gagctggaga acggccgcaa gcgcatgctg gccagcgccg gcgagctgca gaagggcaac 4080 gagctggccc tgcccagcaa gtacgtgaac ttcctgtacc tggccagcca ctacgagaag 4140 ctgaagggca gccccgagga caacgagcag aagcagctgt tcgtggagca gcacaagcac 4200 tacctggacg agatcatcga gcagatcagc gagttcagca agcgcgtgat cctggccgac 4260 gccaacctgg acaaggtgct gagcgcctac aacaagcacc gcgacaagcc catccgcgag 4320 caggccgaga acatcatcca cctgttcacc ctgaccaacc tgggcgcccc cgccgccttc 4380 aagtacttcg acaccaccat cgaccgcaag cgctacacca gcaccaagga ggtgctggac 4440 gccaccctga tccaccagag catcaccggt ctgtacgaga cccgcatcga cctgagccag 4500 ctgggcggcg acgcggccgc actcgacctg caggtgaaac aacagataca acttcgtcgc 4560 cgtgaagtcg atgaaacggc agacttgccc gctgaattgc ctcccttgct gcgccgttta 4620 tacgccagcc ggggagtacg cagtgcgcaa gaactggaac gcagtgttaa aggtatgctg 4680 ccctggcagc aactgagcgg cgtcgaaaag gccgttgaga tcctttacaa cgcttttcgc 4740 gaaggaacgc ggattattgt ggtcggtgat ttcgacgccg acggcgcgac cagcacggct 4800 ctaagcgtgc tggcgatgcg ctcgcttggt tgcagcaata tcgactacct ggtaccaaac 4860 cgtttcgaag acggttacgg cttaagcccg gaagtggtcg atcaggccca tgcccgtggc 4920 gcgcagttaa ttgtcacggt ggataacggt atttcctccc atgcgggggt tgagcacgct 4980 cgctcgttgg gcatcccggt tattgttacc gatcaccatt tgccaggcga cacattaccc 5040 gcagcggaag cgatcattaa ccctaacttg cgcgactgta atttcccgtc gaaatcactg 5100 gcaggcgtgg gtgtggcgtt ttatctgatg ctggcgctgc gcaccttttt gcgcgatcag 5160 ggctggtttg atgagcgtaa catcgcaatt cctaacctgg cagaactgct ggatctggtc 5220 gcgctgggga cagtggcgga cgtcgtgccg ctggacgcta ataatcgcat tctgacctgg 5280 caggggatga gtcgcatccg agccggaaag tgccgtccgg ggattaaagc gctgcttgaa 5340 gtggcaaacc gtgatgcaca aaaactcgcc gccagcgatt taggttttgc gctggggcca 5400 cgtctcaatg ctgccggacg actggacgat atgtccgtcg gtgtggcgct gttgttgtgc 5460 gacaacatcg gcgaagcgcg cgtgctggca aatgaactcg atgcgctaaa ccagacgcga 5520 aaagagatcg aacaaggaat gcaaattgaa gccctgaccc tgtgcgagaa actggagcgc 5580 agccgtgaca cgctacccgg cgggctggca atgtatcacc ccgaatggca tcagggcgtt 5640 gtcggtattc tggcttcgcg catcaaagag cgttttcacc gtccggttat cgcgtttgcg 5700 ccagcaggtg acggtacgct gaaaggttcc ggtcgctcca ttcaggggct gcatatgcgt 5760 gatgcgctgg agcgattaga cacactctac cctggcatga tgctgaagtt tggcggtcat 5820 gcgatggcgg cgggtttgtc gctggaagag gataaattca aactctttca acaacggttt 5880 ggcgaactgg ttactgagtg gctggaccct tcgctattgc aaggcgaagt ggtatcagac 5940 ggtccgttaa gcccggccga aatgaccatg gaagtggcgc agctgctgcg cgatgctggc 6000 ccgtgggggc agatgttccc ggagccgctg tttgacggtc atttccgtct gctgcaacag 6060 cggctggtgg gcgaacgtca tttgaaggtg atggtcgaac cggtcggcgg cggtccactg 6120 ctggatggta ttgcttttaa tgtcgatacc gccctctggc cggataacgg cgtgcgcgaa 6180 gtgcaactgg cttataagct cgatatcaac gagtttcgcg gcaaccgcag cctgcaaatt 6240 atcatcgaca atatctggcc aattctgcag aaaaggccgg cggccacgaa aaaggccggc 6300 caggcaaaaa agaaaaagca ccaccaccac caccactga 6339 <210> 55 <211> 1379 <212> PRT <213> Artificial Sequence <220> <223> FnCpf1 <400> 55 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Ser Ile 35 40 45 Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe 50 55 60 Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly 65 70 75 80 Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys 85 90 95 Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser 100 105 110 Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr 115 120 125 Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys 130 135 140 Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp 145 150 155 160 Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys 165 170 175 Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp 180 185 190 Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp 195 200 205 Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe 210 215 220 Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile 225 230 235 240 Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe 245 250 255 Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu 260 265 270 Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr 275 280 285 Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser 290 295 300 Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln 305 310 315 320 Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn 325 330 335 Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr 340 345 350 Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val 355 360 365 Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile 370 375 380 Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe 385 390 395 400 Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys 405 410 415 Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp 420 425 430 Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser 435 440 445 Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu 450 455 460 Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys 465 470 475 480 Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu 485 490 495 Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg 500 505 510 Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala 515 520 525 Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu 530 535 540 Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu 545 550 555 560 Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp 565 570 575 Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln 580 585 590 Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu 595 600 605 Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr 610 615 620 Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys 625 630 635 640 Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys 645 650 655 Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys 660 665 670 Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp 675 680 685 Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr 690 695 700 Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser 705 710 715 720 Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile 725 730 735 Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr 740 745 750 Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe 755 760 765 Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe 770 775 780 Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg 785 790 795 800 Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu 805 810 815 Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr Leu Phe Gln 820 825 830 Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg Pro Asn Leu 835 840 845 His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp 850 855 860 Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln 865 870 875 880 Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn 885 890 895 Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu 900 905 910 Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro 915 920 925 Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu 930 935 940 Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His Ile Leu Ser 945 950 955 960 Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu Val Asp Gly 965 970 975 Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp 980 985 990 Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp 995 1000 1005 Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu 1010 1015 1020 Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile Ala Lys Leu 1025 1030 1035 1040 Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly 1045 1050 1055 Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys Leu 1060 1065 1070 Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe Lys Asp Asn 1075 1080 1085 Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala 1090 1095 1100 Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr 1105 1110 1115 1120 Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe 1125 1130 1135 Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu 1140 1145 1150 Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr 1155 1160 1165 Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys 1170 1175 1180 Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg 1185 1190 1195 1200 Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr 1205 1210 1215 Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His 1220 1225 1230 Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1235 1240 1245 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn 1250 1255 1260 Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp 1265 1270 1275 1280 Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro 1285 1290 1295 Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys Gly Leu 1300 1305 1310 Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys Lys Leu Asn 1315 1320 1325 Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn 1330 1335 1340 Asn Gln Ala Ala Ala Leu Glu Lys Arg Pro Ala Ala Thr Lys Lys Ala 1345 1350 1355 1360 Gly Gln Ala Lys Lys Lys Lys Ser Thr Pro Pro Pro Pro Pro Leu Arg 1365 1370 1375 Ser Gly Cys <210> 56 <211> 4140 <212> DNA <213> Artificial Sequence <220> <223> FnCpf1 <400> 56 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgtc aatttatcaa gaatttgtta ataaatatag tttaagtaaa 180 actctaagat ttgagttaat cccacagggt aaaacacttg aaaacataaa agcaagaggt 240 ttgattttag atgatgagaa aagagctaaa gactacaaaa aggctaaaca aataattgat 300 aaatatcatc agttttttat agaggagata ttaagttcgg tttgtattag cgaagattta 360 ttacaaaact attctgatgt ttattttaaa cttaaaaaga gtgatgatga taatctacaa 420 aaagatttta aaagtgcaaa agatacgata aagaaacaaa tatctgaata tataaaggac 480 tcagagaaat ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa agggcaagag 540 tcagatttaa ttctatggct aaagcaatct aaggataatg gtatagaact atttaaagcc 600 aatagtgata tcacagatat agatgaggcg ttagaaataa tcaaatcttt taaaggttgg 660 acaacttatt ttaagggttt tcatgaaaat agaaaaaatg tttatagtag caatgatatt 720 cctacatcta ttatttatag gatagtagat gataatttgc ctaaatttct agaaaataaa 780 gctaagtatg agagtttaaa agacaaagct ccagaagcta taaactatga acaaattaaa 840 aaagatttgg cagaagagct aacctttgat attgactaca aaacatctga agttaatcaa 900 agagtttttt cacttgatga agtttttgag atagcaaact ttaataatta tctaaatcaa 960 agtggtatta ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg tgaaaataca 1020 aagagaaaag gtataaatga atatataaat ctatactcac agcaaataaa tgataaaaca 1080 ctcaaaaaat ataaaatgag tgttttattt aagcaaattt taagtgatac agaatctaaa 1140 tcttttgtaa ttgataagtt agaagatgat agtgatgtag ttacaacgat gcaaagtttt 1200 tatgagcaaa tagcagcttt taaaacagta gaagaaaaat ctattaaaga aacactatct 1260 ttattatttg atgatttaaa agctcaaaaa cttgatttga gtaaaattta ttttaaaaat 1320 gataaatctc ttactgatct atcacaacaa gtttttgatg attatagtgt tattggtaca 1380 gcggtactag aatatataac tcaacaaata gcacctaaaa atcttgataa ccctagtaag 1440 aaagagcaag aattaatagc caaaaaaact gaaaaagcaa aatacttatc tctagaaact 1500 ataaagcttg ccttagaaga atttaataag catagagata tagataaaca gtgtaggttt 1560 gaagaaatac ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat agctcaaaac 1620 aaagacaatt tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa agacctactt 1680 caagctagtg cggaagatga tgttaaagct atcaaggatc ttttagatca aactaataat 1740 ctcttacata aactaaaaat atttcatatt agtcagtcag aagataaggc aaatatttta 1800 gacaaggatg agcattttta tctagtattt gaggagtgct actttgagct agcgaatata 1860 gtgcctcttt ataacaaaat tagaaactat ataactcaaa agccatatag tgatgagaaa 1920 tttaagctca attttgagaa ctcgactttg gctaatggtt gggataaaaa taaagagcct 1980 gacaatacgg caattttatt tatcaaagat gataaatatt atctgggtgt gatgaataag 2040 aaaaataaca aaatatttga tgataaagct atcaaagaaa ataaaggcga gggttataaa 2100 aaaattgttt ataaactttt acctggcgca aataaaatgt tacctaaggt tttcttttct 2160 gctaaatcta taaaatttta taatcctagt gaagatatac ttagaataag aaatcattcc 2220 acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt taatattgaa 2280 gattgccgaa aatttataga tttttataaa cagtctataa gtaagcatcc ggagtggaaa 2340 gattttggat ttagattttc tgatactcaa agatataatt ctatagatga attttataga 2400 gaagttgaaa atcaaggcta caaactaact tttgaaaata tatcagagag ctatattgat 2460 agcgtagtta atcagggtaa attgtaccta ttccaaatct ataataaaga tttttcagct 2520 tatagcaaag ggcgaccaaa tctacatact ttatattgga aagcgctgtt tgatgagaga 2580 aatcttcaag atgtggttta taagctaaat ggtgaggcag agctttttta tcgtaaacaa 2640 tcaataccta aaaaaatcac tcacccagct aaagaggcaa tagctaataa aaacaaagat 2700 aatcctaaaa aagagagtgt ttttgaatat gatttaatca aagataaacg ctttactgaa 2760 gataagtttt tctttcactg tcctattaca atcaatttta aatctagtgg agctaataag 2820 tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa atgatgttca tatattaagt 2880 atagatagag gtgaaagaca tttagcttac tatactttgg tagatggtaa aggcaatatc 2940 atcaaacaag atactttcaa catcattggt aatgatagaa tgaaaacaaa ctaccatgat 3000 aagcttgctg caatagagaa agatagggat tcagctagga aagactggaa aaagataaat 3060 aacatcaaag agatgaaaga gggctatcta tctcaggtag ttcatgaaat agctaagcta 3120 gttatagagt ataatgctat tgtggttttt gaggatttaa attttggatt taaaagaggg 3180 cgtttcaagg tagagaagca ggtctatcaa aagttagaaa aaatgctaat tgagaaacta 3240 aactatctag ttttcaaaga taatgagttt gataaaactg ggggagtgct tagagcttat 3300 cagctaacag caccttttga gacttttaaa aagatgggta aacaaacagg tattatctac 3360 tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt aaatcagtta 3420 tatcctaagt atgaaagtgt cagcaaatct caagagttct ttagtaagtt tgacaagatt 3480 tgttataacc ttgataaggg ctattttgag tttagttttg attataaaaa ctttggtgac 3540 aaggctgcca aaggcaagtg gactatagct agctttggga gtagattgat taactttaga 3600 aattcagata aaaatcataa ttgggatact cgagaagttt atccaactaa agagttggag 3660 aaattgctaa aagattattc tatcgaatat gggcatggcg aatgtatcaa agcagctatt 3720 tgcggtgaga gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa tactatctta 3780 caaatgcgta actcaaaaac aggtactgag ttagattatc taatttcacc agtagcagat 3840 gtaaatggca atttctttga ttcgcgacag gcgccaaaaa atatgcctca agatgctgat 3900 gccaatggtg cttatcatat tgggctaaaa ggtctgatgc tactaggtag gatcaaaaat 3960 aatcaagagg gcaaaaaact caatttggtt atcaaaaatg aagagtattt tgagttcgtg 4020 cagaatagga ataaccaagc ggccgcactc gagaaaaggc cggcggccac gaaaaaggcc 4080 ggccaggcaa aaaagaaaaa gtcgacacca ccaccaccac cactgagatc cggctgctaa 4140 4140 <210> 57 <211> 1954 <212> PRT <213> Artificial Sequence <220> <223> FnCpf1-RecJ <400> 57 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Ser Ile 35 40 45 Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe 50 55 60 Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly 65 70 75 80 Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys 85 90 95 Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser 100 105 110 Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr 115 120 125 Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys 130 135 140 Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp 145 150 155 160 Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys 165 170 175 Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp 180 185 190 Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp 195 200 205 Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe 210 215 220 Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile 225 230 235 240 Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe 245 250 255 Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu 260 265 270 Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr 275 280 285 Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser 290 295 300 Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln 305 310 315 320 Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn 325 330 335 Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr 340 345 350 Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val 355 360 365 Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile 370 375 380 Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe 385 390 395 400 Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys 405 410 415 Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp 420 425 430 Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser 435 440 445 Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu 450 455 460 Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys 465 470 475 480 Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu 485 490 495 Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg 500 505 510 Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala 515 520 525 Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu 530 535 540 Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu 545 550 555 560 Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp 565 570 575 Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln 580 585 590 Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu 595 600 605 Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr 610 615 620 Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys 625 630 635 640 Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys 645 650 655 Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys 660 665 670 Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp 675 680 685 Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr 690 695 700 Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser 705 710 715 720 Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile 725 730 735 Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr 740 745 750 Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe 755 760 765 Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe 770 775 780 Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg 785 790 795 800 Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu 805 810 815 Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr Leu Phe Gln 820 825 830 Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg Pro Asn Leu 835 840 845 His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp 850 855 860 Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln 865 870 875 880 Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn 885 890 895 Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu 900 905 910 Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro 915 920 925 Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu 930 935 940 Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His Ile Leu Ser 945 950 955 960 Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu Val Asp Gly 965 970 975 Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp 980 985 990 Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp 995 1000 1005 Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu 1010 1015 1020 Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile Ala Lys Leu 1025 1030 1035 1040 Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly 1045 1050 1055 Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys Leu 1060 1065 1070 Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe Lys Asp Asn 1075 1080 1085 Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala 1090 1095 1100 Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr 1105 1110 1115 1120 Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe 1125 1130 1135 Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu 1140 1145 1150 Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr 1155 1160 1165 Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys 1170 1175 1180 Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg 1185 1190 1195 1200 Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr 1205 1210 1215 Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His 1220 1225 1230 Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1235 1240 1245 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn 1250 1255 1260 Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp 1265 1270 1275 1280 Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro 1285 1290 1295 Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys Gly Leu 1300 1305 1310 Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys Lys Leu Asn 1315 1320 1325 Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn 1330 1335 1340 Asn Gln Ala Ala Ala Leu Asp Leu Gln Val Lys Gln Gln Ile Gln Leu 1345 1350 1355 1360 Arg Arg Arg Glu Val Asp Glu Thr Ala Asp Leu Pro Ala Glu Leu Pro 1365 1370 1375 Pro Leu Leu Arg Arg Leu Tyr Ala Ser Arg Gly Val Arg Ser Ala Gln 1380 1385 1390 Glu Leu Glu Arg Ser Val Lys Gly Met Leu Pro Trp Gln Gln Leu Ser 1395 1400 1405 Gly Val Glu Lys Ala Val Glu Ile Leu Tyr Asn Ala Phe Arg Glu Gly 1410 1415 1420 Thr Arg Ile Ile Val Val Gly Asp Phe Asp Ala Asp Gly Ala Thr Ser 1425 1430 1435 1440 Thr Ala Leu Ser Val Leu Ala Met Arg Ser Leu Gly Cys Ser Asn Ile 1445 1450 1455 Asp Tyr Leu Val Pro Asn Arg Phe Glu Asp Gly Tyr Gly Leu Ser Pro 1460 1465 1470 Glu Val Val Asp Gln Ala His Ala Arg Gly Ala Gln Leu Ile Val Thr 1475 1480 1485 Val Asp Asn Gly Ile Ser Ser His Ala Gly Val Glu His Ala Arg Ser 1490 1495 1500 Leu Gly Ile Pro Val Ile Val Thr Asp His His Leu Pro Gly Glu Thr 1505 1510 1515 1520 Leu Pro Ala Ala Glu Ala Ile Ile Asn Pro Asn Leu Arg Asp Cys Asn 1525 1530 1535 Phe Pro Ser Lys Ser Leu Ala Gly Val Gly Val Ala Phe Tyr Leu Met 1540 1545 1550 Leu Ala Leu Arg Thr Phe Leu Arg Asp Gln Gly Trp Phe Asp Glu Arg 1555 1560 1565 Gly Ile Ala Ile Pro Asn Leu Ala Glu Leu Leu Asp Leu Val Ala Leu 1570 1575 1580 Gly Thr Val Ala Asp Val Val Pro Leu Asp Ala Asn Asn Arg Ile Leu 1585 1590 1595 1600 Thr Trp Gln Gly Met Ser Arg Ile Arg Ala Gly Lys Cys Arg Pro Gly 1605 1610 1615 Ile Lys Ala Leu Leu Glu Val Ala Asn Arg Asp Ala Gln Lys Leu Ala 1620 1625 1630 Ala Ser Asp Leu Gly Phe Ala Leu Gly Pro Arg Leu Asn Ala Ala Gly 1635 1640 1645 Arg Leu Asp Asp Met Ser Val Gly Val Ala Leu Leu Leu Cys Asp Asn 1650 1655 1660 Ile Gly Glu Ala Arg Val Leu Ala Asn Glu Leu Asp Ala Leu Asn Gln 1665 1670 1675 1680 Thr Arg Lys Glu Ile Glu Gln Gly Met Gln Val Glu Ala Leu Thr Leu 1685 1690 1695 Cys Glu Lys Leu Glu Arg Ser Arg Asp Thr Leu Pro Gly Gly Leu Ala 1700 1705 1710 Met Tyr His Pro Glu Trp His Gln Gly Val Val Gly Ile Leu Ala Ser 1715 1720 1725 Arg Ile Lys Glu Arg Phe His Arg Pro Val Ile Ala Phe Ala Pro Ala 1730 1735 1740 Gly Asp Gly Thr Leu Lys Gly Ser Gly Arg Ser Ile Gln Gly Leu His 1745 1750 1755 1760 Met Arg Asp Ala Leu Glu Arg Leu Asp Thr Leu Tyr Pro Gly Met Ile 1765 1770 1775 Leu Lys Phe Gly Gly His Ala Met Ala Ala Gly Leu Ser Leu Glu Glu 1780 1785 1790 Asp Lys Phe Glu Leu Phe Gln Gln Arg Phe Gly Glu Leu Val Thr Glu 1795 1800 1805 Trp Leu Asp Pro Ser Leu Leu Gln Gly Glu Val Val Ser Asp Gly Pro 1810 1815 1820 Leu Ser Pro Ala Glu Met Thr Met Glu Val Ala Gln Leu Leu Arg Asp 1825 1830 1835 1840 Ala Gly Pro Trp Gly Gln Met Phe Pro Glu Pro Leu Phe Asp Gly His 1845 1850 1855 Phe Arg Leu Leu Gln Gln Arg Leu Val Gly Glu Arg His Leu Lys Val 1860 1865 1870 Met Val Glu Pro Val Gly Gly Gly Pro Leu Leu Asp Gly Ile Ala Phe 1875 1880 1885 Asn Val Asp Thr Ala Leu Trp Pro Asp Asn Gly Val Arg Glu Val Gln 1890 1895 1900 Leu Ala Tyr Lys Leu Asp Ile Asn Glu Phe Arg Gly Asn Arg Ser Leu 1905 1910 1915 1920 Gln Ile Ile Ile Asp Asn Ile Trp Pro Ile Leu Gln Lys Arg Pro Ala 1925 1930 1935 Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys His His His His 1940 1945 1950 His His <210> 58 <211> 5865 <212> DNA <213> Artificial Sequence <220> <223> FnCpf1-RecJ <400> 58 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgtc aatttatcaa gaatttgtta ataaatatag tttaagtaaa 180 actctaagat ttgagttaat cccacagggt aaaacacttg aaaacataaa agcaagaggt 240 ttgattttag atgatgagaa aagagctaaa gactacaaaa aggctaaaca aataattgat 300 aaatatcatc agttttttat agaggagata ttaagttcgg tttgtattag cgaagattta 360 ttacaaaact attctgatgt ttattttaaa cttaaaaaga gtgatgatga taatctacaa 420 aaagatttta aaagtgcaaa agatacgata aagaaacaaa tatctgaata tataaaggac 480 tcagagaaat ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa agggcaagag 540 tcagatttaa ttctatggct aaagcaatct aaggataatg gtatagaact atttaaagcc 600 aatagtgata tcacagatat agatgaggcg ttagaaataa tcaaatcttt taaaggttgg 660 acaacttatt ttaagggttt tcatgaaaat agaaaaaatg tttatagtag caatgatatt 720 cctacatcta ttatttatag gatagtagat gataatttgc ctaaatttct agaaaataaa 780 gctaagtatg agagtttaaa agacaaagct ccagaagcta taaactatga acaaattaaa 840 aaagatttgg cagaagagct aacctttgat attgactaca aaacatctga agttaatcaa 900 agagtttttt cacttgatga agtttttgag atagcaaact ttaataatta tctaaatcaa 960 agtggtatta ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg tgaaaataca 1020 aagagaaaag gtataaatga atatataaat ctatactcac agcaaataaa tgataaaaca 1080 ctcaaaaaat ataaaatgag tgttttattt aagcaaattt taagtgatac agaatctaaa 1140 tcttttgtaa ttgataagtt agaagatgat agtgatgtag ttacaacgat gcaaagtttt 1200 tatgagcaaa tagcagcttt taaaacagta gaagaaaaat ctattaaaga aacactatct 1260 ttattatttg atgatttaaa agctcaaaaa cttgatttga gtaaaattta ttttaaaaat 1320 gataaatctc ttactgatct atcacaacaa gtttttgatg attatagtgt tattggtaca 1380 gcggtactag aatatataac tcaacaaata gcacctaaaa atcttgataa ccctagtaag 1440 aaagagcaag aattaatagc caaaaaaact gaaaaagcaa aatacttatc tctagaaact 1500 ataaagcttg ccttagaaga atttaataag catagagata tagataaaca gtgtaggttt 1560 gaagaaatac ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat agctcaaaac 1620 aaagacaatt tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa agacctactt 1680 caagctagtg cggaagatga tgttaaagct atcaaggatc ttttagatca aactaataat 1740 ctcttacata aactaaaaat atttcatatt agtcagtcag aagataaggc aaatatttta 1800 gacaaggatg agcattttta tctagtattt gaggagtgct actttgagct agcgaatata 1860 gtgcctcttt ataacaaaat tagaaactat ataactcaaa agccatatag tgatgagaaa 1920 tttaagctca attttgagaa ctcgactttg gctaatggtt gggataaaaa taaagagcct 1980 gacaatacgg caattttatt tatcaaagat gataaatatt atctgggtgt gatgaataag 2040 aaaaataaca aaatatttga tgataaagct atcaaagaaa ataaaggcga gggttataaa 2100 aaaattgttt ataaactttt acctggcgca aataaaatgt tacctaaggt tttcttttct 2160 gctaaatcta taaaatttta taatcctagt gaagatatac ttagaataag aaatcattcc 2220 acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt taatattgaa 2280 gattgccgaa aatttataga tttttataaa cagtctataa gtaagcatcc ggagtggaaa 2340 gattttggat ttagattttc tgatactcaa agatataatt ctatagatga attttataga 2400 gaagttgaaa atcaaggcta caaactaact tttgaaaata tatcagagag ctatattgat 2460 agcgtagtta atcagggtaa attgtaccta ttccaaatct ataataaaga tttttcagct 2520 tatagcaaag ggcgaccaaa tctacatact ttatattgga aagcgctgtt tgatgagaga 2580 aatcttcaag atgtggttta taagctaaat ggtgaggcag agctttttta tcgtaaacaa 2640 tcaataccta aaaaaatcac tcacccagct aaagaggcaa tagctaataa aaacaaagat 2700 aatcctaaaa aagagagtgt ttttgaatat gatttaatca aagataaacg ctttactgaa 2760 gataagtttt tctttcactg tcctattaca atcaatttta aatctagtgg agctaataag 2820 tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa atgatgttca tatattaagt 2880 atagatagag gtgaaagaca tttagcttac tatactttgg tagatggtaa aggcaatatc 2940 atcaaacaag atactttcaa catcattggt aatgatagaa tgaaaacaaa ctaccatgat 3000 aagcttgctg caatagagaa agatagggat tcagctagga aagactggaa aaagataaat 3060 aacatcaaag agatgaaaga gggctatcta tctcaggtag ttcatgaaat agctaagcta 3120 gttatagagt ataatgctat tgtggttttt gaggatttaa attttggatt taaaagaggg 3180 cgtttcaagg tagagaagca ggtctatcaa aagttagaaa aaatgctaat tgagaaacta 3240 aactatctag ttttcaaaga taatgagttt gataaaactg ggggagtgct tagagcttat 3300 cagctaacag caccttttga gacttttaaa aagatgggta aacaaacagg tattatctac 3360 tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt aaatcagtta 3420 tatcctaagt atgaaagtgt cagcaaatct caagagttct ttagtaagtt tgacaagatt 3480 tgttataacc ttgataaggg ctattttgag tttagttttg attataaaaa ctttggtgac 3540 aaggctgcca aaggcaagtg gactatagct agctttggga gtagattgat taactttaga 3600 aattcagata aaaatcataa ttgggatact cgagaagttt atccaactaa agagttggag 3660 aaattgctaa aagattattc tatcgaatat gggcatggcg aatgtatcaa agcagctatt 3720 tgcggtgaga gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa tactatctta 3780 caaatgcgta actcaaaaac aggtactgag ttagattatc taatttcacc agtagcagat 3840 gtaaatggca atttctttga ttcgcgacag gcgccaaaaa atatgcctca agatgctgat 3900 gccaatggtg cttatcatat tgggctaaaa ggtctgatgc tactaggtag gatcaaaaat 3960 aatcaagagg gcaaaaaact caatttggtt atcaaaaatg aagagtattt tgagttcgtg 4020 cagaatagga ataaccaagc ggccgcactc gacctgcagg tgaaacaaca gatacaactt 4080 cgtcgccgtg aagtcgatga aacggcagac ttgcccgctg aattgcctcc cttgctgcgc 4140 cgtttatacg ccagccgggg cgtgcgcagt gcgcaagaac tggaacgcag tgttaaaggt 4200 atgttgccct ggcagcaact gagcggcgtc gaaaaggccg ttgagatcct ttacaacgct 4260 tttcgcgaag gaacgcggat tattgtggtc ggcgattttg acgccgacgg cgcgaccagc 4320 acggctctaa gcgtgctggc gatgcgctcg cttggttgca gcaatatcga ctatctggta 4380 ccaaaccgtt tcgaagacgg ttacggctta agcccggaag tagtcgatca ggcccatgcc 4440 cgtggcgcgc agttaattgt cacggtggat aacggtattt cctcccatgc gggcgttgaa 4500 cacgctcgct cgttgggcat tccggttatt gttaccgatc accatttgcc gggcgaaaca 4560 ttacccgcag cggaagcgat cattaaccct aacttgcgcg actgtaattt cccgtcgaaa 4620 tcactggcag gcgtgggtgt ggcgttttat ctgatgctgg cgctgcgcac ctttttgcgc 4680 gatcagggct ggtttgatga gcgtggcatc gcaattccta acctggcaga actgctggat 4740 ctggtcgcgc tgggaacagt ggcggacgtc gtgccgctgg acgctaataa tcgcattctg 4800 acctggcagg ggatgagtcg catccgtgcc ggaaagtgcc gtccagggat taaagcgctg 4860 ctggaagtgg caaaccgtga tgcacaaaaa ctcgccgcca gcgatttagg ttttgcgctg 4920 gggccacgtc tcaatgctgc cggacgactg gacgatatgt ccgtcggtgt ggcgctcttg 4980 ctgtgcgaca acatcggcga agcgcgcgtg ctggcaaatg aactcgatgc gctaaaccag 5040 acgcgaaaag agatcgaaca aggaatgcaa gttgaagccc tgaccctgtg cgagaaactg 5100 gagcgaagtc gcgacacgct acccggcggg ctggcaatgt atcaccccga atggcatcag 5160 ggcgttgtcg gtattctggc ttcgcgcatc aaagagcgtt ttcaccgtcc ggttatcgcc 5220 tttgcgccag caggtgatgg tacgctgaaa ggttcaggtc gctccattca ggggctgcat 5280 atgcgtgatg cactggagcg attagacaca ctctaccctg gcatgatact gaagtttggc 5340 ggtcatgcga tggcggcggg tttgtcgctg gaagaggata aattcgaact ctttcaacaa 5400 cggtttggcg agctggttac cgagtggctg gacccttcgc tattgcaagg cgaagtggtg 5460 tcagacggcc cgttaagccc ggccgaaatg accatggaag tggcgcagct gctgcgcgat 5520 gctggcccgt gggggcagat gttcccggag ccgctgtttg atggtcattt ccgtctgctg 5580 caacagcggc tggtgggcga acgtcatttg aaagtcatgg tcgaaccggt cggcggcggt 5640 ccgctgctgg atggtattgc ttttaatgtc gataccgccc tctggccgga taacggcgtg 5700 cgcgaagtgc aactggctta caagctcgat atcaacgagt ttcgcggcaa ccgcagcctg 5760 caaattatca tcgacaatat ctggccaatt ctgcagaaaa ggccggcggc cacgaaaaag 5820 gccggccagg caaaaaagaa aaagcaccac caccaccacc actga 5865 <210> 59 <211> 2243 <212> PRT <213> Artificial Sequence <220> <223> FnCpf1-RecE <400> 59 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Ser Ile 35 40 45 Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe 50 55 60 Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly 65 70 75 80 Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys 85 90 95 Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser 100 105 110 Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr 115 120 125 Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys 130 135 140 Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp 145 150 155 160 Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys 165 170 175 Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp 180 185 190 Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp 195 200 205 Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe 210 215 220 Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile 225 230 235 240 Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe 245 250 255 Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu 260 265 270 Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr 275 280 285 Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser 290 295 300 Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln 305 310 315 320 Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn 325 330 335 Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr 340 345 350 Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val 355 360 365 Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile 370 375 380 Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe 385 390 395 400 Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys 405 410 415 Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp 420 425 430 Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser 435 440 445 Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu 450 455 460 Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys 465 470 475 480 Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu 485 490 495 Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg 500 505 510 Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala 515 520 525 Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu 530 535 540 Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu 545 550 555 560 Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp 565 570 575 Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln 580 585 590 Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu 595 600 605 Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr 610 615 620 Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys 625 630 635 640 Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys 645 650 655 Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys 660 665 670 Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp 675 680 685 Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr 690 695 700 Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser 705 710 715 720 Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile 725 730 735 Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr 740 745 750 Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe 755 760 765 Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe 770 775 780 Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg 785 790 795 800 Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu 805 810 815 Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr Leu Phe Gln 820 825 830 Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg Pro Asn Leu 835 840 845 His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp 850 855 860 Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln 865 870 875 880 Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn 885 890 895 Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu 900 905 910 Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro 915 920 925 Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu 930 935 940 Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His Ile Leu Ser 945 950 955 960 Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu Val Asp Gly 965 970 975 Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp 980 985 990 Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp 995 1000 1005 Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu 1010 1015 1020 Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile Ala Lys Leu 1025 1030 1035 1040 Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly 1045 1050 1055 Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys Leu 1060 1065 1070 Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe Lys Asp Asn 1075 1080 1085 Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala 1090 1095 1100 Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr 1105 1110 1115 1120 Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe 1125 1130 1135 Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu 1140 1145 1150 Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr 1155 1160 1165 Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys 1170 1175 1180 Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg 1185 1190 1195 1200 Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr 1205 1210 1215 Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His 1220 1225 1230 Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1235 1240 1245 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn 1250 1255 1260 Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp 1265 1270 1275 1280 Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro 1285 1290 1295 Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys Gly Leu 1300 1305 1310 Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys Lys Leu Asn 1315 1320 1325 Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn 1330 1335 1340 Asn Gln Ala Ala Ala Leu Asp Leu Gln Met Ser Thr Lys Pro Leu Phe 1345 1350 1355 1360 Leu Leu Arg Lys Ala Lys Lys Ser Ser Gly Glu Pro Asp Val Val Leu 1365 1370 1375 Trp Ala Ser Asn Asp Phe Glu Ser Thr Cys Ala Thr Leu Asp Tyr Leu 1380 1385 1390 Ile Val Lys Ser Gly Lys Lys Leu Ser Ser Tyr Phe Lys Ala Val Ala 1395 1400 1405 Thr Asn Phe Pro Val Val Asn Asp Leu Pro Ala Glu Gly Glu Ile Asp 1410 1415 1420 Phe Thr Trp Ser Glu Arg Tyr Gln Leu Ser Lys Asp Ser Met Thr Trp 1425 1430 1435 1440 Glu Leu Lys Pro Gly Ala Ala Pro Asp Asn Ala His Tyr Gln Gly Asn 1445 1450 1455 Thr Asn Val Asn Gly Glu Asp Met Thr Glu Ile Glu Glu Asn Met Leu 1460 1465 1470 Leu Pro Ile Ser Gly Gln Glu Leu Pro Ile Arg Trp Leu Ala Gln His 1475 1480 1485 Gly Ser Glu Lys Pro Val Thr His Val Ser Arg Asp Gly Leu Gln Ala 1490 1495 1500 Leu His Ile Ala Arg Ala Glu Glu Leu Pro Ala Val Thr Ala Leu Ala 1505 1510 1515 1520 Val Ser His Lys Thr Ser Leu Leu Asp Pro Leu Glu Ile Arg Glu Leu 1525 1530 1535 His Lys Leu Val Arg Asp Thr Asp Lys Val Phe Pro Asn Pro Gly Asn 1540 1545 1550 Ser Asn Leu Gly Leu Ile Thr Ala Phe Phe Glu Ala Tyr Leu Asn Ala 1555 1560 1565 Asp Tyr Thr Asp Arg Gly Leu Leu Thr Lys Glu Trp Met Lys Gly Asn 1570 1575 1580 Arg Val Ser His Ile Thr Arg Thr Ala Ser Gly Ala Asn Ala Gly Gly 1585 1590 1595 1600 Gly Asn Leu Thr Asp Arg Gly Glu Gly Phe Val His Asp Leu Thr Ser 1605 1610 1615 Leu Ala Arg Asp Val Ala Thr Gly Val Leu Ala Arg Ser Met Asp Leu 1620 1625 1630 Asp Ile Tyr Asn Leu His Pro Ala His Ala Lys Arg Ile Glu Glu Ile 1635 1640 1645 Ile Ala Glu Asn Lys Pro Pro Phe Ser Val Phe Arg Asp Lys Phe Ile 1650 1655 1660 Thr Met Pro Gly Gly Leu Asp Tyr Ser Arg Ala Ile Val Val Ala Ser 1665 1670 1675 1680 Val Lys Glu Ala Pro Ile Gly Ile Glu Val Ile Pro Ala His Val Thr 1685 1690 1695 Glu Tyr Leu Asn Lys Val Leu Thr Glu Thr Asp His Ala Asn Pro Asp 1700 1705 1710 Pro Glu Ile Val Asp Ile Ala Cys Gly Arg Ser Ser Ala Pro Met Pro 1715 1720 1725 Gln Arg Val Thr Glu Glu Gly Lys Gln Asp Asp Glu Glu Lys Pro Gln 1730 1735 1740 Pro Ser Gly Thr Thr Ala Val Glu Gln Gly Glu Ala Glu Thr Met Glu 1745 1750 1755 1760 Pro Asp Ala Thr Glu His His Gln Asp Thr Gln Pro Leu Asp Ala Gln 1765 1770 1775 Ser Gln Val Asn Ser Val Asp Ala Lys Tyr Gln Glu Leu Arg Ala Glu 1780 1785 1790 Leu His Glu Ala Arg Lys Asn Ile Pro Ser Lys Asn Pro Val Asp Asp 1795 1800 1805 Asp Lys Leu Leu Ala Ala Ser Arg Gly Glu Phe Val Asp Gly Ile Ser 1810 1815 1820 Asp Pro Asn Asp Pro Lys Trp Val Lys Gly Ile Gln Thr Arg Asp Cys 1825 1830 1835 1840 Val Tyr Gln Asn Gln Pro Glu Thr Glu Lys Thr Ser Pro Asp Met Asn 1845 1850 1855 Gln Pro Glu Pro Val Val Gln Gln Glu Pro Glu Ile Ala Cys Asn Ala 1860 1865 1870 Cys Gly Gln Thr Gly Gly Asp Asn Cys Pro Asp Cys Gly Ala Val Met 1875 1880 1885 Gly Asp Ala Thr Tyr Gln Glu Thr Phe Asp Glu Glu Ser Gln Val Glu 1890 1895 1900 Ala Lys Glu Asn Asp Pro Glu Glu Met Glu Gly Ala Glu His Pro His 1905 1910 1915 1920 Asn Glu Asn Ala Gly Ser Asp Pro His Arg Asp Cys Ser Asp Glu Thr 1925 1930 1935 Gly Glu Val Ala Asp Pro Val Ile Val Glu Asp Ile Glu Pro Gly Ile 1940 1945 1950 Tyr Tyr Gly Ile Ser Asn Glu Asn Tyr His Ala Gly Pro Gly Ile Ser 1955 1960 1965 Lys Ser Gln Leu Asp Asp Ile Ala Asp Thr Pro Ala Leu Tyr Leu Trp 1970 1975 1980 Arg Lys Asn Ala Pro Val Asp Thr Thr Lys Thr Lys Thr Leu Asp Leu 1985 1990 1995 2000 Gly Thr Ala Phe His Cys Arg Val Leu Glu Pro Glu Glu Phe Ser Asn 2005 2010 2015 Arg Phe Ile Val Ala Pro Glu Phe Asn Arg Arg Thr Asn Ala Gly Lys 2020 2025 2030 Glu Glu Glu Lys Ala Phe Leu Met Glu Cys Ala Ser Thr Gly Lys Thr 2035 2040 2045 Val Ile Thr Ala Glu Glu Gly Arg Lys Ile Glu Leu Met Tyr Gln Ser 2050 2055 2060 Val Met Ala Leu Pro Leu Gly Gln Trp Leu Val Glu Ser Ala Gly His 2065 2070 2075 2080 Ala Glu Ser Ser Ile Tyr Trp Glu Asp Pro Glu Thr Gly Ile Leu Cys 2085 2090 2095 Arg Cys Arg Pro Asp Lys Ile Ile Pro Glu Phe His Trp Ile Met Asp 2100 2105 2110 Val Lys Thr Thr Ala Asp Ile Gln Arg Phe Lys Thr Ala Tyr Tyr Asp 2115 2120 2125 Tyr Arg Tyr His Val Gln Asp Ala Phe Tyr Ser Asp Gly Tyr Glu Ala 2130 2135 2140 Gln Phe Gly Val Gln Pro Thr Phe Val Phe Leu Val Ala Ser Thr Thr 2145 2150 2155 2160 Ile Glu Cys Gly Arg Tyr Pro Val Glu Ile Phe Met Met Gly Glu Glu 2165 2170 2175 Ala Lys Leu Ala Gly Gln Gln Glu Tyr His Arg Asn Leu Arg Thr Leu 2180 2185 2190 Ser Asp Cys Leu Asn Thr Asp Glu Trp Pro Ala Ile Lys Thr Leu Ser 2195 2200 2205 Leu Pro Arg Trp Ala Lys Glu Tyr Ala Asn Asp Leu Gln Lys Arg Pro 2210 2215 2220 Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys His His His 2225 2230 2235 2240 His His His <210> 60 <211> 6732 <212> DNA <213> Artificial Sequence <220> <223> FnCpf1-RecE <400> 60 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgtc aatttatcaa gaatttgtta ataaatatag tttaagtaaa 180 actctaagat ttgagttaat cccacagggt aaaacacttg aaaacataaa agcaagaggt 240 ttgattttag atgatgagaa aagagctaaa gactacaaaa aggctaaaca aataattgat 300 aaatatcatc agttttttat agaggagata ttaagttcgg tttgtattag cgaagattta 360 ttacaaaact attctgatgt ttattttaaa cttaaaaaga gtgatgatga taatctacaa 420 aaagatttta aaagtgcaaa agatacgata aagaaacaaa tatctgaata tataaaggac 480 tcagagaaat ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa agggcaagag 540 tcagatttaa ttctatggct aaagcaatct aaggataatg gtatagaact atttaaagcc 600 aatagtgata tcacagatat agatgaggcg ttagaaataa tcaaatcttt taaaggttgg 660 acaacttatt ttaagggttt tcatgaaaat agaaaaaatg tttatagtag caatgatatt 720 cctacatcta ttatttatag gatagtagat gataatttgc ctaaatttct agaaaataaa 780 gctaagtatg agagtttaaa agacaaagct ccagaagcta taaactatga acaaattaaa 840 aaagatttgg cagaagagct aacctttgat attgactaca aaacatctga agttaatcaa 900 agagtttttt cacttgatga agtttttgag atagcaaact ttaataatta tctaaatcaa 960 agtggtatta ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg tgaaaataca 1020 aagagaaaag gtataaatga atatataaat ctatactcac agcaaataaa tgataaaaca 1080 ctcaaaaaat ataaaatgag tgttttattt aagcaaattt taagtgatac agaatctaaa 1140 tcttttgtaa ttgataagtt agaagatgat agtgatgtag ttacaacgat gcaaagtttt 1200 tatgagcaaa tagcagcttt taaaacagta gaagaaaaat ctattaaaga aacactatct 1260 ttattatttg atgatttaaa agctcaaaaa cttgatttga gtaaaattta ttttaaaaat 1320 gataaatctc ttactgatct atcacaacaa gtttttgatg attatagtgt tattggtaca 1380 gcggtactag aatatataac tcaacaaata gcacctaaaa atcttgataa ccctagtaag 1440 aaagagcaag aattaatagc caaaaaaact gaaaaagcaa aatacttatc tctagaaact 1500 ataaagcttg ccttagaaga atttaataag catagagata tagataaaca gtgtaggttt 1560 gaagaaatac ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat agctcaaaac 1620 aaagacaatt tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa agacctactt 1680 caagctagtg cggaagatga tgttaaagct atcaaggatc ttttagatca aactaataat 1740 ctcttacata aactaaaaat atttcatatt agtcagtcag aagataaggc aaatatttta 1800 gacaaggatg agcattttta tctagtattt gaggagtgct actttgagct agcgaatata 1860 gtgcctcttt ataacaaaat tagaaactat ataactcaaa agccatatag tgatgagaaa 1920 tttaagctca attttgagaa ctcgactttg gctaatggtt gggataaaaa taaagagcct 1980 gacaatacgg caattttatt tatcaaagat gataaatatt atctgggtgt gatgaataag 2040 aaaaataaca aaatatttga tgataaagct atcaaagaaa ataaaggcga gggttataaa 2100 aaaattgttt ataaactttt acctggcgca aataaaatgt tacctaaggt tttcttttct 2160 gctaaatcta taaaatttta taatcctagt gaagatatac ttagaataag aaatcattcc 2220 acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt taatattgaa 2280 gattgccgaa aatttataga tttttataaa cagtctataa gtaagcatcc ggagtggaaa 2340 gattttggat ttagattttc tgatactcaa agatataatt ctatagatga attttataga 2400 gaagttgaaa atcaaggcta caaactaact tttgaaaata tatcagagag ctatattgat 2460 agcgtagtta atcagggtaa attgtaccta ttccaaatct ataataaaga tttttcagct 2520 tatagcaaag ggcgaccaaa tctacatact ttatattgga aagcgctgtt tgatgagaga 2580 aatcttcaag atgtggttta taagctaaat ggtgaggcag agctttttta tcgtaaacaa 2640 tcaataccta aaaaaatcac tcacccagct aaagaggcaa tagctaataa aaacaaagat 2700 aatcctaaaa aagagagtgt ttttgaatat gatttaatca aagataaacg ctttactgaa 2760 gataagtttt tctttcactg tcctattaca atcaatttta aatctagtgg agctaataag 2820 tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa atgatgttca tatattaagt 2880 atagatagag gtgaaagaca tttagcttac tatactttgg tagatggtaa aggcaatatc 2940 atcaaacaag atactttcaa catcattggt aatgatagaa tgaaaacaaa ctaccatgat 3000 aagcttgctg caatagagaa agatagggat tcagctagga aagactggaa aaagataaat 3060 aacatcaaag agatgaaaga gggctatcta tctcaggtag ttcatgaaat agctaagcta 3120 gttatagagt ataatgctat tgtggttttt gaggatttaa attttggatt taaaagaggg 3180 cgtttcaagg tagagaagca ggtctatcaa aagttagaaa aaatgctaat tgagaaacta 3240 aactatctag ttttcaaaga taatgagttt gataaaactg ggggagtgct tagagcttat 3300 cagctaacag caccttttga gacttttaaa aagatgggta aacaaacagg tattatctac 3360 tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt aaatcagtta 3420 tatcctaagt atgaaagtgt cagcaaatct caagagttct ttagtaagtt tgacaagatt 3480 tgttataacc ttgataaggg ctattttgag tttagttttg attataaaaa ctttggtgac 3540 aaggctgcca aaggcaagtg gactatagct agctttggga gtagattgat taactttaga 3600 aattcagata aaaatcataa ttgggatact cgagaagttt atccaactaa agagttggag 3660 aaattgctaa aagattattc tatcgaatat gggcatggcg aatgtatcaa agcagctatt 3720 tgcggtgaga gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa tactatctta 3780 caaatgcgta actcaaaaac aggtactgag ttagattatc taatttcacc agtagcagat 3840 gtaaatggca atttctttga ttcgcgacag gcgccaaaaa atatgcctca agatgctgat 3900 gccaatggtg cttatcatat tgggctaaaa ggtctgatgc tactaggtag gatcaaaaat 3960 aatcaagagg gcaaaaaact caatttggtt atcaaaaatg aagagtattt tgagttcgtg 4020 cagaatagga ataaccaagc ggccgcactc gacctgcaga tgagcacaaa accactcttc 4080 ctgttacgga aagcgaaaaa atcatccggt gaacctgacg tcgtcctgtg ggcaagcaac 4140 gattttgaat cgacctgtgc cactctggac tacctgatcg ttaagtcagg taaaaaactg 4200 agcagctatt ttaaagctgt tgccacgaat tttcctgtcg ttaatgacct gcccgctgaa 4260 ggtgagatcg attttacctg gagtgaacgc tatcaactca gcaaagactc catgacatgg 4320 gaactaaaac cgggagcagc accagacaac gctcactatc aaggcaatac caacgtcaac 4380 ggcgaagaca tgactgagat tgaggagaat atgctactcc caatttctgg ccaggaactg 4440 cccattcgtt ggcttgctca acacggcagc gaaaaaccgg taacgcacgt ttcacgcgac 4500 ggactccagg cattacacat tgctcgggct gaagaactac cggctgttac tgccctggct 4560 gtttcccaca aaaccagcct gctcgacccg ctggaaattc gcgaactcca caaactggtt 4620 cgtgacactg acaaagtttt ccctaatcct ggtaattcaa acctgggact gataactgct 4680 tttttcgaag catacctgaa cgctgactac accgatcgag gactgctgac aaaagagtgg 4740 atgaagggta atcgtgtttc acacatcact cgcacggctt ccggtgctaa tgctggcggc 4800 ggaaacctca ccgatcgcgg cgaaggtttc gtacacgatc tgacgtcact ggcgcgcgac 4860 gtagccactg gcgtactggc ccgttcaatg gatctggaca tctataacct tcatccggca 4920 cacgctaaac gcattgagga aattatcgct gaaaataaac cgcccttttc tgttttccgc 4980 gacaaattca tcaccatgcc tggcgggctg gattattccc gcgccatcgt ggttgcgtcc 5040 gtaaaagaag caccaattgg gatcgaggtc atccccgcgc acgtcactga atatctgaac 5100 aaagtactga ctgaaaccga tcatgccaac cctgatccgg aaatcgtgga tattgcctgc 5160 ggtcgctcct ctgccccgat gccgcagcga gtaacagaag aaggaaaaca ggatgatgaa 5220 gaaaaaccgc aaccatctgg aacaacggca gttgaacagg gagaggctga aacaatggaa 5280 ccggacgcaa ctgaacatca tcaggacacg cagccgctgg atgctcagtc acaggtaaat 5340 tctgttgatg cgaaatatca ggaactgcgg gcagaactcc atgaagcccg gaaaaacatt 5400 ccatcaaaaa atcctgtcga tgacgataaa ttgcttgctg catcacgtgg tgaatttgtt 5460 gacggaatta gcgacccgaa cgatccgaaa tgggtaaagg ggatccagac tcgcgattgt 5520 gtgtaccaga accagccaga aacggaaaaa accagcccag atatgaatca acctgagcca 5580 gtagtgcaac aggaaccgga aatagcctgc aatgcctgcg gccagactgg cggggataac 5640 tgccctgact gtggtgcggt gatgggcgac gcaacatacc aggaaacatt cgatgaagag 5700 agtcaggttg aagctaagga aaatgatccg gaggaaatgg aaggcgctga acatccgcac 5760 aatgagaatg ctggcagcga tccgcatcgc gattgcagtg atgaaactgg cgaagtcgca 5820 gatcccgtaa tcgtagaaga catagagcca ggtatttatt acggaatttc gaatgagaat 5880 taccacgcgg gtcccggtat cagtaagtct cagctcgatg acattgctga tactccggca 5940 ctatatttgt ggcgtaaaaa tgcccccgtg gacaccacaa agacaaaaac gctcgattta 6000 ggaactgctt tccactgccg ggtacttgaa ccggaagaat tcagtaaccg ctttatcgta 6060 gcacctgaat ttaaccgccg tacaaacgcc ggaaaagaag aagagaaagc gtttctgatg 6120 gaatgcgcaa gcacaggaaa aacggttatc actgcggaag aaggccggaa aattgaactc 6180 atgtatcaaa gcgttatggc tttgccgctg gggcaatggc ttgttgaaag cgccggacac 6240 gctgaatcat caatttactg ggaagatcct gaaacaggaa ttttgtgtcg gtgccgtccg 6300 gacaaaatta tccctgaatt tcactggatc atggacgtga aaactacggc ggatattcaa 6360 cgattcaaaa ccgcttatta cgactaccgc tatcacgttc aggatgcatt ctacagtgac 6420 ggttatgaag cacagtttgg agtgcagcca actttcgttt ttctggttgc cagcacaact 6480 attgaatgcg gacgttatcc ggttgaaatt ttcatgatgg gcgaagaagc aaaactggca 6540 ggtcaacagg aatatcaccg caatctgcga accctgtctg actgcctgaa taccgatgaa 6600 tggccagcta ttaagacatt atcactgccc cgctgggcta aggaatatgc aaatgacctg 6660 cagaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gcaccaccac 6720 caccaccact ga 6732 <210> 61 <211> 1885 <212> PRT <213> Artificial Sequence <220> <223> FnCpf1-hTdT <400> 61 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Ser Ile 35 40 45 Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe 50 55 60 Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly 65 70 75 80 Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys 85 90 95 Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser 100 105 110 Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr 115 120 125 Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys 130 135 140 Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp 145 150 155 160 Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys 165 170 175 Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp 180 185 190 Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp 195 200 205 Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe 210 215 220 Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile 225 230 235 240 Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe 245 250 255 Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu 260 265 270 Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr 275 280 285 Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser 290 295 300 Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln 305 310 315 320 Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn 325 330 335 Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr 340 345 350 Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val 355 360 365 Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile 370 375 380 Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe 385 390 395 400 Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys 405 410 415 Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp 420 425 430 Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser 435 440 445 Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu 450 455 460 Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys 465 470 475 480 Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu 485 490 495 Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg 500 505 510 Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala 515 520 525 Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu 530 535 540 Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu 545 550 555 560 Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp 565 570 575 Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln 580 585 590 Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu 595 600 605 Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr 610 615 620 Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys 625 630 635 640 Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys 645 650 655 Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys 660 665 670 Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp 675 680 685 Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr 690 695 700 Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser 705 710 715 720 Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile 725 730 735 Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr 740 745 750 Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe 755 760 765 Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe 770 775 780 Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg 785 790 795 800 Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu 805 810 815 Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr Leu Phe Gln 820 825 830 Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg Pro Asn Leu 835 840 845 His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp 850 855 860 Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln 865 870 875 880 Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn 885 890 895 Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu 900 905 910 Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro 915 920 925 Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu 930 935 940 Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His Ile Leu Ser 945 950 955 960 Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu Val Asp Gly 965 970 975 Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp 980 985 990 Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp 995 1000 1005 Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu 1010 1015 1020 Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile Ala Lys Leu 1025 1030 1035 1040 Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly 1045 1050 1055 Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys Leu 1060 1065 1070 Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe Lys Asp Asn 1075 1080 1085 Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala 1090 1095 1100 Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr 1105 1110 1115 1120 Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe 1125 1130 1135 Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu 1140 1145 1150 Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr 1155 1160 1165 Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys 1170 1175 1180 Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg 1185 1190 1195 1200 Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr 1205 1210 1215 Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His 1220 1225 1230 Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1235 1240 1245 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn 1250 1255 1260 Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp 1265 1270 1275 1280 Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro 1285 1290 1295 Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys Gly Leu 1300 1305 1310 Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys Lys Leu Asn 1315 1320 1325 Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn 1330 1335 1340 Asn Gln Ala Ala Ala Leu Asp Leu Glu Met Asp Pro Pro Arg Ala Ser 1345 1350 1355 1360 His Leu Ser Pro Arg Lys Lys Arg Pro Arg Gln Thr Gly Ala Leu Met 1365 1370 1375 Ala Ser Ser Pro Gln Asp Ile Lys Phe Gln Asp Leu Val Val Phe Ile 1380 1385 1390 Leu Glu Lys Lys Met Gly Thr Thr Arg Arg Ala Phe Leu Met Glu Leu 1395 1400 1405 Ala Arg Arg Lys Gly Phe Arg Val Glu Asn Glu Leu Ser Asp Ser Val 1410 1415 1420 Thr His Ile Val Ala Glu Asn Asn Ser Gly Ser Asp Val Leu Glu Trp 1425 1430 1435 1440 Leu Gln Ala Gln Lys Val Gln Val Ser Ser Gln Pro Glu Leu Leu Asp 1445 1450 1455 Val Ser Trp Leu Ile Glu Cys Ile Gly Ala Gly Lys Pro Val Glu Met 1460 1465 1470 Thr Gly Lys His Gln Leu Val Val Arg Arg Asp Tyr Ser Asp Ser Thr 1475 1480 1485 Asn Pro Gly Pro Pro Lys Thr Pro Pro Ile Ala Val Gln Lys Ile Ser 1490 1495 1500 Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn Cys Asn Gln Ile 1505 1510 1515 1520 Phe Thr Asp Ala Phe Asp Ile Leu Ala Glu Asn Cys Glu Phe Arg Glu 1525 1530 1535 Asn Glu Asp Ser Cys Val Thr Phe Met Arg Ala Ala Ser Val Leu Lys 1540 1545 1550 Ser Leu Pro Phe Thr Ile Ile Ser Met Lys Asp Thr Glu Gly Ile Pro 1555 1560 1565 Cys Leu Gly Ser Lys Val Lys Gly Ile Ile Glu Glu Ile Ile Glu Asp 1570 1575 1580 Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu Arg Tyr Gln 1585 1590 1595 1600 Ser Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Leu Lys Thr Ser 1605 1610 1615 Glu Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Ser Lys Val Arg Ser 1620 1625 1630 Asp Lys Ser Leu Lys Phe Thr Arg Met Gln Lys Ala Gly Phe Leu Tyr 1635 1640 1645 Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu Ala Glu Ala Val 1650 1655 1660 Ser Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro Asp Ala Phe 1665 1670 1675 1680 Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys Met Gly His Asp 1685 1690 1695 Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Thr Glu Asp Glu Glu Gln 1700 1705 1710 Leu Leu Gln Lys Val Met Asn Leu Trp Glu Lys Lys Gly Leu Leu Leu 1715 1720 1725 Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Leu Arg Leu Pro Ser 1730 1735 1740 Arg Lys Val Asp Ala Leu Asp His Phe Gln Lys Cys Phe Leu Ile Phe 1745 1750 1755 1760 Lys Leu Pro Arg Gln Arg Val Asp Ser Asp Gln Ser Ser Trp Gln Glu 1765 1770 1775 Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Leu Cys Pro Tyr 1780 1785 1790 Glu Arg Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe 1795 1800 1805 Glu Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys Met Ile Leu 1810 1815 1820 Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Ile Phe Leu Lys Ala 1825 1830 1835 1840 Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Ile Glu 1845 1850 1855 Pro Trp Glu Arg Asn Ala Leu Glu Lys Arg Pro Ala Ala Thr Lys Lys 1860 1865 1870 Ala Gly Gln Ala Lys Lys Lys Lys His His His His His 1875 1880 1885 <210> 62 <211> 5658 <212> DNA <213> Artificial Sequence <220> <223> FnCpf1-hTdT <400> 62 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgtc aatttatcaa gaatttgtta ataaatatag tttaagtaaa 180 actctaagat ttgagttaat cccacagggt aaaacacttg aaaacataaa agcaagaggt 240 ttgattttag atgatgagaa aagagctaaa gactacaaaa aggctaaaca aataattgat 300 aaatatcatc agttttttat agaggagata ttaagttcgg tttgtattag cgaagattta 360 ttacaaaact attctgatgt ttattttaaa cttaaaaaga gtgatgatga taatctacaa 420 aaagatttta aaagtgcaaa agatacgata aagaaacaaa tatctgaata tataaaggac 480 tcagagaaat ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa agggcaagag 540 tcagatttaa ttctatggct aaagcaatct aaggataatg gtatagaact atttaaagcc 600 aatagtgata tcacagatat agatgaggcg ttagaaataa tcaaatcttt taaaggttgg 660 acaacttatt ttaagggttt tcatgaaaat agaaaaaatg tttatagtag caatgatatt 720 cctacatcta ttatttatag gatagtagat gataatttgc ctaaatttct agaaaataaa 780 gctaagtatg agagtttaaa agacaaagct ccagaagcta taaactatga acaaattaaa 840 aaagatttgg cagaagagct aacctttgat attgactaca aaacatctga agttaatcaa 900 agagtttttt cacttgatga agtttttgag atagcaaact ttaataatta tctaaatcaa 960 agtggtatta ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg tgaaaataca 1020 aagagaaaag gtataaatga atatataaat ctatactcac agcaaataaa tgataaaaca 1080 ctcaaaaaat ataaaatgag tgttttattt aagcaaattt taagtgatac agaatctaaa 1140 tcttttgtaa ttgataagtt agaagatgat agtgatgtag ttacaacgat gcaaagtttt 1200 tatgagcaaa tagcagcttt taaaacagta gaagaaaaat ctattaaaga aacactatct 1260 ttattatttg atgatttaaa agctcaaaaa cttgatttga gtaaaattta ttttaaaaat 1320 gataaatctc ttactgatct atcacaacaa gtttttgatg attatagtgt tattggtaca 1380 gcggtactag aatatataac tcaacaaata gcacctaaaa atcttgataa ccctagtaag 1440 aaagagcaag aattaatagc caaaaaaact gaaaaagcaa aatacttatc tctagaaact 1500 ataaagcttg ccttagaaga atttaataag catagagata tagataaaca gtgtaggttt 1560 gaagaaatac ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat agctcaaaac 1620 aaagacaatt tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa agacctactt 1680 caagctagtg cggaagatga tgttaaagct atcaaggatc ttttagatca aactaataat 1740 ctcttacata aactaaaaat atttcatatt agtcagtcag aagataaggc aaatatttta 1800 gacaaggatg agcattttta tctagtattt gaggagtgct actttgagct agcgaatata 1860 gtgcctcttt ataacaaaat tagaaactat ataactcaaa agccatatag tgatgagaaa 1920 tttaagctca attttgagaa ctcgactttg gctaatggtt gggataaaaa taaagagcct 1980 gacaatacgg caattttatt tatcaaagat gataaatatt atctgggtgt gatgaataag 2040 aaaaataaca aaatatttga tgataaagct atcaaagaaa ataaaggcga gggttataaa 2100 aaaattgttt ataaactttt acctggcgca aataaaatgt tacctaaggt tttcttttct 2160 gctaaatcta taaaatttta taatcctagt gaagatatac ttagaataag aaatcattcc 2220 acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt taatattgaa 2280 gattgccgaa aatttataga tttttataaa cagtctataa gtaagcatcc ggagtggaaa 2340 gattttggat ttagattttc tgatactcaa agatataatt ctatagatga attttataga 2400 gaagttgaaa atcaaggcta caaactaact tttgaaaata tatcagagag ctatattgat 2460 agcgtagtta atcagggtaa attgtaccta ttccaaatct ataataaaga tttttcagct 2520 tatagcaaag ggcgaccaaa tctacatact ttatattgga aagcgctgtt tgatgagaga 2580 aatcttcaag atgtggttta taagctaaat ggtgaggcag agctttttta tcgtaaacaa 2640 tcaataccta aaaaaatcac tcacccagct aaagaggcaa tagctaataa aaacaaagat 2700 aatcctaaaa aagagagtgt ttttgaatat gatttaatca aagataaacg ctttactgaa 2760 gataagtttt tctttcactg tcctattaca atcaatttta aatctagtgg agctaataag 2820 tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa atgatgttca tatattaagt 2880 atagatagag gtgaaagaca tttagcttac tatactttgg tagatggtaa aggcaatatc 2940 atcaaacaag atactttcaa catcattggt aatgatagaa tgaaaacaaa ctaccatgat 3000 aagcttgctg caatagagaa agatagggat tcagctagga aagactggaa aaagataaat 3060 aacatcaaag agatgaaaga gggctatcta tctcaggtag ttcatgaaat agctaagcta 3120 gttatagagt ataatgctat tgtggttttt gaggatttaa attttggatt taaaagaggg 3180 cgtttcaagg tagagaagca ggtctatcaa aagttagaaa aaatgctaat tgagaaacta 3240 aactatctag ttttcaaaga taatgagttt gataaaactg ggggagtgct tagagcttat 3300 cagctaacag caccttttga gacttttaaa aagatgggta aacaaacagg tattatctac 3360 tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt aaatcagtta 3420 tatcctaagt atgaaagtgt cagcaaatct caagagttct ttagtaagtt tgacaagatt 3480 tgttataacc ttgataaggg ctattttgag tttagttttg attataaaaa ctttggtgac 3540 aaggctgcca aaggcaagtg gactatagct agctttggga gtagattgat taactttaga 3600 aattcagata aaaatcataa ttgggatact cgagaagttt atccaactaa agagttggag 3660 aaattgctaa aagattattc tatcgaatat gggcatggcg aatgtatcaa agcagctatt 3720 tgcggtgaga gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa tactatctta 3780 caaatgcgta actcaaaaac aggtactgag ttagattatc taatttcacc agtagcagat 3840 gtaaatggca atttctttga ttcgcgacag gcgccaaaaa atatgcctca agatgctgat 3900 gccaatggtg cttatcatat tgggctaaaa ggtctgatgc tactaggtag gatcaaaaat 3960 aatcaagagg gcaaaaaact caatttggtt atcaaaaatg aagagtattt tgagttcgtg 4020 cagaatagga ataaccaagc ggccgcactc gacctcgaga tggatccacc acgagcgtcc 4080 cacttgagcc ctcggaagaa gagaccccgg cagacgggtg ccttgatggc ctcctctcct 4140 caagacatca aatttcaaga tttggtcgtc ttcattttgg agaagaaaat gggaaccacc 4200 cgcagagcgt tcctcatgga gctggcccgc aggaaagggt tcagggttga aaatgagctc 4260 agtgattctg tcacccacat tgtagcagag aacaactcgg gttcggatgt tctggagtgg 4320 cttcaagcac agaaagtaca agtcagctca caaccagagc tcctcgatgt ctcctggctg 4380 atcgaatgca taggagcagg gaaaccggtg gaaatgacag gaaaacacca gcttgttgtg 4440 agaagagact attcagatag caccaaccca ggccccccga agactccacc aattgctgta 4500 caaaagatct cccagtatgc gtgtcagaga agaaccactt taaacaactg taaccagata 4560 ttcacggatg cctttgatat actggctgaa aactgtgagt ttagagaaaa tgaagactcc 4620 tgtgtgacat ttatgagagc agcttctgta ttgaaatctc tgccattcac aatcatcagt 4680 atgaaggaca cagaaggaat tccctgcctg gggtccaagg tgaagggtat catagaggag 4740 attattgaag atggagaaag ttctgaagtt aaagctgtgt taaatgatga acgatatcaa 4800 tccttcaaac tctttacttc tgtatttgga gtggggctga agacttctga gaagtggttc 4860 aggatgggtt tcagaactct gagtaaagta aggtcggaca aaagcctgaa atttacacga 4920 atgcagaaag caggatttct gtattatgaa gaccttgtca gctgtgtgac cagggcagaa 4980 gcagaggccg tcagtgtgct ggttaaagag gctgtctggg catttcttcc ggatgctttc 5040 gtcaccatga caggagggtt ccggaggggt aagaagatgg ggcatgatgt agatttttta 5100 attaccagcc caggatcaac agaggatgaa gagcaacttt tacagaaagt gatgaactta 5160 tgggaaaaga agggattact tttatattat gaccttgtgg agtcaacatt tgaaaagctc 5220 aggttgccta gcaggaaggt tgatgctttg gatcattttc aaaagtgctt tctgattttc 5280 aaattgcctc gtcaaagagt ggacagtgac cagtccagct ggcaggaagg aaagacctgg 5340 aaggccatcc gtgtggattt agttctgtgc ccctacgagc gtcgtgcctt tgccctgttg 5400 ggatggactg gctcccggca gtttgagaga gacctccggc gctatgccac acatgagcgg 5460 aagatgattc tggataacca tgctttatat gacaagacca agaggatatt cctcaaagca 5520 gaaagtgaag aagaaatttt tgcgcatctg ggattggatt atattgaacc gtgggaaaga 5580 aatgccctcg agaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag 5640 caccaccacc accactga 5658 <210> 63 <211> 1652 <212> PRT <213> Artificial Sequence <220> <223> FnCpf1-T5 <400> 63 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Ser Ile 35 40 45 Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe 50 55 60 Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly 65 70 75 80 Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys 85 90 95 Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser 100 105 110 Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr 115 120 125 Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys 130 135 140 Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp 145 150 155 160 Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys 165 170 175 Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp 180 185 190 Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp 195 200 205 Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe 210 215 220 Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile 225 230 235 240 Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe 245 250 255 Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu 260 265 270 Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr 275 280 285 Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser 290 295 300 Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln 305 310 315 320 Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn 325 330 335 Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr 340 345 350 Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val 355 360 365 Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile 370 375 380 Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe 385 390 395 400 Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys 405 410 415 Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp 420 425 430 Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser 435 440 445 Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu 450 455 460 Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys 465 470 475 480 Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu 485 490 495 Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg 500 505 510 Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala 515 520 525 Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu 530 535 540 Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu 545 550 555 560 Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp 565 570 575 Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln 580 585 590 Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu 595 600 605 Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr 610 615 620 Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys 625 630 635 640 Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys 645 650 655 Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys 660 665 670 Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp 675 680 685 Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr 690 695 700 Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser 705 710 715 720 Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile 725 730 735 Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr 740 745 750 Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe 755 760 765 Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe 770 775 780 Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg 785 790 795 800 Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu 805 810 815 Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr Leu Phe Gln 820 825 830 Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg Pro Asn Leu 835 840 845 His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp 850 855 860 Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln 865 870 875 880 Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn 885 890 895 Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu 900 905 910 Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro 915 920 925 Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu 930 935 940 Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His Ile Leu Ser 945 950 955 960 Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu Val Asp Gly 965 970 975 Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp 980 985 990 Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp 995 1000 1005 Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu 1010 1015 1020 Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile Ala Lys Leu 1025 1030 1035 1040 Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly 1045 1050 1055 Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys Leu 1060 1065 1070 Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe Lys Asp Asn 1075 1080 1085 Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala 1090 1095 1100 Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr 1105 1110 1115 1120 Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe 1125 1130 1135 Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu 1140 1145 1150 Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr 1155 1160 1165 Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys 1170 1175 1180 Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg 1185 1190 1195 1200 Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr 1205 1210 1215 Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His 1220 1225 1230 Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1235 1240 1245 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn 1250 1255 1260 Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp 1265 1270 1275 1280 Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro 1285 1290 1295 Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys Gly Leu 1300 1305 1310 Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys Lys Leu Asn 1315 1320 1325 Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn 1330 1335 1340 Asn Gln Ala Ala Ala Leu Asp Leu Gln Met Ala Ser Arg Arg Asn Leu 1345 1350 1355 1360 Met Ile Val Asp Gly Thr Asn Leu Gly Phe Arg Phe Lys His Asn Asn 1365 1370 1375 Ser Lys Lys Pro Phe Ala Ser Ser Tyr Val Ser Thr Ile Gln Ser Leu 1380 1385 1390 Ala Lys Ser Tyr Ser Ala Arg Thr Thr Ile Val Leu Gly Asp Lys Gly 1395 1400 1405 Lys Ser Val Phe Arg Leu Glu His Leu Pro Glu Tyr Lys Gly Asn Arg 1410 1415 1420 Asp Glu Lys Tyr Ala Gln Arg Thr Glu Glu Glu Lys Ala Leu Asp Glu 1425 1430 1435 1440 Gln Phe Phe Glu Tyr Leu Lys Asp Ala Phe Glu Leu Cys Lys Thr Thr 1445 1450 1455 Phe Pro Thr Phe Thr Ile Arg Gly Val Glu Ala Asp Asp Met Ala Ala 1460 1465 1470 Tyr Ile Val Lys Leu Ile Gly His Leu Tyr Asp His Val Trp Leu Ile 1475 1480 1485 Ser Thr Asp Gly Asp Trp Asp Thr Leu Leu Thr Asp Lys Val Ser Arg 1490 1495 1500 Phe Ser Phe Thr Thr Arg Arg Glu Tyr His Leu Arg Asp Met Tyr Glu 1505 1510 1515 1520 His His Asn Val Asp Asp Val Glu Gln Phe Ile Ser Leu Lys Ala Ile 1525 1530 1535 Met Gly Asp Leu Gly Asp Asn Ile Arg Gly Val Glu Gly Ile Gly Ala 1540 1545 1550 Lys Arg Gly Tyr Asn Ile Ile Arg Glu Phe Gly Asn Val Leu Asp Ile 1555 1560 1565 Ile Asp Gln Leu Pro Leu Pro Gly Lys Gln Lys Tyr Ile Gln Asn Leu 1570 1575 1580 Asn Ala Ser Glu Glu Leu Leu Phe Arg Asn Leu Ile Leu Val Asp Leu 1585 1590 1595 1600 Pro Thr Tyr Cys Val Asp Ala Ile Ala Ala Val Gly Gln Asp Val Leu 1605 1610 1615 Asp Lys Phe Thr Lys Asp Ile Leu Glu Ile Ala Glu Gln Leu Gln Lys 1620 1625 1630 Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys His 1635 1640 1645 His His His His 1650 <210> 64 <211> 4959 <212> DNA <213> Artificial Sequence <220> <223> FnCpf1-T5 <400> 64 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgtc aatttatcaa gaatttgtta ataaatatag tttaagtaaa 180 actctaagat ttgagttaat cccacagggt aaaacacttg aaaacataaa agcaagaggt 240 ttgattttag atgatgagaa aagagctaaa gactacaaaa aggctaaaca aataattgat 300 aaatatcatc agttttttat agaggagata ttaagttcgg tttgtattag cgaagattta 360 ttacaaaact attctgatgt ttattttaaa cttaaaaaga gtgatgatga taatctacaa 420 aaagatttta aaagtgcaaa agatacgata aagaaacaaa tatctgaata tataaaggac 480 tcagagaaat ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa agggcaagag 540 tcagatttaa ttctatggct aaagcaatct aaggataatg gtatagaact atttaaagcc 600 aatagtgata tcacagatat agatgaggcg ttagaaataa tcaaatcttt taaaggttgg 660 acaacttatt ttaagggttt tcatgaaaat agaaaaaatg tttatagtag caatgatatt 720 cctacatcta ttatttatag gatagtagat gataatttgc ctaaatttct agaaaataaa 780 gctaagtatg agagtttaaa agacaaagct ccagaagcta taaactatga acaaattaaa 840 aaagatttgg cagaagagct aacctttgat attgactaca aaacatctga agttaatcaa 900 agagtttttt cacttgatga agtttttgag atagcaaact ttaataatta tctaaatcaa 960 agtggtatta ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg tgaaaataca 1020 aagagaaaag gtataaatga atatataaat ctatactcac agcaaataaa tgataaaaca 1080 ctcaaaaaat ataaaatgag tgttttattt aagcaaattt taagtgatac agaatctaaa 1140 tcttttgtaa ttgataagtt agaagatgat agtgatgtag ttacaacgat gcaaagtttt 1200 tatgagcaaa tagcagcttt taaaacagta gaagaaaaat ctattaaaga aacactatct 1260 ttattatttg atgatttaaa agctcaaaaa cttgatttga gtaaaattta ttttaaaaat 1320 gataaatctc ttactgatct atcacaacaa gtttttgatg attatagtgt tattggtaca 1380 gcggtactag aatatataac tcaacaaata gcacctaaaa atcttgataa ccctagtaag 1440 aaagagcaag aattaatagc caaaaaaact gaaaaagcaa aatacttatc tctagaaact 1500 ataaagcttg ccttagaaga atttaataag catagagata tagataaaca gtgtaggttt 1560 gaagaaatac ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat agctcaaaac 1620 aaagacaatt tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa agacctactt 1680 caagctagtg cggaagatga tgttaaagct atcaaggatc ttttagatca aactaataat 1740 ctcttacata aactaaaaat atttcatatt agtcagtcag aagataaggc aaatatttta 1800 gacaaggatg agcattttta tctagtattt gaggagtgct actttgagct agcgaatata 1860 gtgcctcttt ataacaaaat tagaaactat ataactcaaa agccatatag tgatgagaaa 1920 tttaagctca attttgagaa ctcgactttg gctaatggtt gggataaaaa taaagagcct 1980 gacaatacgg caattttatt tatcaaagat gataaatatt atctgggtgt gatgaataag 2040 aaaaataaca aaatatttga tgataaagct atcaaagaaa ataaaggcga gggttataaa 2100 aaaattgttt ataaactttt acctggcgca aataaaatgt tacctaaggt tttcttttct 2160 gctaaatcta taaaatttta taatcctagt gaagatatac ttagaataag aaatcattcc 2220 acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt taatattgaa 2280 gattgccgaa aatttataga tttttataaa cagtctataa gtaagcatcc ggagtggaaa 2340 gattttggat ttagattttc tgatactcaa agatataatt ctatagatga attttataga 2400 gaagttgaaa atcaaggcta caaactaact tttgaaaata tatcagagag ctatattgat 2460 agcgtagtta atcagggtaa attgtaccta ttccaaatct ataataaaga tttttcagct 2520 tatagcaaag ggcgaccaaa tctacatact ttatattgga aagcgctgtt tgatgagaga 2580 aatcttcaag atgtggttta taagctaaat ggtgaggcag agctttttta tcgtaaacaa 2640 tcaataccta aaaaaatcac tcacccagct aaagaggcaa tagctaataa aaacaaagat 2700 aatcctaaaa aagagagtgt ttttgaatat gatttaatca aagataaacg ctttactgaa 2760 gataagtttt tctttcactg tcctattaca atcaatttta aatctagtgg agctaataag 2820 tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa atgatgttca tatattaagt 2880 atagatagag gtgaaagaca tttagcttac tatactttgg tagatggtaa aggcaatatc 2940 atcaaacaag atactttcaa catcattggt aatgatagaa tgaaaacaaa ctaccatgat 3000 aagcttgctg caatagagaa agatagggat tcagctagga aagactggaa aaagataaat 3060 aacatcaaag agatgaaaga gggctatcta tctcaggtag ttcatgaaat agctaagcta 3120 gttatagagt ataatgctat tgtggttttt gaggatttaa attttggatt taaaagaggg 3180 cgtttcaagg tagagaagca ggtctatcaa aagttagaaa aaatgctaat tgagaaacta 3240 aactatctag ttttcaaaga taatgagttt gataaaactg ggggagtgct tagagcttat 3300 cagctaacag caccttttga gacttttaaa aagatgggta aacaaacagg tattatctac 3360 tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt aaatcagtta 3420 tatcctaagt atgaaagtgt cagcaaatct caagagttct ttagtaagtt tgacaagatt 3480 tgttataacc ttgataaggg ctattttgag tttagttttg attataaaaa ctttggtgac 3540 aaggctgcca aaggcaagtg gactatagct agctttggga gtagattgat taactttaga 3600 aattcagata aaaatcataa ttgggatact cgagaagttt atccaactaa agagttggag 3660 aaattgctaa aagattattc tatcgaatat gggcatggcg aatgtatcaa agcagctatt 3720 tgcggtgaga gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa tactatctta 3780 caaatgcgta actcaaaaac aggtactgag ttagattatc taatttcacc agtagcagat 3840 gtaaatggca atttctttga ttcgcgacag gcgccaaaaa atatgcctca agatgctgat 3900 gccaatggtg cttatcatat tgggctaaaa ggtctgatgc tactaggtag gatcaaaaat 3960 aatcaagagg gcaaaaaact caatttggtt atcaaaaatg aagagtattt tgagttcgtg 4020 cagaatagga ataaccaagc ggccgcactc gacctgcaga tggcttcccg tcgtaatcta 4080 atgattgtcg atggaactaa cttaggcttt cgcttcaaac ataacaatag taaaaaacca 4140 tttgcctcaa gttatgtttc aactattcaa tctctggcaa aatcctactc tgccagaact 4200 acgattgttc taggtgataa gggaaaatct gtatttcgtc tagaacatct accagagtat 4260 aaaggtaatc gtgatgaaaa gtacgcacaa cgtacggaag aggagaaagc gctagatgag 4320 cagttctttg agtatttgaa ggatgctttc gagttgtgta aaactacatt cccaactttt 4380 accattcgtg gtgtagaagc agacgatatg gcagcttata ttgttaagct catcgggcat 4440 ctttatgatc acgtttggct aatatctaca gatggtgact gggatacttt attaacggat 4500 aaagtttctc gtttttcttt cacaacacgt cgtgagtatc atcttcgtga tatgtatgaa 4560 catcataatg ttgatgatgt tgagcagttt atctccctga aagcaattat gggagatcta 4620 ggagataata ttcgtggtgt tgaaggaata ggagcaaaac gcggatataa tattattcgt 4680 gagtttggta acgtactgga tattattgat cagcttccac tgcctggaaa gcagaaatat 4740 atacagaacc tgaatgcatc ggaagaactg cttttccgaa acttgattct ggttgattta 4800 cctacctact gtgtggatgc tattgctgct gtaggtcaag atgtgttaga taagtttaca 4860 aaagatattt tggagattgc agaacaactg cagaaaaggc cggcggccac gaaaaaggcc 4920 ggccaggcaa aaaagaaaaa gcaccaccac caccactga 4959 <210> 65 <211> 1602 <212> PRT <213> Artificial Sequence <220> <223> FnCpf1-lambda <400> 65 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Ser Ile 35 40 45 Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe 50 55 60 Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly 65 70 75 80 Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys 85 90 95 Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser 100 105 110 Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr 115 120 125 Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys 130 135 140 Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp 145 150 155 160 Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys 165 170 175 Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp 180 185 190 Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp 195 200 205 Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe 210 215 220 Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile 225 230 235 240 Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe 245 250 255 Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu 260 265 270 Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr 275 280 285 Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser 290 295 300 Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln 305 310 315 320 Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn 325 330 335 Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr 340 345 350 Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val 355 360 365 Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile 370 375 380 Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe 385 390 395 400 Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys 405 410 415 Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp 420 425 430 Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser 435 440 445 Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu 450 455 460 Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys 465 470 475 480 Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu 485 490 495 Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg 500 505 510 Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala 515 520 525 Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu 530 535 540 Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu 545 550 555 560 Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp 565 570 575 Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln 580 585 590 Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu 595 600 605 Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr 610 615 620 Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys 625 630 635 640 Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys 645 650 655 Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys 660 665 670 Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp 675 680 685 Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr 690 695 700 Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser 705 710 715 720 Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile 725 730 735 Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr 740 745 750 Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe 755 760 765 Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe 770 775 780 Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg 785 790 795 800 Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu 805 810 815 Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr Leu Phe Gln 820 825 830 Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg Pro Asn Leu 835 840 845 His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp 850 855 860 Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln 865 870 875 880 Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn 885 890 895 Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu 900 905 910 Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro 915 920 925 Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu 930 935 940 Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His Ile Leu Ser 945 950 955 960 Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu Val Asp Gly 965 970 975 Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp 980 985 990 Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp 995 1000 1005 Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu 1010 1015 1020 Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile Ala Lys Leu 1025 1030 1035 1040 Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly 1045 1050 1055 Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys Leu 1060 1065 1070 Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe Lys Asp Asn 1075 1080 1085 Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala 1090 1095 1100 Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr 1105 1110 1115 1120 Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe 1125 1130 1135 Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu 1140 1145 1150 Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr 1155 1160 1165 Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys 1170 1175 1180 Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg 1185 1190 1195 1200 Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr 1205 1210 1215 Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His 1220 1225 1230 Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1235 1240 1245 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn 1250 1255 1260 Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp 1265 1270 1275 1280 Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro 1285 1290 1295 Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys Gly Leu 1300 1305 1310 Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys Lys Leu Asn 1315 1320 1325 Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn 1330 1335 1340 Asn Gln Ala Ala Ala Leu Asp Leu Glu Met Thr Pro Asp Ile Ile Leu 1345 1350 1355 1360 Gln Arg Thr Gly Ile Asp Val Arg Ala Val Glu Gln Gly Asp Asp Ala 1365 1370 1375 Trp His Lys Leu Arg Leu Gly Val Ile Thr Ala Ser Glu Val His Asn 1380 1385 1390 Val Ile Ala Lys Pro Arg Ser Gly Lys Lys Trp Pro Asp Met Lys Met 1395 1400 1405 Ser Tyr Phe His Thr Leu Leu Ala Glu Val Cys Thr Gly Val Ala Pro 1410 1415 1420 Glu Val Asn Ala Lys Ala Leu Ala Trp Gly Lys Gln Tyr Glu Asn Asp 1425 1430 1435 1440 Ala Arg Thr Leu Phe Glu Phe Thr Ser Gly Val Asn Val Thr Glu Ser 1445 1450 1455 Pro Ile Ile Tyr Arg Asp Glu Ser Met Arg Thr Ala Cys Ser Pro Asp 1460 1465 1470 Gly Leu Cys Ser Asp Gly Asn Gly Leu Glu Leu Lys Cys Pro Phe Thr 1475 1480 1485 Ser Arg Asp Phe Met Lys Phe Arg Leu Gly Gly Phe Glu Ala Ile Lys 1490 1495 1500 Ser Ala Tyr Met Ala Gln Val Gln Tyr Ser Met Trp Val Thr Arg Lys 1505 1510 1515 1520 Asn Ala Trp Tyr Phe Ala Asn Tyr Asp Pro Arg Met Lys Arg Glu Gly 1525 1530 1535 Leu His Tyr Val Val Ile Glu Arg Asp Glu Lys Tyr Met Ala Ser Phe 1540 1545 1550 Asp Glu Ile Val Pro Glu Phe Ile Glu Lys Met Asp Glu Ala Leu Ala 1555 1560 1565 Glu Ile Gly Phe Val Phe Gly Glu Gln Trp Arg Leu Glu Lys Arg Pro 1570 1575 1580 Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys His His His 1585 1590 1595 1600 His His <210> 66 <211> 4809 <212> DNA <213> Artificial Sequence <220> <223> FnCpf1-lambda <400> 66 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgtc aatttatcaa gaatttgtta ataaatatag tttaagtaaa 180 actctaagat ttgagttaat cccacagggt aaaacacttg aaaacataaa agcaagaggt 240 ttgattttag atgatgagaa aagagctaaa gactacaaaa aggctaaaca aataattgat 300 aaatatcatc agttttttat agaggagata ttaagttcgg tttgtattag cgaagattta 360 ttacaaaact attctgatgt ttattttaaa cttaaaaaga gtgatgatga taatctacaa 420 aaagatttta aaagtgcaaa agatacgata aagaaacaaa tatctgaata tataaaggac 480 tcagagaaat ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa agggcaagag 540 tcagatttaa ttctatggct aaagcaatct aaggataatg gtatagaact atttaaagcc 600 aatagtgata tcacagatat agatgaggcg ttagaaataa tcaaatcttt taaaggttgg 660 acaacttatt ttaagggttt tcatgaaaat agaaaaaatg tttatagtag caatgatatt 720 cctacatcta ttatttatag gatagtagat gataatttgc ctaaatttct agaaaataaa 780 gctaagtatg agagtttaaa agacaaagct ccagaagcta taaactatga acaaattaaa 840 aaagatttgg cagaagagct aacctttgat attgactaca aaacatctga agttaatcaa 900 agagtttttt cacttgatga agtttttgag atagcaaact ttaataatta tctaaatcaa 960 agtggtatta ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg tgaaaataca 1020 aagagaaaag gtataaatga atatataaat ctatactcac agcaaataaa tgataaaaca 1080 ctcaaaaaat ataaaatgag tgttttattt aagcaaattt taagtgatac agaatctaaa 1140 tcttttgtaa ttgataagtt agaagatgat agtgatgtag ttacaacgat gcaaagtttt 1200 tatgagcaaa tagcagcttt taaaacagta gaagaaaaat ctattaaaga aacactatct 1260 ttattatttg atgatttaaa agctcaaaaa cttgatttga gtaaaattta ttttaaaaat 1320 gataaatctc ttactgatct atcacaacaa gtttttgatg attatagtgt tattggtaca 1380 gcggtactag aatatataac tcaacaaata gcacctaaaa atcttgataa ccctagtaag 1440 aaagagcaag aattaatagc caaaaaaact gaaaaagcaa aatacttatc tctagaaact 1500 ataaagcttg ccttagaaga atttaataag catagagata tagataaaca gtgtaggttt 1560 gaagaaatac ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat agctcaaaac 1620 aaagacaatt tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa agacctactt 1680 caagctagtg cggaagatga tgttaaagct atcaaggatc ttttagatca aactaataat 1740 ctcttacata aactaaaaat atttcatatt agtcagtcag aagataaggc aaatatttta 1800 gacaaggatg agcattttta tctagtattt gaggagtgct actttgagct agcgaatata 1860 gtgcctcttt ataacaaaat tagaaactat ataactcaaa agccatatag tgatgagaaa 1920 tttaagctca attttgagaa ctcgactttg gctaatggtt gggataaaaa taaagagcct 1980 gacaatacgg caattttatt tatcaaagat gataaatatt atctgggtgt gatgaataag 2040 aaaaataaca aaatatttga tgataaagct atcaaagaaa ataaaggcga gggttataaa 2100 aaaattgttt ataaactttt acctggcgca aataaaatgt tacctaaggt tttcttttct 2160 gctaaatcta taaaatttta taatcctagt gaagatatac ttagaataag aaatcattcc 2220 acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt taatattgaa 2280 gattgccgaa aatttataga tttttataaa cagtctataa gtaagcatcc ggagtggaaa 2340 gattttggat ttagattttc tgatactcaa agatataatt ctatagatga attttataga 2400 gaagttgaaa atcaaggcta caaactaact tttgaaaata tatcagagag ctatattgat 2460 agcgtagtta atcagggtaa attgtaccta ttccaaatct ataataaaga tttttcagct 2520 tatagcaaag ggcgaccaaa tctacatact ttatattgga aagcgctgtt tgatgagaga 2580 aatcttcaag atgtggttta taagctaaat ggtgaggcag agctttttta tcgtaaacaa 2640 tcaataccta aaaaaatcac tcacccagct aaagaggcaa tagctaataa aaacaaagat 2700 aatcctaaaa aagagagtgt ttttgaatat gatttaatca aagataaacg ctttactgaa 2760 gataagtttt tctttcactg tcctattaca atcaatttta aatctagtgg agctaataag 2820 tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa atgatgttca tatattaagt 2880 atagatagag gtgaaagaca tttagcttac tatactttgg tagatggtaa aggcaatatc 2940 atcaaacaag atactttcaa catcattggt aatgatagaa tgaaaacaaa ctaccatgat 3000 aagcttgctg caatagagaa agatagggat tcagctagga aagactggaa aaagataaat 3060 aacatcaaag agatgaaaga gggctatcta tctcaggtag ttcatgaaat agctaagcta 3120 gttatagagt ataatgctat tgtggttttt gaggatttaa attttggatt taaaagaggg 3180 cgtttcaagg tagagaagca ggtctatcaa aagttagaaa aaatgctaat tgagaaacta 3240 aactatctag ttttcaaaga taatgagttt gataaaactg ggggagtgct tagagcttat 3300 cagctaacag caccttttga gacttttaaa aagatgggta aacaaacagg tattatctac 3360 tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt aaatcagtta 3420 tatcctaagt atgaaagtgt cagcaaatct caagagttct ttagtaagtt tgacaagatt 3480 tgttataacc ttgataaggg ctattttgag tttagttttg attataaaaa ctttggtgac 3540 aaggctgcca aaggcaagtg gactatagct agctttggga gtagattgat taactttaga 3600 aattcagata aaaatcataa ttgggatact cgagaagttt atccaactaa agagttggag 3660 aaattgctaa aagattattc tatcgaatat gggcatggcg aatgtatcaa agcagctatt 3720 tgcggtgaga gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa tactatctta 3780 caaatgcgta actcaaaaac aggtactgag ttagattatc taatttcacc agtagcagat 3840 gtaaatggca atttctttga ttcgcgacag gcgccaaaaa atatgcctca agatgctgat 3900 gccaatggtg cttatcatat tgggctaaaa ggtctgatgc tactaggtag gatcaaaaat 3960 aatcaagagg gcaaaaaact caatttggtt atcaaaaatg aagagtattt tgagttcgtg 4020 cagaatagga ataaccaagc ggccgcactc gacctcgaga tgacaccgga cattatcctg 4080 cagcgtaccg ggatcgatgt gagagctgtc gaacaggggg atgatgcgtg gcacaaatta 4140 cggctcggcg tcatcaccgc ttcagaagtt cacaacgtga tagcaaaacc ccgctccgga 4200 aagaagtggc ctgacatgaa aatgtcctac ttccacaccc tgcttgctga ggtttgcacc 4260 ggtgtggctc cggaagttaa cgctaaagca ctggcctggg gaaaacagta cgagaacgac 4320 gccagaaccc tgtttgaatt cacttccggc gtgaatgtta ctgaatcccc gatcatctat 4380 cgcgacgaaa gtatgcgtac cgcctgctct cccgatggtt tatgcagtga cggcaacggc 4440 cttgaactga aatgcccgtt tacctcccgg gatttcatga agttccggct cggtggtttc 4500 gaggccataa agtcagctta catggcccag gtgcagtaca gcatgtgggt gacgcgaaaa 4560 aatgcctggt actttgccaa ctatgacccg cgtatgaagc gtgaaggcct gcattatgtc 4620 gtgattgagc gggatgaaaa gtacatggcg agttttgacg agatcgtgcc ggagttcatc 4680 gaaaaaatgg acgaggcact ggctgaaatt ggttttgtat ttggggagca atggcgactc 4740 gagaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gcaccaccac 4800 caccactga 4809 <210> 67 <211> 1731 <212> PRT <213> Artificial Sequence <220> <223> FnCpf1-mungbean <400> 67 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Ser Ile 35 40 45 Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe 50 55 60 Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly 65 70 75 80 Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys 85 90 95 Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser 100 105 110 Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr 115 120 125 Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys 130 135 140 Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp 145 150 155 160 Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys 165 170 175 Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp 180 185 190 Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp 195 200 205 Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe 210 215 220 Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile 225 230 235 240 Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe 245 250 255 Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu 260 265 270 Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr 275 280 285 Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser 290 295 300 Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln 305 310 315 320 Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn 325 330 335 Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr 340 345 350 Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val 355 360 365 Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile 370 375 380 Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe 385 390 395 400 Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys 405 410 415 Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp 420 425 430 Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser 435 440 445 Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu 450 455 460 Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys 465 470 475 480 Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu 485 490 495 Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg 500 505 510 Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala 515 520 525 Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu 530 535 540 Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu 545 550 555 560 Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp 565 570 575 Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln 580 585 590 Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu 595 600 605 Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr 610 615 620 Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys 625 630 635 640 Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys 645 650 655 Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys 660 665 670 Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp 675 680 685 Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr 690 695 700 Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser 705 710 715 720 Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile 725 730 735 Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr 740 745 750 Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe 755 760 765 Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe 770 775 780 Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg 785 790 795 800 Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu 805 810 815 Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr Leu Phe Gln 820 825 830 Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg Pro Asn Leu 835 840 845 His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp 850 855 860 Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln 865 870 875 880 Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn 885 890 895 Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu 900 905 910 Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro 915 920 925 Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu 930 935 940 Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His Ile Leu Ser 945 950 955 960 Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu Val Asp Gly 965 970 975 Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp 980 985 990 Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp 995 1000 1005 Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu 1010 1015 1020 Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile Ala Lys Leu 1025 1030 1035 1040 Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly 1045 1050 1055 Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys Leu 1060 1065 1070 Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe Lys Asp Asn 1075 1080 1085 Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala 1090 1095 1100 Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr 1105 1110 1115 1120 Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe 1125 1130 1135 Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu 1140 1145 1150 Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr 1155 1160 1165 Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys 1170 1175 1180 Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg 1185 1190 1195 1200 Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr 1205 1210 1215 Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His 1220 1225 1230 Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1235 1240 1245 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn 1250 1255 1260 Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp 1265 1270 1275 1280 Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro 1285 1290 1295 Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys Gly Leu 1300 1305 1310 Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys Lys Leu Asn 1315 1320 1325 Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn 1330 1335 1340 Asn Gln Ala Ala Ala Leu Asp Leu Gln Met Gln Thr Leu Gln Met Ser 1345 1350 1355 1360 Leu Leu Thr Gln Pro Tyr Val Gln Pro Arg Phe Pro Cys Lys Arg Tyr 1365 1370 1375 Pro Thr Phe Ser Ala Ser Cys Arg Thr Gln Lys Thr Ala Ile Thr Lys 1380 1385 1390 Thr Glu Lys Val Phe Phe Ser Glu Ser Phe Asp Gln Thr Arg Cys Thr 1395 1400 1405 Gln Pro Leu Ser Glu Lys Lys Lys Arg Val Phe Phe Leu Asp Val Asn 1410 1415 1420 Pro Leu Cys Tyr Glu Gly Ser Lys Pro Ser Leu Arg Ser Phe Gly Arg 1425 1430 1435 1440 Trp Leu Ser Leu Phe Leu His Gln Val Ser Leu Thr Asp Pro Val Ile 1445 1450 1455 Ala Val Ile Asp Gly Glu Gly Gly Ser Glu His Arg Arg Lys Leu Leu 1460 1465 1470 Pro Ser Tyr Lys Ala His Arg Lys Lys Phe Met Arg His Met Ser Ser 1475 1480 1485 Gly His Val Gly Arg Ser His Gln Val Ile Asn Asp Val Leu Gly Lys 1490 1495 1500 Cys Asn Val Pro Val Ile Lys Val Ala Gly His Glu Ala Asp Asp Val 1505 1510 1515 1520 Val Ala Thr Leu Ala Gly Gln Val Val Asn Lys Gly Phe Arg Val Val 1525 1530 1535 Ile Gly Ser Pro Asp Lys Asp Phe Lys Gln Leu Ile Ser Glu Asp Val 1540 1545 1550 Gln Ile Val Met Pro Leu Pro Glu Leu Gln Arg Trp Ser Phe Tyr Thr 1555 1560 1565 Leu Arg His Tyr Arg Asp Gln Tyr Asn Cys Asp Pro Glu Ser Asp Leu 1570 1575 1580 Ser Phe Arg Cys Ile Val Gly Asp Glu Val Asp Gly Val Pro Gly Ile 1585 1590 1595 1600 Gln His Leu Val Pro Ser Phe Gly Arg Lys Thr Ala Met Lys Leu Ile 1605 1610 1615 Lys Lys His Gly Ser Leu Glu Thr Leu Leu Asn Ala Ala Ala Ile Arg 1620 1625 1630 Thr Val Gly Arg Pro Tyr Ala Gln Asp Ala Leu Lys Asn His Ala Asp 1635 1640 1645 Tyr Leu Arg Arg Asn Tyr Glu Val Leu Ala Leu Lys Arg Asp Val Asn 1650 1655 1660 Ile Gln Leu Tyr Asp Glu Trp Leu Val Lys Arg Asp Asn His Asn Asp 1665 1670 1675 1680 Lys Thr Ala Leu Ser Ser Phe Phe Lys Tyr Leu Gly Glu Ser Lys Glu 1685 1690 1695 Leu Ser Tyr Asn Gly Arg Pro Ile Ser Tyr Asn Gly Leu Gln Lys Arg 1700 1705 1710 Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys His His 1715 1720 1725 His His His 1730 <210> 68 <211> 5196 <212> DNA <213> Artificial Sequence <220> <223> FnCpf1-mungbean <400> 68 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgtc aatttatcaa gaatttgtta ataaatatag tttaagtaaa 180 actctaagat ttgagttaat cccacagggt aaaacacttg aaaacataaa agcaagaggt 240 ttgattttag atgatgagaa aagagctaaa gactacaaaa aggctaaaca aataattgat 300 aaatatcatc agttttttat agaggagata ttaagttcgg tttgtattag cgaagattta 360 ttacaaaact attctgatgt ttattttaaa cttaaaaaga gtgatgatga taatctacaa 420 aaagatttta aaagtgcaaa agatacgata aagaaacaaa tatctgaata tataaaggac 480 tcagagaaat ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa agggcaagag 540 tcagatttaa ttctatggct aaagcaatct aaggataatg gtatagaact atttaaagcc 600 aatagtgata tcacagatat agatgaggcg ttagaaataa tcaaatcttt taaaggttgg 660 acaacttatt ttaagggttt tcatgaaaat agaaaaaatg tttatagtag caatgatatt 720 cctacatcta ttatttatag gatagtagat gataatttgc ctaaatttct agaaaataaa 780 gctaagtatg agagtttaaa agacaaagct ccagaagcta taaactatga acaaattaaa 840 aaagatttgg cagaagagct aacctttgat attgactaca aaacatctga agttaatcaa 900 agagtttttt cacttgatga agtttttgag atagcaaact ttaataatta tctaaatcaa 960 agtggtatta ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg tgaaaataca 1020 aagagaaaag gtataaatga atatataaat ctatactcac agcaaataaa tgataaaaca 1080 ctcaaaaaat ataaaatgag tgttttattt aagcaaattt taagtgatac agaatctaaa 1140 tcttttgtaa ttgataagtt agaagatgat agtgatgtag ttacaacgat gcaaagtttt 1200 tatgagcaaa tagcagcttt taaaacagta gaagaaaaat ctattaaaga aacactatct 1260 ttattatttg atgatttaaa agctcaaaaa cttgatttga gtaaaattta ttttaaaaat 1320 gataaatctc ttactgatct atcacaacaa gtttttgatg attatagtgt tattggtaca 1380 gcggtactag aatatataac tcaacaaata gcacctaaaa atcttgataa ccctagtaag 1440 aaagagcaag aattaatagc caaaaaaact gaaaaagcaa aatacttatc tctagaaact 1500 ataaagcttg ccttagaaga atttaataag catagagata tagataaaca gtgtaggttt 1560 gaagaaatac ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat agctcaaaac 1620 aaagacaatt tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa agacctactt 1680 caagctagtg cggaagatga tgttaaagct atcaaggatc ttttagatca aactaataat 1740 ctcttacata aactaaaaat atttcatatt agtcagtcag aagataaggc aaatatttta 1800 gacaaggatg agcattttta tctagtattt gaggagtgct actttgagct agcgaatata 1860 gtgcctcttt ataacaaaat tagaaactat ataactcaaa agccatatag tgatgagaaa 1920 tttaagctca attttgagaa ctcgactttg gctaatggtt gggataaaaa taaagagcct 1980 gacaatacgg caattttatt tatcaaagat gataaatatt atctgggtgt gatgaataag 2040 aaaaataaca aaatatttga tgataaagct atcaaagaaa ataaaggcga gggttataaa 2100 aaaattgttt ataaactttt acctggcgca aataaaatgt tacctaaggt tttcttttct 2160 gctaaatcta taaaatttta taatcctagt gaagatatac ttagaataag aaatcattcc 2220 acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt taatattgaa 2280 gattgccgaa aatttataga tttttataaa cagtctataa gtaagcatcc ggagtggaaa 2340 gattttggat ttagattttc tgatactcaa agatataatt ctatagatga attttataga 2400 gaagttgaaa atcaaggcta caaactaact tttgaaaata tatcagagag ctatattgat 2460 agcgtagtta atcagggtaa attgtaccta ttccaaatct ataataaaga tttttcagct 2520 tatagcaaag ggcgaccaaa tctacatact ttatattgga aagcgctgtt tgatgagaga 2580 aatcttcaag atgtggttta taagctaaat ggtgaggcag agctttttta tcgtaaacaa 2640 tcaataccta aaaaaatcac tcacccagct aaagaggcaa tagctaataa aaacaaagat 2700 aatcctaaaa aagagagtgt ttttgaatat gatttaatca aagataaacg ctttactgaa 2760 gataagtttt tctttcactg tcctattaca atcaatttta aatctagtgg agctaataag 2820 tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa atgatgttca tatattaagt 2880 atagatagag gtgaaagaca tttagcttac tatactttgg tagatggtaa aggcaatatc 2940 atcaaacaag atactttcaa catcattggt aatgatagaa tgaaaacaaa ctaccatgat 3000 aagcttgctg caatagagaa agatagggat tcagctagga aagactggaa aaagataaat 3060 aacatcaaag agatgaaaga gggctatcta tctcaggtag ttcatgaaat agctaagcta 3120 gttatagagt ataatgctat tgtggttttt gaggatttaa attttggatt taaaagaggg 3180 cgtttcaagg tagagaagca ggtctatcaa aagttagaaa aaatgctaat tgagaaacta 3240 aactatctag ttttcaaaga taatgagttt gataaaactg ggggagtgct tagagcttat 3300 cagctaacag caccttttga gacttttaaa aagatgggta aacaaacagg tattatctac 3360 tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt aaatcagtta 3420 tatcctaagt atgaaagtgt cagcaaatct caagagttct ttagtaagtt tgacaagatt 3480 tgttataacc ttgataaggg ctattttgag tttagttttg attataaaaa ctttggtgac 3540 aaggctgcca aaggcaagtg gactatagct agctttggga gtagattgat taactttaga 3600 aattcagata aaaatcataa ttgggatact cgagaagttt atccaactaa agagttggag 3660 aaattgctaa aagattattc tatcgaatat gggcatggcg aatgtatcaa agcagctatt 3720 tgcggtgaga gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa tactatctta 3780 caaatgcgta actcaaaaac aggtactgag ttagattatc taatttcacc agtagcagat 3840 gtaaatggca atttctttga ttcgcgacag gcgccaaaaa atatgcctca agatgctgat 3900 gccaatggtg cttatcatat tgggctaaaa ggtctgatgc tactaggtag gatcaaaaat 3960 aatcaagagg gcaaaaaact caatttggtt atcaaaaatg aagagtattt tgagttcgtg 4020 cagaatagga ataaccaagc ggccgcactc gacctgcaga tgcaaacgtt acagatgagt 4080 ctgttgacac aaccttacgt tcagcctcgt ttcccttgca agcgttaccc gaccttctcc 4140 gcatcctgca gaactcaaaa gacagcgatc acgaaaacag agaaggtgtt tttcagtgag 4200 tcatttgatc aaacacgttg cacgcagcct ctctcggaaa agaagaagag ggtgttcttt 4260 ttggacgtta acccgctctg ttacgaagga agcaagccca gcttgcgctc cttcgggcgg 4320 tggctctctc tgtttctcca tcaagtcagc ctcactgacc ccgtcattgc tgttattgat 4380 ggagaaggag gcagcgagca tcgcagaaag ttgctacctt catataaagc acataggaaa 4440 aagttcatga gacacatgtc aagtggccat gttgggaggt ctcatcaagt tataaatgat 4500 gttcttggaa aatgcaacgt gccagttata aaggttgctg gtcatgaagc tgatgatgtt 4560 gtagctactc tagctggaca agttgtcaat aaagggtttc gagtggtcat tggctcccct 4620 gataaggatt ttaagcagct tatatctgaa gatgtgcaaa tagttatgcc tttgccagag 4680 ttacaaaggt ggtccttcta cactctgagg cactacaggg atcagtataa ttgtgatcca 4740 gaatctgatc tgagctttag atgcattgta ggtgatgaag tagacggcgt tcctggtatc 4800 cagcatttgg tccctagttt tggtcggaag actgctatga aacttattaa aaaacatggt 4860 tccttggaaa ctttattaaa tgcggctgca ataaggactg taggcagacc atatgcacag 4920 gatgccctca aaaaccatgc tgattacctt cggagaaact atgaagttct tgccttgaaa 4980 agggatgtaa atatccaact ttatgatgag tggttggtta agagagacaa tcacaatgat 5040 aaaactgcac tatcttcctt cttcaaatat ttgggagaaa gtaaggagct cagttacaat 5100 ggcagaccta tctcttacaa tggtctgcag aaaaggccgg cggccacgaa aaaggccggc 5160 caggcaaaaa agaaaaagca ccaccaccac cactga 5196 <210> 69 <211> 1614 <212> PRT <213> Artificial Sequence <220> <223> FnCpf1-GFP <400> 69 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Ser Ile 35 40 45 Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe 50 55 60 Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly 65 70 75 80 Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys 85 90 95 Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser 100 105 110 Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr 115 120 125 Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys 130 135 140 Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp 145 150 155 160 Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys 165 170 175 Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp 180 185 190 Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp 195 200 205 Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe 210 215 220 Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile 225 230 235 240 Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe 245 250 255 Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu 260 265 270 Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr 275 280 285 Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser 290 295 300 Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln 305 310 315 320 Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn 325 330 335 Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr 340 345 350 Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val 355 360 365 Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile 370 375 380 Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe 385 390 395 400 Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys 405 410 415 Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp 420 425 430 Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser 435 440 445 Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu 450 455 460 Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys 465 470 475 480 Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu 485 490 495 Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg 500 505 510 Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala 515 520 525 Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu 530 535 540 Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu 545 550 555 560 Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp 565 570 575 Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln 580 585 590 Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu 595 600 605 Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr 610 615 620 Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys 625 630 635 640 Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys 645 650 655 Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys 660 665 670 Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp 675 680 685 Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr 690 695 700 Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser 705 710 715 720 Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile 725 730 735 Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr 740 745 750 Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe 755 760 765 Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe 770 775 780 Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg 785 790 795 800 Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu 805 810 815 Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr Leu Phe Gln 820 825 830 Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg Pro Asn Leu 835 840 845 His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp 850 855 860 Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln 865 870 875 880 Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn 885 890 895 Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu 900 905 910 Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro 915 920 925 Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu 930 935 940 Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His Ile Leu Ser 945 950 955 960 Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu Val Asp Gly 965 970 975 Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp 980 985 990 Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp 995 1000 1005 Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu 1010 1015 1020 Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile Ala Lys Leu 1025 1030 1035 1040 Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly 1045 1050 1055 Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys Leu 1060 1065 1070 Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe Lys Asp Asn 1075 1080 1085 Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala 1090 1095 1100 Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr 1105 1110 1115 1120 Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe 1125 1130 1135 Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu 1140 1145 1150 Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr 1155 1160 1165 Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys 1170 1175 1180 Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg 1185 1190 1195 1200 Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr 1205 1210 1215 Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His 1220 1225 1230 Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1235 1240 1245 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn 1250 1255 1260 Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp 1265 1270 1275 1280 Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro 1285 1290 1295 Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys Gly Leu 1300 1305 1310 Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys Lys Leu Asn 1315 1320 1325 Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn 1330 1335 1340 Asn Gln Ala Ala Ala Leu Asp Leu Glu Met Ser Lys Gly Glu Glu Leu 1345 1350 1355 1360 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 1365 1370 1375 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 1380 1385 1390 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val 1395 1400 1405 Pro Trp Pro Thr Leu Val Thr Thr Phe Ser Tyr Gly Val Gln Cys Phe 1410 1415 1420 Ser Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe Lys Ser Ala 1425 1430 1435 1440 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Ser Phe Lys Asp Asp 1445 1450 1455 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 1460 1465 1470 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 1475 1480 1485 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr 1490 1495 1500 Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile 1505 1510 1515 1520 Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln 1525 1530 1535 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 1540 1545 1550 Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 1555 1560 1565 Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr His 1570 1575 1580 Gly Met Asp Glu Leu Tyr Lys Leu Glu Lys Arg Pro Ala Ala Thr Lys 1585 1590 1595 1600 Lys Ala Gly Gln Ala Lys Lys Lys Lys His His His His His 1605 1610 <210> 70 <211> 4845 <212> DNA <213> Artificial Sequence <220> <223> FnCpf1-GFP <400> 70 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga 120 caagcttgcg gccgcatgtc aatttatcaa gaatttgtta ataaatatag tttaagtaaa 180 actctaagat ttgagttaat cccacagggt aaaacacttg aaaacataaa agcaagaggt 240 ttgattttag atgatgagaa aagagctaaa gactacaaaa aggctaaaca aataattgat 300 aaatatcatc agttttttat agaggagata ttaagttcgg tttgtattag cgaagattta 360 ttacaaaact attctgatgt ttattttaaa cttaaaaaga gtgatgatga taatctacaa 420 aaagatttta aaagtgcaaa agatacgata aagaaacaaa tatctgaata tataaaggac 480 tcagagaaat ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa agggcaagag 540 tcagatttaa ttctatggct aaagcaatct aaggataatg gtatagaact atttaaagcc 600 aatagtgata tcacagatat agatgaggcg ttagaaataa tcaaatcttt taaaggttgg 660 acaacttatt ttaagggttt tcatgaaaat agaaaaaatg tttatagtag caatgatatt 720 cctacatcta ttatttatag gatagtagat gataatttgc ctaaatttct agaaaataaa 780 gctaagtatg agagtttaaa agacaaagct ccagaagcta taaactatga acaaattaaa 840 aaagatttgg cagaagagct aacctttgat attgactaca aaacatctga agttaatcaa 900 agagtttttt cacttgatga agtttttgag atagcaaact ttaataatta tctaaatcaa 960 agtggtatta ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg tgaaaataca 1020 aagagaaaag gtataaatga atatataaat ctatactcac agcaaataaa tgataaaaca 1080 ctcaaaaaat ataaaatgag tgttttattt aagcaaattt taagtgatac agaatctaaa 1140 tcttttgtaa ttgataagtt agaagatgat agtgatgtag ttacaacgat gcaaagtttt 1200 tatgagcaaa tagcagcttt taaaacagta gaagaaaaat ctattaaaga aacactatct 1260 ttattatttg atgatttaaa agctcaaaaa cttgatttga gtaaaattta ttttaaaaat 1320 gataaatctc ttactgatct atcacaacaa gtttttgatg attatagtgt tattggtaca 1380 gcggtactag aatatataac tcaacaaata gcacctaaaa atcttgataa ccctagtaag 1440 aaagagcaag aattaatagc caaaaaaact gaaaaagcaa aatacttatc tctagaaact 1500 ataaagcttg ccttagaaga atttaataag catagagata tagataaaca gtgtaggttt 1560 gaagaaatac ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat agctcaaaac 1620 aaagacaatt tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa agacctactt 1680 caagctagtg cggaagatga tgttaaagct atcaaggatc ttttagatca aactaataat 1740 ctcttacata aactaaaaat atttcatatt agtcagtcag aagataaggc aaatatttta 1800 gacaaggatg agcattttta tctagtattt gaggagtgct actttgagct agcgaatata 1860 gtgcctcttt ataacaaaat tagaaactat ataactcaaa agccatatag tgatgagaaa 1920 tttaagctca attttgagaa ctcgactttg gctaatggtt gggataaaaa taaagagcct 1980 gacaatacgg caattttatt tatcaaagat gataaatatt atctgggtgt gatgaataag 2040 aaaaataaca aaatatttga tgataaagct atcaaagaaa ataaaggcga gggttataaa 2100 aaaattgttt ataaactttt acctggcgca aataaaatgt tacctaaggt tttcttttct 2160 gctaaatcta taaaatttta taatcctagt gaagatatac ttagaataag aaatcattcc 2220 acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt taatattgaa 2280 gattgccgaa aatttataga tttttataaa cagtctataa gtaagcatcc ggagtggaaa 2340 gattttggat ttagattttc tgatactcaa agatataatt ctatagatga attttataga 2400 gaagttgaaa atcaaggcta caaactaact tttgaaaata tatcagagag ctatattgat 2460 agcgtagtta atcagggtaa attgtaccta ttccaaatct ataataaaga tttttcagct 2520 tatagcaaag ggcgaccaaa tctacatact ttatattgga aagcgctgtt tgatgagaga 2580 aatcttcaag atgtggttta taagctaaat ggtgaggcag agctttttta tcgtaaacaa 2640 tcaataccta aaaaaatcac tcacccagct aaagaggcaa tagctaataa aaacaaagat 2700 aatcctaaaa aagagagtgt ttttgaatat gatttaatca aagataaacg ctttactgaa 2760 gataagtttt tctttcactg tcctattaca atcaatttta aatctagtgg agctaataag 2820 tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa atgatgttca tatattaagt 2880 atagatagag gtgaaagaca tttagcttac tatactttgg tagatggtaa aggcaatatc 2940 atcaaacaag atactttcaa catcattggt aatgatagaa tgaaaacaaa ctaccatgat 3000 aagcttgctg caatagagaa agatagggat tcagctagga aagactggaa aaagataaat 3060 aacatcaaag agatgaaaga gggctatcta tctcaggtag ttcatgaaat agctaagcta 3120 gttatagagt ataatgctat tgtggttttt gaggatttaa attttggatt taaaagaggg 3180 cgtttcaagg tagagaagca ggtctatcaa aagttagaaa aaatgctaat tgagaaacta 3240 aactatctag ttttcaaaga taatgagttt gataaaactg ggggagtgct tagagcttat 3300 cagctaacag caccttttga gacttttaaa aagatgggta aacaaacagg tattatctac 3360 tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt aaatcagtta 3420 tatcctaagt atgaaagtgt cagcaaatct caagagttct ttagtaagtt tgacaagatt 3480 tgttataacc ttgataaggg ctattttgag tttagttttg attataaaaa ctttggtgac 3540 aaggctgcca aaggcaagtg gactatagct agctttggga gtagattgat taactttaga 3600 aattcagata aaaatcataa ttgggatact cgagaagttt atccaactaa agagttggag 3660 aaattgctaa aagattattc tatcgaatat gggcatggcg aatgtatcaa agcagctatt 3720 tgcggtgaga gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa tactatctta 3780 caaatgcgta actcaaaaac aggtactgag ttagattatc taatttcacc agtagcagat 3840 gtaaatggca atttctttga ttcgcgacag gcgccaaaaa atatgcctca agatgctgat 3900 gccaatggtg cttatcatat tgggctaaaa ggtctgatgc tactaggtag gatcaaaaat 3960 aatcaagagg gcaaaaaact caatttggtt atcaaaaatg aagagtattt tgagttcgtg 4020 cagaatagga ataaccaagc ggccgcactc gacctcgaga tgagtaaagg agaagaactt 4080 ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt 4140 tctgtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt 4200 tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactacttt ctcttatggt 4260 gttcaatgct tttcaagata cccagatcat atgaagcggc acgacttctt caagagcgcc 4320 atgcctgagg gatacgtgca ggagaggacc atctctttca aggacgacgg gaactacaag 4380 acacgtgctg aagtcaagtt tgagggagac accctcgtca acaggatcga gcttaaggga 4440 atcgatttca aggaggacgg aaacatcctc ggccacaagt tggaatacaa ctacaactcc 4500 cacaacgtat acatcacggc agacaaacaa aagaatggaa tcaaagctaa cttcaaaatt 4560 agacacaaca ttgaagatgg aagcgttcaa ctagcagacc attatcaaca aaatactcca 4620 attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt 4680 tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct 4740 gggattacac atggcatgga tgaactatac aaactcgaga aaaggccggc ggccacgaaa 4800 aaggccggcc aggcaaaaaa gaaaaagcac caccaccacc actga 4845 <210> 71 <211> 1958 <212> PRT <213> Artificial Sequence <220> <223> RecJ-FnCpf1 <400> 71 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Val Lys Gln Gln Ile Gln Leu Arg Arg Arg Glu Val Asp Glu 35 40 45 Thr Ala Asp Leu Pro Ala Glu Leu Pro Pro Leu Leu Arg Arg Leu Tyr 50 55 60 Ala Ser Arg Gly Val Arg Ser Ala Gln Glu Leu Glu Arg Ser Val Lys 65 70 75 80 Gly Met Leu Pro Trp Gln Gln Leu Ser Gly Val Glu Lys Ala Val Glu 85 90 95 Ile Leu Tyr Asn Ala Phe Arg Glu Gly Thr Arg Ile Ile Val Val Gly 100 105 110 Asp Phe Asp Ala Asp Gly Ala Thr Ser Thr Ala Leu Ser Val Leu Ala 115 120 125 Met Arg Ser Leu Gly Cys Ser Asn Ile Asp Tyr Leu Val Pro Asn Arg 130 135 140 Phe Glu Asp Gly Tyr Gly Leu Ser Pro Glu Val Val Asp Gln Ala His 145 150 155 160 Ala Arg Gly Ala Gln Leu Ile Val Thr Val Asp Asn Gly Ile Ser Ser 165 170 175 His Ala Gly Val Glu His Ala Arg Ser Leu Gly Ile Pro Val Ile Val 180 185 190 Thr Asp His His Leu Pro Gly Glu Thr Leu Pro Ala Ala Glu Ala Ile 195 200 205 Ile Asn Pro Asn Leu Arg Asp Cys Asn Phe Pro Ser Lys Ser Leu Ala 210 215 220 Gly Val Gly Val Ala Phe Tyr Leu Met Leu Ala Leu Arg Thr Phe Leu 225 230 235 240 Arg Asp Gln Gly Trp Phe Asp Glu Arg Gly Ile Ala Ile Pro Asn Leu 245 250 255 Ala Glu Leu Leu Asp Leu Val Ala Leu Gly Thr Val Ala Asp Val Val 260 265 270 Pro Leu Asp Ala Asn Asn Arg Ile Leu Thr Trp Gln Gly Met Ser Arg 275 280 285 Ile Arg Ala Gly Lys Cys Arg Pro Gly Ile Lys Ala Leu Leu Glu Val 290 295 300 Ala Asn Arg Asp Ala Gln Lys Leu Ala Ala Ser Asp Leu Gly Phe Ala 305 310 315 320 Leu Gly Pro Arg Leu Asn Ala Ala Gly Arg Leu Asp Asp Met Ser Val 325 330 335 Gly Val Ala Leu Leu Leu Cys Asp Asn Ile Gly Glu Ala Arg Val Leu 340 345 350 Ala Asn Glu Leu Asp Ala Leu Asn Gln Thr Arg Lys Glu Ile Glu Gln 355 360 365 Gly Met Gln Val Glu Ala Leu Thr Leu Cys Glu Lys Leu Glu Arg Ser 370 375 380 Arg Asp Thr Leu Pro Gly Gly Leu Ala Met Tyr His Pro Glu Trp His 385 390 395 400 Gln Gly Val Val Gly Ile Leu Ala Ser Arg Ile Lys Glu Arg Phe His 405 410 415 Arg Pro Val Ile Ala Phe Ala Pro Ala Gly Asp Gly Thr Leu Lys Gly 420 425 430 Ser Gly Arg Ser Ile Gln Gly Leu His Met Arg Asp Ala Leu Glu Arg 435 440 445 Leu Asp Thr Leu Tyr Pro Gly Met Ile Leu Lys Phe Gly Gly His Ala 450 455 460 Met Ala Ala Gly Leu Ser Leu Glu Glu Asp Lys Phe Glu Leu Phe Gln 465 470 475 480 Gln Arg Phe Gly Glu Leu Val Thr Glu Trp Leu Asp Pro Ser Leu Leu 485 490 495 Gln Gly Glu Val Val Ser Asp Gly Pro Leu Ser Pro Ala Glu Met Thr 500 505 510 Met Glu Val Ala Gln Leu Leu Arg Asp Ala Gly Pro Trp Gly Gln Met 515 520 525 Phe Pro Glu Pro Leu Phe Asp Gly His Phe Arg Leu Leu Gln Gln Arg 530 535 540 Leu Val Gly Glu Arg His Leu Lys Val Met Val Glu Pro Val Gly Gly 545 550 555 560 Gly Pro Leu Leu Asp Gly Ile Ala Phe Asn Val Asp Thr Ala Leu Trp 565 570 575 Pro Asp Asn Gly Val Arg Glu Val Gln Leu Ala Tyr Lys Leu Asp Ile 580 585 590 Asn Glu Phe Arg Gly Asn Arg Ser Leu Gln Ile Ile Ile Asp Asn Ile 595 600 605 Trp Pro Ile Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg 610 615 620 Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr 625 630 635 640 Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys 645 650 655 Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys 660 665 670 Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu 675 680 685 Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser 690 695 700 Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys 705 710 715 720 Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr 725 730 735 Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile 740 745 750 Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln 755 760 765 Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr 770 775 780 Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr 785 790 795 800 Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser 805 810 815 Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu 820 825 830 Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys 835 840 845 Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu 850 855 860 Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg 865 870 875 880 Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr 885 890 895 Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys 900 905 910 Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile 915 920 925 Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys 930 935 940 Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser 945 950 955 960 Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met 965 970 975 Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys 980 985 990 Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln 995 1000 1005 Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr 1010 1015 1020 Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala 1025 1030 1035 1040 Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn 1045 1050 1055 Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala 1060 1065 1070 Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn 1075 1080 1085 Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala 1090 1095 1100 Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys 1105 1110 1115 1120 Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys 1125 1130 1135 Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp 1140 1145 1150 Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His 1155 1160 1165 Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His 1170 1175 1180 Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val 1185 1190 1195 1200 Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser 1205 1210 1215 Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly 1220 1225 1230 Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys 1235 1240 1245 Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile 1250 1255 1260 Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys 1265 1270 1275 1280 Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val 1285 1290 1295 Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile 1300 1305 1310 Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln 1315 1320 1325 Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe 1330 1335 1340 Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp 1345 1350 1355 1360 Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu 1365 1370 1375 Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn 1380 1385 1390 Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr 1395 1400 1405 Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg 1410 1415 1420 Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn 1425 1430 1435 1440 Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr 1445 1450 1455 Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala 1460 1465 1470 Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu 1475 1480 1485 Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe 1490 1495 1500 His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe 1505 1510 1515 1520 Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His 1525 1530 1535 Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu 1540 1545 1550 Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile 1555 1560 1565 Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile 1570 1575 1580 Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn 1585 1590 1595 1600 Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile 1605 1610 1615 Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu 1620 1625 1630 Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr 1635 1640 1645 Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe 1650 1655 1660 Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln 1665 1670 1675 1680 Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly 1685 1690 1695 Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val 1700 1705 1710 Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys 1715 1720 1725 Ser Gln Glu Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp 1730 1735 1740 Lys Gly Tyr Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys 1745 1750 1755 1760 Ala Ala Lys Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile 1765 1770 1775 Asn Phe Arg Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val 1780 1785 1790 Tyr Pro Thr Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu 1795 1800 1805 Tyr Gly His Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp 1810 1815 1820 Lys Lys Phe Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln 1825 1830 1835 1840 Met Arg Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro 1845 1850 1855 Val Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys 1860 1865 1870 Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu 1875 1880 1885 Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys 1890 1895 1900 Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln 1905 1910 1915 1920 Asn Arg Asn Asn Gln Ala Ala Ala Leu Glu Lys Arg Pro Ala Ala Thr 1925 1930 1935 Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Ser Thr Pro Pro Pro Pro 1940 1945 1950 Pro Leu Arg Ser Gly Cys 1955 <210> 72 <211> 5877 <212> DNA <213> Artificial Sequence <220> <223> RecJ-FnCpf1 <400> 72 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgtgaaaca acagatacaa 120 cttcgtcgcc gtgaagtcga tgaaacggca gacttgcccg ctgaattgcc tcccttgctg 180 cgccgtttat acgccagccg gggcgtgcgc agtgcgcaag aactggaacg cagtgttaaa 240 ggtatgttgc cctggcagca actgagcggc gtcgaaaagg ccgttgagat cctttacaac 300 gcttttcgcg aaggaacgcg gattattgtg gtcggcgatt ttgacgccga cggcgcgacc 360 agcacggctc taagcgtgct ggcgatgcgc tcgcttggtt gcagcaatat cgactatctg 420 gtaccaaacc gtttcgaaga cggttacggc ttaagcccgg aagtagtcga tcaggcccat 480 gcccgtggcg cgcagttaat tgtcacggtg gataacggta tttcctccca tgcgggcgtt 540 gaacacgctc gctcgttggg cattccggtt attgttaccg atcaccattt gccgggcgaa 600 acattacccg cagcggaagc gatcattaac cctaacttgc gcgactgtaa tttcccgtcg 660 aaatcactgg caggcgtggg tgtggcgttt tatctgatgc tggcgctgcg cacctttttg 720 cgcgatcagg gctggtttga tgagcgtggc atcgcaattc ctaacctggc agaactgctg 780 gatctggtcg cgctgggaac agtggcggac gtcgtgccgc tggacgctaa taatcgcatt 840 ctgacctggc aggggatgag tcgcatccgt gccggaaagt gccgtccagg gattaaagcg 900 ctgctggaag tggcaaaccg tgatgcacaa aaactcgccg ccagcgattt aggttttgcg 960 ctggggccac gtctcaatgc tgccggacga ctggacgata tgtccgtcgg tgtggcgctc 1020 ttgctgtgcg acaacatcgg cgaagcgcgc gtgctggcaa atgaactcga tgcgctaaac 1080 cagacgcgaa aagagatcga acaaggaatg caagttgaag ccctgaccct gtgcgagaaa 1140 ctggagcgaa gtcgcgacac gctacccggc gggctggcaa tgtatcaccc cgaatggcat 1200 cagggcgttg tcggtattct ggcttcgcgc atcaaagagc gttttcaccg tccggttatc 1260 gcctttgcgc cagcaggtga tggtacgctg aaaggttcag gtcgctccat tcaggggctg 1320 catatgcgtg atgcactgga gcgattagac acactctacc ctggcatgat actgaagttt 1380 ggcggtcatg cgatggcggc gggtttgtcg ctggaagagg ataaattcga actctttcaa 1440 caacggtttg gcgagctggt taccgagtgg ctggaccctt cgctattgca aggcgaagtg 1500 gtgtcagacg gcccgttaag cccggccgaa atgaccatgg aagtggcgca gctgctgcgc 1560 gatgctggcc cgtgggggca gatgttcccg gagccgctgt ttgatggtca tttccgtctg 1620 ctgcaacagc ggctggtggg cgaacgtcat ttgaaagtca tggtcgaacc ggtcggcggc 1680 ggtccgctgc tggatggtat tgcttttaat gtcgataccg ccctctggcc ggataacggc 1740 gtgcgcgaag tgcaactggc ttacaagctc gatatcaacg agtttcgcgg caaccgcagc 1800 ctgcaaatta tcatcgacaa tatctggcca attggatccg aattcgagct ccgtcgacaa 1860 gcttgcggcc gcatgtcaat ttatcaagaa tttgttaata aatatagttt aagtaaaact 1920 ctaagatttg agttaatccc acagggtaaa acacttgaaa acataaaagc aagaggtttg 1980 attttagatg atgagaaaag agctaaagac tacaaaaagg ctaaacaaat aattgataaa 2040 tatcatcagt tttttataga ggagatatta agttcggttt gtattagcga agatttatta 2100 caaaactatt ctgatgttta ttttaaactt aaaaagagtg atgatgataa tctacaaaaa 2160 gattttaaaa gtgcaaaaga tacgataaag aaacaaatat ctgaatatat aaaggactca 2220 gagaaattta agaatttgtt taatcaaaac cttatcgatg ctaaaaaagg gcaagagtca 2280 gatttaattc tatggctaaa gcaatctaag gataatggta tagaactatt taaagccaat 2340 agtgatatca cagatataga tgaggcgtta gaaataatca aatcttttaa aggttggaca 2400 acttatttta agggttttca tgaaaataga aaaaatgttt atagtagcaa tgatattcct 2460 acatctatta tttataggat agtagatgat aatttgccta aatttctaga aaataaagct 2520 aagtatgaga gtttaaaaga caaagctcca gaagctataa actatgaaca aattaaaaaa 2580 gatttggcag aagagctaac ctttgatatt gactacaaaa catctgaagt taatcaaaga 2640 gttttttcac ttgatgaagt ttttgagata gcaaacttta ataattatct aaatcaaagt 2700 ggtattacta aatttaatac tattattggt ggtaaatttg taaatggtga aaatacaaag 2760 agaaaaggta taaatgaata tataaatcta tactcacagc aaataaatga taaaacactc 2820 aaaaaatata aaatgagtgt tttatttaag caaattttaa gtgatacaga atctaaatct 2880 tttgtaattg ataagttaga agatgatagt gatgtagtta caacgatgca aagtttttat 2940 gagcaaatag cagcttttaa aacagtagaa gaaaaatcta ttaaagaaac actatcttta 3000 ttatttgatg atttaaaagc tcaaaaactt gatttgagta aaatttattt taaaaatgat 3060 aaatctctta ctgatctatc acaacaagtt tttgatgatt atagtgttat tggtacagcg 3120 gtactagaat atataactca acaaatagca cctaaaaatc ttgataaccc tagtaagaaa 3180 gagcaagaat taatagccaa aaaaactgaa aaagcaaaat acttatctct agaaactata 3240 aagcttgcct tagaagaatt taataagcat agagatatag ataaacagtg taggtttgaa 3300 gaaatacttg caaactttgc ggctattccg atgatatttg atgaaatagc tcaaaacaaa 3360 gacaatttgg cacagatatc tatcaaatat caaaatcaag gtaaaaaaga cctacttcaa 3420 gctagtgcgg aagatgatgt taaagctatc aaggatcttt tagatcaaac taataatctc 3480 ttacataaac taaaaatatt tcatattagt cagtcagaag ataaggcaaa tattttagac 3540 aaggatgagc atttttatct agtatttgag gagtgctact ttgagctagc gaatatagtg 3600 cctctttata acaaaattag aaactatata actcaaaagc catatagtga tgagaaattt 3660 aagctcaatt ttgagaactc gactttggct aatggttggg ataaaaataa agagcctgac 3720 aatacggcaa ttttatttat caaagatgat aaatattatc tgggtgtgat gaataagaaa 3780 aataacaaaa tatttgatga taaagctatc aaagaaaata aaggcgaggg ttataaaaaa 3840 attgtttata aacttttacc tggcgcaaat aaaatgttac ctaaggtttt cttttctgct 3900 aaatctataa aattttataa tcctagtgaa gatatactta gaataagaaa tcattccaca 3960 catacaaaaa atggtagtcc tcaaaaagga tatgaaaaat ttgagtttaa tattgaagat 4020 tgccgaaaat ttatagattt ttataaacag tctataagta agcatccgga gtggaaagat 4080 tttggattta gattttctga tactcaaaga tataattcta tagatgaatt ttatagagaa 4140 gttgaaaatc aaggctacaa actaactttt gaaaatatat cagagagcta tattgatagc 4200 gtagttaatc agggtaaatt gtacctattc caaatctata ataaagattt ttcagcttat 4260 agcaaagggc gaccaaatct acatacttta tattggaaag cgctgtttga tgagagaaat 4320 cttcaagatg tggtttataa gctaaatggt gaggcagagc ttttttatcg taaacaatca 4380 atacctaaaa aaatcactca cccagctaaa gaggcaatag ctaataaaaa caaagataat 4440 cctaaaaaag agagtgtttt tgaatatgat ttaatcaaag ataaacgctt tactgaagat 4500 aagtttttct ttcactgtcc tattacaatc aattttaaat ctagtggagc taataagttt 4560 aatgatgaaa tcaatttatt gctaaaagaa aaagcaaatg atgttcatat attaagtata 4620 gatagaggtg aaagacattt agcttactat actttggtag atggtaaagg caatatcatc 4680 aaacaagata ctttcaacat cattggtaat gatagaatga aaacaaacta ccatgataag 4740 cttgctgcaa tagagaaaga tagggattca gctaggaaag actggaaaaa gataaataac 4800 atcaaagaga tgaaagaggg ctatctatct caggtagttc atgaaatagc taagctagtt 4860 atagagtata atgctattgt ggtttttgag gatttaaatt ttggatttaa aagagggcgt 4920 ttcaaggtag agaagcaggt ctatcaaaag ttagaaaaaa tgctaattga gaaactaaac 4980 tatctagttt tcaaagataa tgagtttgat aaaactgggg gagtgcttag agcttatcag 5040 ctaacagcac cttttgagac ttttaaaaag atgggtaaac aaacaggtat tatctactat 5100 gtaccagctg gttttacttc aaaaatttgt cctgtaactg gttttgtaaa tcagttatat 5160 cctaagtatg aaagtgtcag caaatctcaa gagttcttta gtaagtttga caagatttgt 5220 tataaccttg ataagggcta ttttgagttt agttttgatt ataaaaactt tggtgacaag 5280 gctgccaaag gcaagtggac tatagctagc tttgggagta gattgattaa ctttagaaat 5340 tcagataaaa atcataattg ggatactcga gaagtttatc caactaaaga gttggagaaa 5400 ttgctaaaag attattctat cgaatatggg catggcgaat gtatcaaagc agctatttgc 5460 ggtgagagcg acaaaaagtt ttttgctaag ctaactagtg tcctaaatac tatcttacaa 5520 atgcgtaact caaaaacagg tactgagtta gattatctaa tttcaccagt agcagatgta 5580 aatggcaatt tctttgattc gcgacaggcg ccaaaaaata tgcctcaaga tgctgatgcc 5640 aatggtgctt atcatattgg gctaaaaggt ctgatgctac taggtaggat caaaaataat 5700 caagagggca aaaaactcaa tttggttatc aaaaatgaag agtattttga gttcgtgcag 5760 aataggaata accaagcggc cgcactcgag aaaaggccgg cggccacgaa aaaggccggc 5820 caggcaaaaa agaaaaagtc gacaccacca ccaccaccac tgagatccgg ctgctaa 5877 <210> 73 <211> 1619 <212> PRT <213> Artificial Sequence <220> <223> GFP-FnCpf1 <400> 73 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile 35 40 45 Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser 50 55 60 Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe 65 70 75 80 Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr 85 90 95 Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met 100 105 110 Lys Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln 115 120 125 Glu Arg Thr Ile Ser Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala 130 135 140 Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys 145 150 155 160 Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu 165 170 175 Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys 180 185 190 Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly 195 200 205 Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp 210 215 220 Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala 225 230 235 240 Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu 245 250 255 Phe Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 260 265 270 Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala Cys Gly Arg Met Ser Ile 275 280 285 Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe 290 295 300 Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly 305 310 315 320 Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys 325 330 335 Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser 340 345 350 Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr 355 360 365 Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys 370 375 380 Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp 385 390 395 400 Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys 405 410 415 Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp 420 425 430 Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp 435 440 445 Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe 450 455 460 Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile 465 470 475 480 Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe 485 490 495 Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu 500 505 510 Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr 515 520 525 Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser 530 535 540 Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln 545 550 555 560 Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn 565 570 575 Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr 580 585 590 Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val 595 600 605 Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile 610 615 620 Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe 625 630 635 640 Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys 645 650 655 Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp 660 665 670 Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser 675 680 685 Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu 690 695 700 Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys 705 710 715 720 Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu 725 730 735 Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg 740 745 750 Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala 755 760 765 Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu 770 775 780 Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu 785 790 795 800 Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp 805 810 815 Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His Ile Ser Gln 820 825 830 Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu 835 840 845 Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr 850 855 860 Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys 865 870 875 880 Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys 885 890 895 Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys 900 905 910 Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp 915 920 925 Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr 930 935 940 Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser 945 950 955 960 Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile 965 970 975 Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr 980 985 990 Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe 995 1000 1005 Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe 1010 1015 1020 Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg 1025 1030 1035 1040 Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu 1045 1050 1055 Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr Leu Phe Gln 1060 1065 1070 Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg Pro Asn Leu 1075 1080 1085 His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp 1090 1095 1100 Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln 1105 1110 1115 1120 Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn 1125 1130 1135 Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu 1140 1145 1150 Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro 1155 1160 1165 Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu 1170 1175 1180 Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His Ile Leu Ser 1185 1190 1195 1200 Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu Val Asp Gly 1205 1210 1215 Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp 1220 1225 1230 Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp 1235 1240 1245 Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu 1250 1255 1260 Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile Ala Lys Leu 1265 1270 1275 1280 Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly 1285 1290 1295 Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr Gln Lys Leu 1300 1305 1310 Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe Lys Asp Asn 1315 1320 1325 Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala 1330 1335 1340 Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr 1345 1350 1355 1360 Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe 1365 1370 1375 Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu 1380 1385 1390 Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr 1395 1400 1405 Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys 1410 1415 1420 Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg 1425 1430 1435 1440 Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr 1445 1450 1455 Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His 1460 1465 1470 Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1475 1480 1485 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn 1490 1495 1500 Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp 1505 1510 1515 1520 Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro 1525 1530 1535 Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys Gly Leu 1540 1545 1550 Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys Lys Leu Asn 1555 1560 1565 Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn 1570 1575 1580 Asn Gln Ala Ala Ala Leu Glu Lys Arg Pro Ala Ala Thr Lys Lys Ala 1585 1590 1595 1600 Gly Gln Ala Lys Lys Lys Lys Ser Thr Pro Pro Pro Pro Pro Leu Arg 1605 1610 1615 Ser Gly Cys <210> 74 <211> 4860 <212> DNA <213> Artificial Sequence <220> <223> GFP-FnCpf1 <400> 74 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgagtaa aggagaagaa 120 cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa 180 ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt 240 atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac tttctcttat 300 ggtgttcaat gcttttcaag atacccagat catatgaagc ggcacgactt cttcaagagc 360 gccatgcctg agggatacgt gcaggagagg accatctctt tcaaggacga cgggaactac 420 aagacacgtg ctgaagtcaa gtttgaggga gacaccctcg tcaacaggat cgagcttaag 480 ggaatcgatt tcaaggagga cggaaacatc ctcggccaca agttggaata caactacaac 540 tcccacaacg tatacatcac ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa 600 attagacaca acattgaaga tggaagcgtt caactagcag accattatca acaaaatact 660 ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc 720 ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct 780 gctgggatta cacatggcat ggatgaacta tacaaaggat ccgaattcga gctccgtcga 840 caagcttgcg gccgcatgtc aatttatcaa gaatttgtta ataaatatag tttaagtaaa 900 actctaagat ttgagttaat cccacagggt aaaacacttg aaaacataaa agcaagaggt 960 ttgattttag atgatgagaa aagagctaaa gactacaaaa aggctaaaca aataattgat 1020 aaatatcatc agttttttat agaggagata ttaagttcgg tttgtattag cgaagattta 1080 ttacaaaact attctgatgt ttattttaaa cttaaaaaga gtgatgatga taatctacaa 1140 aaagatttta aaagtgcaaa agatacgata aagaaacaaa tatctgaata tataaaggac 1200 tcagagaaat ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa agggcaagag 1260 tcagatttaa ttctatggct aaagcaatct aaggataatg gtatagaact atttaaagcc 1320 aatagtgata tcacagatat agatgaggcg ttagaaataa tcaaatcttt taaaggttgg 1380 acaacttatt ttaagggttt tcatgaaaat agaaaaaatg tttatagtag caatgatatt 1440 cctacatcta ttatttatag gatagtagat gataatttgc ctaaatttct agaaaataaa 1500 gctaagtatg agagtttaaa agacaaagct ccagaagcta taaactatga acaaattaaa 1560 aaagatttgg cagaagagct aacctttgat attgactaca aaacatctga agttaatcaa 1620 agagtttttt cacttgatga agtttttgag atagcaaact ttaataatta tctaaatcaa 1680 agtggtatta ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg tgaaaataca 1740 aagagaaaag gtataaatga atatataaat ctatactcac agcaaataaa tgataaaaca 1800 ctcaaaaaat ataaaatgag tgttttattt aagcaaattt taagtgatac agaatctaaa 1860 tcttttgtaa ttgataagtt agaagatgat agtgatgtag ttacaacgat gcaaagtttt 1920 tatgagcaaa tagcagcttt taaaacagta gaagaaaaat ctattaaaga aacactatct 1980 ttattatttg atgatttaaa agctcaaaaa cttgatttga gtaaaattta ttttaaaaat 2040 gataaatctc ttactgatct atcacaacaa gtttttgatg attatagtgt tattggtaca 2100 gcggtactag aatatataac tcaacaaata gcacctaaaa atcttgataa ccctagtaag 2160 aaagagcaag aattaatagc caaaaaaact gaaaaagcaa aatacttatc tctagaaact 2220 ataaagcttg ccttagaaga atttaataag catagagata tagataaaca gtgtaggttt 2280 gaagaaatac ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat agctcaaaac 2340 aaagacaatt tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa agacctactt 2400 caagctagtg cggaagatga tgttaaagct atcaaggatc ttttagatca aactaataat 2460 ctcttacata aactaaaaat atttcatatt agtcagtcag aagataaggc aaatatttta 2520 gacaaggatg agcattttta tctagtattt gaggagtgct actttgagct agcgaatata 2580 gtgcctcttt ataacaaaat tagaaactat ataactcaaa agccatatag tgatgagaaa 2640 tttaagctca attttgagaa ctcgactttg gctaatggtt gggataaaaa taaagagcct 2700 gacaatacgg caattttatt tatcaaagat gataaatatt atctgggtgt gatgaataag 2760 aaaaataaca aaatatttga tgataaagct atcaaagaaa ataaaggcga gggttataaa 2820 aaaattgttt ataaactttt acctggcgca aataaaatgt tacctaaggt tttcttttct 2880 gctaaatcta taaaatttta taatcctagt gaagatatac ttagaataag aaatcattcc 2940 acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt taatattgaa 3000 gattgccgaa aatttataga tttttataaa cagtctataa gtaagcatcc ggagtggaaa 3060 gattttggat ttagattttc tgatactcaa agatataatt ctatagatga attttataga 3120 gaagttgaaa atcaaggcta caaactaact tttgaaaata tatcagagag ctatattgat 3180 agcgtagtta atcagggtaa attgtaccta ttccaaatct ataataaaga tttttcagct 3240 tatagcaaag ggcgaccaaa tctacatact ttatattgga aagcgctgtt tgatgagaga 3300 aatcttcaag atgtggttta taagctaaat ggtgaggcag agctttttta tcgtaaacaa 3360 tcaataccta aaaaaatcac tcacccagct aaagaggcaa tagctaataa aaacaaagat 3420 aatcctaaaa aagagagtgt ttttgaatat gatttaatca aagataaacg ctttactgaa 3480 gataagtttt tctttcactg tcctattaca atcaatttta aatctagtgg agctaataag 3540 tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa atgatgttca tatattaagt 3600 atagatagag gtgaaagaca tttagcttac tatactttgg tagatggtaa aggcaatatc 3660 atcaaacaag atactttcaa catcattggt aatgatagaa tgaaaacaaa ctaccatgat 3720 aagcttgctg caatagagaa agatagggat tcagctagga aagactggaa aaagataaat 3780 aacatcaaag agatgaaaga gggctatcta tctcaggtag ttcatgaaat agctaagcta 3840 gttatagagt ataatgctat tgtggttttt gaggatttaa attttggatt taaaagaggg 3900 cgtttcaagg tagagaagca ggtctatcaa aagttagaaa aaatgctaat tgagaaacta 3960 aactatctag ttttcaaaga taatgagttt gataaaactg ggggagtgct tagagcttat 4020 cagctaacag caccttttga gacttttaaa aagatgggta aacaaacagg tattatctac 4080 tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt aaatcagtta 4140 tatcctaagt atgaaagtgt cagcaaatct caagagttct ttagtaagtt tgacaagatt 4200 tgttataacc ttgataaggg ctattttgag tttagttttg attataaaaa ctttggtgac 4260 aaggctgcca aaggcaagtg gactatagct agctttggga gtagattgat taactttaga 4320 aattcagata aaaatcataa ttgggatact cgagaagttt atccaactaa agagttggag 4380 aaattgctaa aagattattc tatcgaatat gggcatggcg aatgtatcaa agcagctatt 4440 tgcggtgaga gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa tactatctta 4500 caaatgcgta actcaaaaac aggtactgag ttagattatc taatttcacc agtagcagat 4560 gtaaatggca atttctttga ttcgcgacag gcgccaaaaa atatgcctca agatgctgat 4620 gccaatggtg cttatcatat tgggctaaaa ggtctgatgc tactaggtag gatcaaaaat 4680 aatcaagagg gcaaaaaact caatttggtt atcaaaaatg aagagtattt tgagttcgtg 4740 cagaatagga ataaccaagc ggccgcactc gagaaaaggc cggcggccac gaaaaaggcc 4800 ggccaggcaa aaaagaaaaa gtcgacacca ccaccaccac cactgagatc cggctgctaa 4860 4860 <210> 75 <211> 1497 <212> PRT <213> Artificial Sequence <220> <223> SSB-FnCpf1 <400> 75 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Ala Ser Arg Gly Val Asn Lys Val Ile Leu Val Gly Asn 35 40 45 Leu Gly Gln Asp Pro Glu Val Arg Tyr Met Pro Asn Gly Gly Ala Val 50 55 60 Ala Asn Ile Thr Leu Ala Thr Ser Glu Ser Trp Arg Asp Lys Ala Thr 65 70 75 80 Gly Glu Met Lys Glu Gln Thr Glu Trp His Arg Val Val Leu Phe Gly 85 90 95 Lys Leu Ala Glu Val Ala Ser Glu Tyr Leu Arg Lys Gly Ser Gln Val 100 105 110 Tyr Ile Glu Gly Gln Leu Arg Thr Arg Lys Trp Thr Asp Gln Ser Gly 115 120 125 Gln Asp Arg Tyr Thr Thr Glu Val Val Val Asn Val Gly Gly Thr Met 130 135 140 Gln Met Leu Gly Gly Arg Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala 145 150 155 160 Cys Gly Arg Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu 165 170 175 Ser Lys Thr Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu 180 185 190 Asn Ile Lys Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys 195 200 205 Asp Tyr Lys Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe 210 215 220 Ile Glu Glu Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln 225 230 235 240 Asn Tyr Ser Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn 245 250 255 Leu Gln Lys Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile 260 265 270 Ser Glu Tyr Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln 275 280 285 Asn Leu Ile Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp 290 295 300 Leu Lys Gln Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser 305 310 315 320 Asp Ile Thr Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys 325 330 335 Gly Trp Thr Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val 340 345 350 Tyr Ser Ser Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp 355 360 365 Asp Asn Leu Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu 370 375 380 Lys Asp Lys Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp 385 390 395 400 Leu Ala Glu Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val 405 410 415 Asn Gln Arg Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe 420 425 430 Asn Asn Tyr Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile 435 440 445 Gly Gly Lys Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn 450 455 460 Glu Tyr Ile Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys 465 470 475 480 Lys Tyr Lys Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu 485 490 495 Ser Lys Ser Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val 500 505 510 Thr Thr Met Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val 515 520 525 Glu Glu Lys Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu 530 535 540 Lys Ala Gln Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys 545 550 555 560 Ser Leu Thr Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile 565 570 575 Gly Thr Ala Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn 580 585 590 Leu Asp Asn Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr 595 600 605 Glu Lys Ala Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu 610 615 620 Glu Phe Asn Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu 625 630 635 640 Ile Leu Ala Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala 645 650 655 Gln Asn Lys Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln 660 665 670 Gly Lys Lys Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala 675 680 685 Ile Lys Asp Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys 690 695 700 Ile Phe His Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys 705 710 715 720 Asp Glu His Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala 725 730 735 Asn Ile Val Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys 740 745 750 Pro Tyr Ser Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu 755 760 765 Ala Asn Gly Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu 770 775 780 Phe Ile Lys Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn 785 790 795 800 Asn Lys Ile Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly 805 810 815 Tyr Lys Lys Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu 820 825 830 Pro Lys Val Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser 835 840 845 Glu Asp Ile Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly 850 855 860 Ser Pro Gln Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys 865 870 875 880 Arg Lys Phe Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu 885 890 895 Trp Lys Asp Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser 900 905 910 Ile Asp Glu Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr 915 920 925 Phe Glu Asn Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly 930 935 940 Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser 945 950 955 960 Lys Gly Arg Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp 965 970 975 Glu Arg Asn Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu 980 985 990 Leu Phe Tyr Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala 995 1000 1005 Lys Glu Ala Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser 1010 1015 1020 Val Phe Glu Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys 1025 1030 1035 1040 Phe Phe Phe His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala 1045 1050 1055 Asn Lys Phe Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn 1060 1065 1070 Asp Val His Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr 1075 1080 1085 Tyr Thr Leu Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe 1090 1095 1100 Asn Ile Ile Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu 1105 1110 1115 1120 Ala Ala Ile Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys 1125 1130 1135 Ile Asn Asn Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val 1140 1145 1150 His Glu Ile Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe 1155 1160 1165 Glu Asp Leu Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys 1170 1175 1180 Gln Val Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr 1185 1190 1195 1200 Leu Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg 1205 1210 1215 Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly Lys 1220 1225 1230 Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile 1235 1240 1245 Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser 1250 1255 1260 Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr 1265 1270 1275 1280 Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe 1285 1290 1295 Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser 1300 1305 1310 Arg Leu Ile Asn Phe Arg Asn Ser Asp Lys Asn His Asn Trp Asp Thr 1315 1320 1325 Arg Glu Val Tyr Pro Thr Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr 1330 1335 1340 Ser Ile Glu Tyr Gly His Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly 1345 1350 1355 1360 Glu Ser Asp Lys Lys Phe Phe Ala Lys Leu Thr Ser Val Leu Asn Thr 1365 1370 1375 Ile Leu Gln Met Arg Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu 1380 1385 1390 Ile Ser Pro Val Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln 1395 1400 1405 Ala Pro Lys Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His 1410 1415 1420 Ile Gly Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln 1425 1430 1435 1440 Glu Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu 1445 1450 1455 Phe Val Gln Asn Arg Asn Asn Gln Ala Ala Ala Leu Glu Lys Arg Pro 1460 1465 1470 Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Ser Thr Pro 1475 1480 1485 Pro Pro Pro Pro Leu Arg Ser Gly Cys 1490 1495 <210> 76 <211> 4494 <212> DNA <213> Artificial Sequence <220> <223> SSB-FnCpf1 <400> 76 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggccag cagaggcgta 120 aacaaggtta ttctcgttgg taatctgggt caggacccgg aagtacgcta catgccaaat 180 ggtggcgcag ttgccaacat tacgctggct acttccgaat cctggcgtga taaagcgacc 240 ggcgagatga aagaacagac tgaatggcac cgcgttgtgc tgttcggcaa actggcagaa 300 gtggcgagcg aatatctgcg taaaggttct caggtttata tcgaaggtca gctgcgtacc 360 cgtaaatgga ccgatcaatc cggtcaggat cgctacacca cagaagtcgt ggtgaacgtt 420 ggcggcacca tgcagatgct gggtggtcgt ggatccgaat tcgagctccg tcgacaagct 480 tgcggccgca tgtcaattta tcaagaattt gttaataaat atagtttaag taaaactcta 540 agatttgagt taatcccaca gggtaaaaca cttgaaaaca taaaagcaag aggtttgatt 600 ttagatgatg agaaaagagc taaagactac aaaaaggcta aacaaataat tgataaatat 660 catcagtttt ttatagagga gatattaagt tcggtttgta ttagcgaaga tttattacaa 720 aactattctg atgtttattt taaacttaaa aagagtgatg atgataatct acaaaaagat 780 tttaaaagtg caaaagatac gataaagaaa caaatatctg aatatataaa ggactcagag 840 aaatttaaga atttgtttaa tcaaaacctt atcgatgcta aaaaagggca agagtcagat 900 ttaattctat ggctaaagca atctaaggat aatggtatag aactatttaa agccaatagt 960 gatatcacag atatagatga ggcgttagaa ataatcaaat cttttaaagg ttggacaact 1020 tattttaagg gttttcatga aaatagaaaa aatgtttata gtagcaatga tattcctaca 1080 tctattattt ataggatagt agatgataat ttgcctaaat ttctagaaaa taaagctaag 1140 tatgagagtt taaaagacaa agctccagaa gctataaact atgaacaaat taaaaaagat 1200 ttggcagaag agctaacctt tgatattgac tacaaaacat ctgaagttaa tcaaagagtt 1260 ttttcacttg atgaagtttt tgagatagca aactttaata attatctaaa tcaaagtggt 1320 attactaaat ttaatactat tattggtggt aaatttgtaa atggtgaaaa tacaaagaga 1380 aaaggtataa atgaatatat aaatctatac tcacagcaaa taaatgataa aacactcaaa 1440 aaatataaaa tgagtgtttt atttaagcaa attttaagtg atacagaatc taaatctttt 1500 gtaattgata agttagaaga tgatagtgat gtagttacaa cgatgcaaag tttttatgag 1560 caaatagcag cttttaaaac agtagaagaa aaatctatta aagaaacact atctttatta 1620 tttgatgatt taaaagctca aaaacttgat ttgagtaaaa tttattttaa aaatgataaa 1680 tctcttactg atctatcaca acaagttttt gatgattata gtgttattgg tacagcggta 1740 ctagaatata taactcaaca aatagcacct aaaaatcttg ataaccctag taagaaagag 1800 caagaattaa tagccaaaaa aactgaaaaa gcaaaatact tatctctaga aactataaag 1860 cttgccttag aagaatttaa taagcataga gatatagata aacagtgtag gtttgaagaa 1920 atacttgcaa actttgcggc tattccgatg atatttgatg aaatagctca aaacaaagac 1980 aatttggcac agatatctat caaatatcaa aatcaaggta aaaaagacct acttcaagct 2040 agtgcggaag atgatgttaa agctatcaag gatcttttag atcaaactaa taatctctta 2100 cataaactaa aaatatttca tattagtcag tcagaagata aggcaaatat tttagacaag 2160 gatgagcatt tttatctagt atttgaggag tgctactttg agctagcgaa tatagtgcct 2220 ctttataaca aaattagaaa ctatataact caaaagccat atagtgatga gaaatttaag 2280 ctcaattttg agaactcgac tttggctaat ggttgggata aaaataaaga gcctgacaat 2340 acggcaattt tatttatcaa agatgataaa tattatctgg gtgtgatgaa taagaaaaat 2400 aacaaaatat ttgatgataa agctatcaaa gaaaataaag gcgagggtta taaaaaaatt 2460 gtttataaac ttttacctgg cgcaaataaa atgttaccta aggttttctt ttctgctaaa 2520 tctataaaat tttataatcc tagtgaagat atacttagaa taagaaatca ttccacacat 2580 acaaaaaatg gtagtcctca aaaaggatat gaaaaatttg agtttaatat tgaagattgc 2640 cgaaaattta tagattttta taaacagtct ataagtaagc atccggagtg gaaagatttt 2700 ggatttagat tttctgatac tcaaagatat aattctatag atgaatttta tagagaagtt 2760 gaaaatcaag gctacaaact aacttttgaa aatatatcag agagctatat tgatagcgta 2820 gttaatcagg gtaaattgta cctattccaa atctataata aagatttttc agcttatagc 2880 aaagggcgac caaatctaca tactttatat tggaaagcgc tgtttgatga gagaaatctt 2940 caagatgtgg tttataagct aaatggtgag gcagagcttt tttatcgtaa acaatcaata 3000 cctaaaaaaa tcactcaccc agctaaagag gcaatagcta ataaaaacaa agataatcct 3060 aaaaaagaga gtgtttttga atatgattta atcaaagata aacgctttac tgaagataag 3120 tttttctttc actgtcctat tacaatcaat tttaaatcta gtggagctaa taagtttaat 3180 gatgaaatca atttattgct aaaagaaaaa gcaaatgatg ttcatatatt aagtatagat 3240 agaggtgaaa gacatttagc ttactatact ttggtagatg gtaaaggcaa tatcatcaaa 3300 caagatactt tcaacatcat tggtaatgat agaatgaaaa caaactacca tgataagctt 3360 gctgcaatag agaaagatag ggattcagct aggaaagact ggaaaaagat aaataacatc 3420 aaagagatga aagagggcta tctatctcag gtagttcatg aaatagctaa gctagttata 3480 gagtataatg ctattgtggt ttttgaggat ttaaattttg gatttaaaag agggcgtttc 3540 aaggtagaga agcaggtcta tcaaaagtta gaaaaaatgc taattgagaa actaaactat 3600 ctagttttca aagataatga gtttgataaa actgggggag tgcttagagc ttatcagcta 3660 acagcacctt ttgagacttt taaaaagatg ggtaaacaaa caggtattat ctactatgta 3720 ccagctggtt ttacttcaaa aatttgtcct gtaactggtt ttgtaaatca gttatatcct 3780 aagtatgaaa gtgtcagcaa atctcaagag ttctttagta agtttgacaa gatttgttat 3840 aaccttgata agggctattt tgagtttagt tttgattata aaaactttgg tgacaaggct 3900 gccaaaggca agtggactat agctagcttt gggagtagat tgattaactt tagaaattca 3960 gataaaaatc ataattggga tactcgagaa gtttatccaa ctaaagagtt ggagaaattg 4020 ctaaaagatt attctatcga atatgggcat ggcgaatgta tcaaagcagc tatttgcggt 4080 gagagcgaca aaaagttttt tgctaagcta actagtgtcc taaatactat cttacaaatg 4140 cgtaactcaa aaacaggtac tgagttagat tatctaattt caccagtagc agatgtaaat 4200 ggcaatttct ttgattcgcg acaggcgcca aaaaatatgc ctcaagatgc tgatgccaat 4260 ggtgcttatc atattgggct aaaaggtctg atgctactag gtaggatcaa aaataatcaa 4320 gagggcaaaa aactcaattt ggttatcaaa aatgaagagt attttgagtt cgtgcagaat 4380 aggaataacc aagcggccgc actcgagaaa aggccggcgg ccacgaaaaa ggccggccag 4440 gcaaaaaaga aaaagtcgac accaccacca ccaccactga gatccggctg ctaa 4494 <210> 77 <211> 2072 <212> PRT <213> Artificial Sequence <220> <223> SSB-FnCpf1-RecJ <400> 77 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Ala Ser Arg Gly Val Asn Lys Val Ile Leu Val Gly Asn 35 40 45 Leu Gly Gln Asp Pro Glu Val Arg Tyr Met Pro Asn Gly Gly Ala Val 50 55 60 Ala Asn Ile Thr Leu Ala Thr Ser Glu Ser Trp Arg Asp Lys Ala Thr 65 70 75 80 Gly Glu Met Lys Glu Gln Thr Glu Trp His Arg Val Val Leu Phe Gly 85 90 95 Lys Leu Ala Glu Val Ala Ser Glu Tyr Leu Arg Lys Gly Ser Gln Val 100 105 110 Tyr Ile Glu Gly Gln Leu Arg Thr Arg Lys Trp Thr Asp Gln Ser Gly 115 120 125 Gln Asp Arg Tyr Thr Thr Glu Val Val Val Asn Val Gly Gly Thr Met 130 135 140 Gln Met Leu Gly Gly Arg Gly Ser Glu Phe Glu Leu Arg Arg Gln Ala 145 150 155 160 Cys Gly Arg Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu 165 170 175 Ser Lys Thr Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu 180 185 190 Asn Ile Lys Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys 195 200 205 Asp Tyr Lys Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe 210 215 220 Ile Glu Glu Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln 225 230 235 240 Asn Tyr Ser Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn 245 250 255 Leu Gln Lys Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile 260 265 270 Ser Glu Tyr Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln 275 280 285 Asn Leu Ile Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp 290 295 300 Leu Lys Gln Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser 305 310 315 320 Asp Ile Thr Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys 325 330 335 Gly Trp Thr Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val 340 345 350 Tyr Ser Ser Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp 355 360 365 Asp Asn Leu Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu 370 375 380 Lys Asp Lys Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp 385 390 395 400 Leu Ala Glu Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val 405 410 415 Asn Gln Arg Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe 420 425 430 Asn Asn Tyr Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile 435 440 445 Gly Gly Lys Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn 450 455 460 Glu Tyr Ile Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys 465 470 475 480 Lys Tyr Lys Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu 485 490 495 Ser Lys Ser Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val 500 505 510 Thr Thr Met Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val 515 520 525 Glu Glu Lys Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu 530 535 540 Lys Ala Gln Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys 545 550 555 560 Ser Leu Thr Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile 565 570 575 Gly Thr Ala Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn 580 585 590 Leu Asp Asn Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr 595 600 605 Glu Lys Ala Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu 610 615 620 Glu Phe Asn Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu 625 630 635 640 Ile Leu Ala Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala 645 650 655 Gln Asn Lys Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln 660 665 670 Gly Lys Lys Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala 675 680 685 Ile Lys Asp Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys 690 695 700 Ile Phe His Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys 705 710 715 720 Asp Glu His Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala 725 730 735 Asn Ile Val Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys 740 745 750 Pro Tyr Ser Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu 755 760 765 Ala Asn Gly Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu 770 775 780 Phe Ile Lys Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn 785 790 795 800 Asn Lys Ile Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly 805 810 815 Tyr Lys Lys Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu 820 825 830 Pro Lys Val Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser 835 840 845 Glu Asp Ile Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly 850 855 860 Ser Pro Gln Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys 865 870 875 880 Arg Lys Phe Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu 885 890 895 Trp Lys Asp Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser 900 905 910 Ile Asp Glu Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr 915 920 925 Phe Glu Asn Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly 930 935 940 Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser 945 950 955 960 Lys Gly Arg Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp 965 970 975 Glu Arg Asn Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu 980 985 990 Leu Phe Tyr Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala 995 1000 1005 Lys Glu Ala Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser 1010 1015 1020 Val Phe Glu Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys 1025 1030 1035 1040 Phe Phe Phe His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala 1045 1050 1055 Asn Lys Phe Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn 1060 1065 1070 Asp Val His Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr 1075 1080 1085 Tyr Thr Leu Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe 1090 1095 1100 Asn Ile Ile Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu 1105 1110 1115 1120 Ala Ala Ile Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys 1125 1130 1135 Ile Asn Asn Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val 1140 1145 1150 His Glu Ile Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe 1155 1160 1165 Glu Asp Leu Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys 1170 1175 1180 Gln Val Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr 1185 1190 1195 1200 Leu Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg 1205 1210 1215 Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly Lys 1220 1225 1230 Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile 1235 1240 1245 Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser 1250 1255 1260 Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr 1265 1270 1275 1280 Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe 1285 1290 1295 Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser 1300 1305 1310 Arg Leu Ile Asn Phe Arg Asn Ser Asp Lys Asn His Asn Trp Asp Thr 1315 1320 1325 Arg Glu Val Tyr Pro Thr Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr 1330 1335 1340 Ser Ile Glu Tyr Gly His Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly 1345 1350 1355 1360 Glu Ser Asp Lys Lys Phe Phe Ala Lys Leu Thr Ser Val Leu Asn Thr 1365 1370 1375 Ile Leu Gln Met Arg Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu 1380 1385 1390 Ile Ser Pro Val Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln 1395 1400 1405 Ala Pro Lys Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His 1410 1415 1420 Ile Gly Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln 1425 1430 1435 1440 Glu Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu 1445 1450 1455 Phe Val Gln Asn Arg Asn Asn Gln Ala Ala Ala Leu Asp Leu Gln Val 1460 1465 1470 Lys Gln Gln Ile Gln Leu Arg Arg Arg Glu Val Asp Glu Thr Ala Asp 1475 1480 1485 Leu Pro Ala Glu Leu Pro Pro Leu Leu Arg Arg Leu Tyr Ala Ser Arg 1490 1495 1500 Gly Val Arg Ser Ala Gln Glu Leu Glu Arg Ser Val Lys Gly Met Leu 1505 1510 1515 1520 Pro Trp Gln Gln Leu Ser Gly Val Glu Lys Ala Val Glu Ile Leu Tyr 1525 1530 1535 Asn Ala Phe Arg Glu Gly Thr Arg Ile Ile Val Val Gly Asp Phe Asp 1540 1545 1550 Ala Asp Gly Ala Thr Ser Thr Ala Leu Ser Val Leu Ala Met Arg Ser 1555 1560 1565 Leu Gly Cys Ser Asn Ile Asp Tyr Leu Val Pro Asn Arg Phe Glu Asp 1570 1575 1580 Gly Tyr Gly Leu Ser Pro Glu Val Val Asp Gln Ala His Ala Arg Gly 1585 1590 1595 1600 Ala Gln Leu Ile Val Thr Val Asp Asn Gly Ile Ser Ser His Ala Gly 1605 1610 1615 Val Glu His Ala Arg Ser Leu Gly Ile Pro Val Ile Val Thr Asp His 1620 1625 1630 His Leu Pro Gly Asp Thr Leu Pro Ala Ala Glu Ala Ile Ile Asn Pro 1635 1640 1645 Asn Leu Arg Asp Cys Asn Phe Pro Ser Lys Ser Leu Ala Gly Val Gly 1650 1655 1660 Val Ala Phe Tyr Leu Met Leu Ala Leu Arg Thr Phe Leu Arg Asp Gln 1665 1670 1675 1680 Gly Trp Phe Asp Glu Arg Asn Ile Ala Ile Pro Asn Leu Ala Glu Leu 1685 1690 1695 Leu Asp Leu Val Ala Leu Gly Thr Val Ala Asp Val Val Pro Leu Asp 1700 1705 1710 Ala Asn Asn Arg Ile Leu Thr Trp Gln Gly Met Ser Arg Ile Arg Ala 1715 1720 1725 Gly Lys Cys Arg Pro Gly Ile Lys Ala Leu Leu Glu Val Ala Asn Arg 1730 1735 1740 Asp Ala Gln Lys Leu Ala Ala Ser Asp Leu Gly Phe Ala Leu Gly Pro 1745 1750 1755 1760 Arg Leu Asn Ala Ala Gly Arg Leu Asp Asp Met Ser Val Gly Val Ala 1765 1770 1775 Leu Leu Leu Cys Asp Asn Ile Gly Glu Ala Arg Val Leu Ala Asn Glu 1780 1785 1790 Leu Asp Ala Leu Asn Gln Thr Arg Lys Glu Ile Glu Gln Gly Met Gln 1795 1800 1805 Ile Glu Ala Leu Thr Leu Cys Glu Lys Leu Glu Arg Ser Arg Asp Thr 1810 1815 1820 Leu Pro Gly Gly Leu Ala Met Tyr His Pro Glu Trp His Gln Gly Val 1825 1830 1835 1840 Val Gly Ile Leu Ala Ser Arg Ile Lys Glu Arg Phe His Arg Pro Val 1845 1850 1855 Ile Ala Phe Ala Pro Ala Gly Asp Gly Thr Leu Lys Gly Ser Gly Arg 1860 1865 1870 Ser Ile Gln Gly Leu His Met Arg Asp Ala Leu Glu Arg Leu Asp Thr 1875 1880 1885 Leu Tyr Pro Gly Met Met Leu Lys Phe Gly Gly His Ala Met Ala Ala 1890 1895 1900 Gly Leu Ser Leu Glu Glu Asp Lys Phe Lys Leu Phe Gln Gln Arg Phe 1905 1910 1915 1920 Gly Glu Leu Val Thr Glu Trp Leu Asp Pro Ser Leu Leu Gln Gly Glu 1925 1930 1935 Val Val Ser Asp Gly Pro Leu Ser Pro Ala Glu Met Thr Met Glu Val 1940 1945 1950 Ala Gln Leu Leu Arg Asp Ala Gly Pro Trp Gly Gln Met Phe Pro Glu 1955 1960 1965 Pro Leu Phe Asp Gly His Phe Arg Leu Leu Gln Gln Arg Leu Val Gly 1970 1975 1980 Glu Arg His Leu Lys Val Met Val Glu Pro Val Gly Gly Gly Pro Leu 1985 1990 1995 2000 Leu Asp Gly Ile Ala Phe Asn Val Asp Thr Ala Leu Trp Pro Asp Asn 2005 2010 2015 Gly Val Arg Glu Val Gln Leu Ala Tyr Lys Leu Asp Ile Asn Glu Phe 2020 2025 2030 Arg Gly Asn Arg Ser Leu Gln Ile Ile Ile Asp Asn Ile Trp Pro Ile 2035 2040 2045 Leu Gln Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys 2050 2055 2060 Lys Lys His His His His His His 2065 2070 <210> 78 <211> 6219 <212> DNA <213> Artificial Sequence <220> <223> SSB-FnCpf1-RecJ <400> 78 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggccag cagaggcgta 120 aacaaggtta ttctcgttgg taatctgggt caggacccgg aagtacgcta catgccaaat 180 ggtggcgcag ttgccaacat tacgctggct acttccgaat cctggcgtga taaagcgacc 240 ggcgagatga aagaacagac tgaatggcac cgcgttgtgc tgttcggcaa actggcagaa 300 gtggcgagcg aatatctgcg taaaggttct caggtttata tcgaaggtca gctgcgtacc 360 cgtaaatgga ccgatcaatc cggtcaggat cgctacacca cagaagtcgt ggtgaacgtt 420 ggcggcacca tgcagatgct gggtggtcgt ggatccgaat tcgagctccg tcgacaagct 480 tgcggccgca tgtcaattta tcaagaattt gttaataaat atagtttaag taaaactcta 540 agatttgagt taatcccaca gggtaaaaca cttgaaaaca taaaagcaag aggtttgatt 600 ttagatgatg agaaaagagc taaagactac aaaaaggcta aacaaataat tgataaatat 660 catcagtttt ttatagagga gatattaagt tcggtttgta ttagcgaaga tttattacaa 720 aactattctg atgtttattt taaacttaaa aagagtgatg atgataatct acaaaaagat 780 tttaaaagtg caaaagatac gataaagaaa caaatatctg aatatataaa ggactcagag 840 aaatttaaga atttgtttaa tcaaaacctt atcgatgcta aaaaagggca agagtcagat 900 ttaattctat ggctaaagca atctaaggat aatggtatag aactatttaa agccaatagt 960 gatatcacag atatagatga ggcgttagaa ataatcaaat cttttaaagg ttggacaact 1020 tattttaagg gttttcatga aaatagaaaa aatgtttata gtagcaatga tattcctaca 1080 tctattattt ataggatagt agatgataat ttgcctaaat ttctagaaaa taaagctaag 1140 tatgagagtt taaaagacaa agctccagaa gctataaact atgaacaaat taaaaaagat 1200 ttggcagaag agctaacctt tgatattgac tacaaaacat ctgaagttaa tcaaagagtt 1260 ttttcacttg atgaagtttt tgagatagca aactttaata attatctaaa tcaaagtggt 1320 attactaaat ttaatactat tattggtggt aaatttgtaa atggtgaaaa tacaaagaga 1380 aaaggtataa atgaatatat aaatctatac tcacagcaaa taaatgataa aacactcaaa 1440 aaatataaaa tgagtgtttt atttaagcaa attttaagtg atacagaatc taaatctttt 1500 gtaattgata agttagaaga tgatagtgat gtagttacaa cgatgcaaag tttttatgag 1560 caaatagcag cttttaaaac agtagaagaa aaatctatta aagaaacact atctttatta 1620 tttgatgatt taaaagctca aaaacttgat ttgagtaaaa tttattttaa aaatgataaa 1680 tctcttactg atctatcaca acaagttttt gatgattata gtgttattgg tacagcggta 1740 ctagaatata taactcaaca aatagcacct aaaaatcttg ataaccctag taagaaagag 1800 caagaattaa tagccaaaaa aactgaaaaa gcaaaatact tatctctaga aactataaag 1860 cttgccttag aagaatttaa taagcataga gatatagata aacagtgtag gtttgaagaa 1920 atacttgcaa actttgcggc tattccgatg atatttgatg aaatagctca aaacaaagac 1980 aatttggcac agatatctat caaatatcaa aatcaaggta aaaaagacct acttcaagct 2040 agtgcggaag atgatgttaa agctatcaag gatcttttag atcaaactaa taatctctta 2100 cataaactaa aaatatttca tattagtcag tcagaagata aggcaaatat tttagacaag 2160 gatgagcatt tttatctagt atttgaggag tgctactttg agctagcgaa tatagtgcct 2220 ctttataaca aaattagaaa ctatataact caaaagccat atagtgatga gaaatttaag 2280 ctcaattttg agaactcgac tttggctaat ggttgggata aaaataaaga gcctgacaat 2340 acggcaattt tatttatcaa agatgataaa tattatctgg gtgtgatgaa taagaaaaat 2400 aacaaaatat ttgatgataa agctatcaaa gaaaataaag gcgagggtta taaaaaaatt 2460 gtttataaac ttttacctgg cgcaaataaa atgttaccta aggttttctt ttctgctaaa 2520 tctataaaat tttataatcc tagtgaagat atacttagaa taagaaatca ttccacacat 2580 acaaaaaatg gtagtcctca aaaaggatat gaaaaatttg agtttaatat tgaagattgc 2640 cgaaaattta tagattttta taaacagtct ataagtaagc atccggagtg gaaagatttt 2700 ggatttagat tttctgatac tcaaagatat aattctatag atgaatttta tagagaagtt 2760 gaaaatcaag gctacaaact aacttttgaa aatatatcag agagctatat tgatagcgta 2820 gttaatcagg gtaaattgta cctattccaa atctataata aagatttttc agcttatagc 2880 aaagggcgac caaatctaca tactttatat tggaaagcgc tgtttgatga gagaaatctt 2940 caagatgtgg tttataagct aaatggtgag gcagagcttt tttatcgtaa acaatcaata 3000 cctaaaaaaa tcactcaccc agctaaagag gcaatagcta ataaaaacaa agataatcct 3060 aaaaaagaga gtgtttttga atatgattta atcaaagata aacgctttac tgaagataag 3120 tttttctttc actgtcctat tacaatcaat tttaaatcta gtggagctaa taagtttaat 3180 gatgaaatca atttattgct aaaagaaaaa gcaaatgatg ttcatatatt aagtatagat 3240 agaggtgaaa gacatttagc ttactatact ttggtagatg gtaaaggcaa tatcatcaaa 3300 caagatactt tcaacatcat tggtaatgat agaatgaaaa caaactacca tgataagctt 3360 gctgcaatag agaaagatag ggattcagct aggaaagact ggaaaaagat aaataacatc 3420 aaagagatga aagagggcta tctatctcag gtagttcatg aaatagctaa gctagttata 3480 gagtataatg ctattgtggt ttttgaggat ttaaattttg gatttaaaag agggcgtttc 3540 aaggtagaga agcaggtcta tcaaaagtta gaaaaaatgc taattgagaa actaaactat 3600 ctagttttca aagataatga gtttgataaa actgggggag tgcttagagc ttatcagcta 3660 acagcacctt ttgagacttt taaaaagatg ggtaaacaaa caggtattat ctactatgta 3720 ccagctggtt ttacttcaaa aatttgtcct gtaactggtt ttgtaaatca gttatatcct 3780 aagtatgaaa gtgtcagcaa atctcaagag ttctttagta agtttgacaa gatttgttat 3840 aaccttgata agggctattt tgagtttagt tttgattata aaaactttgg tgacaaggct 3900 gccaaaggca agtggactat agctagcttt gggagtagat tgattaactt tagaaattca 3960 gataaaaatc ataattggga tactcgagaa gtttatccaa ctaaagagtt ggagaaattg 4020 ctaaaagatt attctatcga atatgggcat ggcgaatgta tcaaagcagc tatttgcggt 4080 gagagcgaca aaaagttttt tgctaagcta actagtgtcc taaatactat cttacaaatg 4140 cgtaactcaa aaacaggtac tgagttagat tatctaattt caccagtagc agatgtaaat 4200 ggcaatttct ttgattcgcg acaggcgcca aaaaatatgc ctcaagatgc tgatgccaat 4260 ggtgcttatc atattgggct aaaaggtctg atgctactag gtaggatcaa aaataatcaa 4320 gagggcaaaa aactcaattt ggttatcaaa aatgaagagt attttgagtt cgtgcagaat 4380 aggaataacc aagcggccgc actcgacctg caggtgaaac aacagataca acttcgtcgc 4440 cgtgaagtcg atgaaacggc agacttgccc gctgaattgc ctcccttgct gcgccgttta 4500 tacgccagcc ggggagtacg cagtgcgcaa gaactggaac gcagtgttaa aggtatgctg 4560 ccctggcagc aactgagcgg cgtcgaaaag gccgttgaga tcctttacaa cgcttttcgc 4620 gaaggaacgc ggattattgt ggtcggtgat ttcgacgccg acggcgcgac cagcacggct 4680 ctaagcgtgc tggcgatgcg ctcgcttggt tgcagcaata tcgactacct ggtaccaaac 4740 cgtttcgaag acggttacgg cttaagcccg gaagtggtcg atcaggccca tgcccgtggc 4800 gcgcagttaa ttgtcacggt ggataacggt atttcctccc atgcgggggt tgagcacgct 4860 cgctcgttgg gcatcccggt tattgttacc gatcaccatt tgccaggcga cacattaccc 4920 gcagcggaag cgatcattaa ccctaacttg cgcgactgta atttcccgtc gaaatcactg 4980 gcaggcgtgg gtgtggcgtt ttatctgatg ctggcgctgc gcaccttttt gcgcgatcag 5040 ggctggtttg atgagcgtaa catcgcaatt cctaacctgg cagaactgct ggatctggtc 5100 gcgctgggga cagtggcgga cgtcgtgccg ctggacgcta ataatcgcat tctgacctgg 5160 caggggatga gtcgcatccg agccggaaag tgccgtccgg ggattaaagc gctgcttgaa 5220 gtggcaaacc gtgatgcaca aaaactcgcc gccagcgatt taggttttgc gctggggcca 5280 cgtctcaatg ctgccggacg actggacgat atgtccgtcg gtgtggcgct gttgttgtgc 5340 gacaacatcg gcgaagcgcg cgtgctggca aatgaactcg atgcgctaaa ccagacgcga 5400 aaagagatcg aacaaggaat gcaaattgaa gccctgaccc tgtgcgagaa actggagcgc 5460 agccgtgaca cgctacccgg cgggctggca atgtatcacc ccgaatggca tcagggcgtt 5520 gtcggtattc tggcttcgcg catcaaagag cgttttcacc gtccggttat cgcgtttgcg 5580 ccagcaggtg acggtacgct gaaaggttcc ggtcgctcca ttcaggggct gcatatgcgt 5640 gatgcgctgg agcgattaga cacactctac cctggcatga tgctgaagtt tggcggtcat 5700 gcgatggcgg cgggtttgtc gctggaagag gataaattca aactctttca acaacggttt 5760 ggcgaactgg ttactgagtg gctggaccct tcgctattgc aaggcgaagt ggtatcagac 5820 ggtccgttaa gcccggccga aatgaccatg gaagtggcgc agctgctgcg cgatgctggc 5880 ccgtgggggc agatgttccc ggagccgctg tttgacggtc atttccgtct gctgcaacag 5940 cggctggtgg gcgaacgtca tttgaaggtg atggtcgaac cggtcggcgg cggtccactg 6000 ctggatggta ttgcttttaa tgtcgatacc gccctctggc cggataacgg cgtgcgcgaa 6060 gtgcaactgg cttataagct cgatatcaac gagtttcgcg gcaaccgcag cctgcaaatt 6120 atcatcgaca atatctggcc aattctgcag aaaaggccgg cggccacgaa aaaggccggc 6180 caggcaaaaa agaaaaagca ccaccaccac caccactga 6219 <210> 79 <211> 1470 <212> PRT <213> Artificial Sequence <220> <223> DSB-FnCpf1 <400> 79 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Met Ala Lys Lys Glu Met Val Glu Phe Asp Glu Ala Ile His 35 40 45 Gly Glu Asp Leu Ala Lys Phe Ile Lys Glu Ala Ser Asp His Lys Leu 50 55 60 Lys Ile Ser Gly Tyr Asn Glu Leu Ile Lys Asp Ile Arg Ile Arg Ala 65 70 75 80 Lys Asp Glu Leu Gly Val Asp Gly Lys Met Phe Asn Arg Leu Leu Ala 85 90 95 Leu Tyr His Lys Asp Asn Arg Asp Val Phe Glu Ala Glu Thr Glu Glu 100 105 110 Val Val Glu Leu Tyr Asp Thr Val Phe Ser Lys Gly Ser Glu Phe Glu 115 120 125 Leu Arg Arg Gln Ala Cys Gly Arg Met Ser Ile Tyr Gln Glu Phe Val 130 135 140 Asn Lys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Glu Leu Ile Pro Gln 145 150 155 160 Gly Lys Thr Leu Glu Asn Ile Lys Ala Arg Gly Leu Ile Leu Asp Asp 165 170 175 Glu Lys Arg Ala Lys Asp Tyr Lys Lys Ala Lys Gln Ile Ile Asp Lys 180 185 190 Tyr His Gln Phe Phe Ile Glu Glu Ile Leu Ser Ser Val Cys Ile Ser 195 200 205 Glu Asp Leu Leu Gln Asn Tyr Ser Asp Val Tyr Phe Lys Leu Lys Lys 210 215 220 Ser Asp Asp Asp Asn Leu Gln Lys Asp Phe Lys Ser Ala Lys Asp Thr 225 230 235 240 Ile Lys Lys Gln Ile Ser Glu Tyr Ile Lys Asp Ser Glu Lys Phe Lys 245 250 255 Asn Leu Phe Asn Gln Asn Leu Ile Asp Ala Lys Lys Gly Gln Glu Ser 260 265 270 Asp Leu Ile Leu Trp Leu Lys Gln Ser Lys Asp Asn Gly Ile Glu Leu 275 280 285 Phe Lys Ala Asn Ser Asp Ile Thr Asp Ile Asp Glu Ala Leu Glu Ile 290 295 300 Ile Lys Ser Phe Lys Gly Trp Thr Thr Tyr Phe Lys Gly Phe His Glu 305 310 315 320 Asn Arg Lys Asn Val Tyr Ser Ser Asn Asp Ile Pro Thr Ser Ile Ile 325 330 335 Tyr Arg Ile Val Asp Asp Asn Leu Pro Lys Phe Leu Glu Asn Lys Ala 340 345 350 Lys Tyr Glu Ser Leu Lys Asp Lys Ala Pro Glu Ala Ile Asn Tyr Glu 355 360 365 Gln Ile Lys Lys Asp Leu Ala Glu Glu Leu Thr Phe Asp Ile Asp Tyr 370 375 380 Lys Thr Ser Glu Val Asn Gln Arg Val Phe Ser Leu Asp Glu Val Phe 385 390 395 400 Glu Ile Ala Asn Phe Asn Asn Tyr Leu Asn Gln Ser Gly Ile Thr Lys 405 410 415 Phe Asn Thr Ile Ile Gly Gly Lys Phe Val Asn Gly Glu Asn Thr Lys 420 425 430 Arg Lys Gly Ile Asn Glu Tyr Ile Asn Leu Tyr Ser Gln Gln Ile Asn 435 440 445 Asp Lys Thr Leu Lys Lys Tyr Lys Met Ser Val Leu Phe Lys Gln Ile 450 455 460 Leu Ser Asp Thr Glu Ser Lys Ser Phe Val Ile Asp Lys Leu Glu Asp 465 470 475 480 Asp Ser Asp Val Val Thr Thr Met Gln Ser Phe Tyr Glu Gln Ile Ala 485 490 495 Ala Phe Lys Thr Val Glu Glu Lys Ser Ile Lys Glu Thr Leu Ser Leu 500 505 510 Leu Phe Asp Asp Leu Lys Ala Gln Lys Leu Asp Leu Ser Lys Ile Tyr 515 520 525 Phe Lys Asn Asp Lys Ser Leu Thr Asp Leu Ser Gln Gln Val Phe Asp 530 535 540 Asp Tyr Ser Val Ile Gly Thr Ala Val Leu Glu Tyr Ile Thr Gln Gln 545 550 555 560 Ile Ala Pro Lys Asn Leu Asp Asn Pro Ser Lys Lys Glu Gln Glu Leu 565 570 575 Ile Ala Lys Lys Thr Glu Lys Ala Lys Tyr Leu Ser Leu Glu Thr Ile 580 585 590 Lys Leu Ala Leu Glu Glu Phe Asn Lys His Arg Asp Ile Asp Lys Gln 595 600 605 Cys Arg Phe Glu Glu Ile Leu Ala Asn Phe Ala Ala Ile Pro Met Ile 610 615 620 Phe Asp Glu Ile Ala Gln Asn Lys Asp Asn Leu Ala Gln Ile Ser Ile 625 630 635 640 Lys Tyr Gln Asn Gln Gly Lys Lys Asp Leu Leu Gln Ala Ser Ala Glu 645 650 655 Asp Asp Val Lys Ala Ile Lys Asp Leu Leu Asp Gln Thr Asn Asn Leu 660 665 670 Leu His Lys Leu Lys Ile Phe His Ile Ser Gln Ser Glu Asp Lys Ala 675 680 685 Asn Ile Leu Asp Lys Asp Glu His Phe Tyr Leu Val Phe Glu Glu Cys 690 695 700 Tyr Phe Glu Leu Ala Asn Ile Val Pro Leu Tyr Asn Lys Ile Arg Asn 705 710 715 720 Tyr Ile Thr Gln Lys Pro Tyr Ser Asp Glu Lys Phe Lys Leu Asn Phe 725 730 735 Glu Asn Ser Thr Leu Ala Asn Gly Trp Asp Lys Asn Lys Glu Pro Asp 740 745 750 Asn Thr Ala Ile Leu Phe Ile Lys Asp Asp Lys Tyr Tyr Leu Gly Val 755 760 765 Met Asn Lys Lys Asn Asn Lys Ile Phe Asp Asp Lys Ala Ile Lys Glu 770 775 780 Asn Lys Gly Glu Gly Tyr Lys Lys Ile Val Tyr Lys Leu Leu Pro Gly 785 790 795 800 Ala Asn Lys Met Leu Pro Lys Val Phe Phe Ser Ala Lys Ser Ile Lys 805 810 815 Phe Tyr Asn Pro Ser Glu Asp Ile Leu Arg Ile Arg Asn His Ser Thr 820 825 830 His Thr Lys Asn Gly Ser Pro Gln Lys Gly Tyr Glu Lys Phe Glu Phe 835 840 845 Asn Ile Glu Asp Cys Arg Lys Phe Ile Asp Phe Tyr Lys Gln Ser Ile 850 855 860 Ser Lys His Pro Glu Trp Lys Asp Phe Gly Phe Arg Phe Ser Asp Thr 865 870 875 880 Gln Arg Tyr Asn Ser Ile Asp Glu Phe Tyr Arg Glu Val Glu Asn Gln 885 890 895 Gly Tyr Lys Leu Thr Phe Glu Asn Ile Ser Glu Ser Tyr Ile Asp Ser 900 905 910 Val Val Asn Gln Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp 915 920 925 Phe Ser Ala Tyr Ser Lys Gly Arg Pro Asn Leu His Thr Leu Tyr Trp 930 935 940 Lys Ala Leu Phe Asp Glu Arg Asn Leu Gln Asp Val Val Tyr Lys Leu 945 950 955 960 Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Gln Ser Ile Pro Lys Lys 965 970 975 Ile Thr His Pro Ala Lys Glu Ala Ile Ala Asn Lys Asn Lys Asp Asn 980 985 990 Pro Lys Lys Glu Ser Val Phe Glu Tyr Asp Leu Ile Lys Asp Lys Arg 995 1000 1005 Phe Thr Glu Asp Lys Phe Phe Phe His Cys Pro Ile Thr Ile Asn Phe 1010 1015 1020 Lys Ser Ser Gly Ala Asn Lys Phe Asn Asp Glu Ile Asn Leu Leu Leu 1025 1030 1035 1040 Lys Glu Lys Ala Asn Asp Val His Ile Leu Ser Ile Asp Arg Gly Glu 1045 1050 1055 Arg His Leu Ala Tyr Tyr Thr Leu Val Asp Gly Lys Gly Asn Ile Ile 1060 1065 1070 Lys Gln Asp Thr Phe Asn Ile Ile Gly Asn Asp Arg Met Lys Thr Asn 1075 1080 1085 Tyr His Asp Lys Leu Ala Ala Ile Glu Lys Asp Arg Asp Ser Ala Arg 1090 1095 1100 Lys Asp Trp Lys Lys Ile Asn Asn Ile Lys Glu Met Lys Glu Gly Tyr 1105 1110 1115 1120 Leu Ser Gln Val Val His Glu Ile Ala Lys Leu Val Ile Glu Tyr Asn 1125 1130 1135 Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly Phe Lys Arg Gly Arg 1140 1145 1150 Phe Lys Val Glu Lys Gln Val Tyr Gln Lys Leu Glu Lys Met Leu Ile 1155 1160 1165 Glu Lys Leu Asn Tyr Leu Val Phe Lys Asp Asn Glu Phe Asp Lys Thr 1170 1175 1180 Gly Gly Val Leu Arg Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe 1185 1190 1195 1200 Lys Lys Met Gly Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly 1205 1210 1215 Phe Thr Ser Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr 1220 1225 1230 Pro Lys Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe 1235 1240 1245 Asp Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe 1250 1255 1260 Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr Ile 1265 1270 1275 1280 Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp Lys Asn 1285 1290 1295 His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu Leu Glu Lys 1300 1305 1310 Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly Glu Cys Ile Lys 1315 1320 1325 Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe Phe Ala Lys Leu Thr 1330 1335 1340 Ser Val Leu Asn Thr Ile Leu Gln Met Arg Asn Ser Lys Thr Gly Thr 1345 1350 1355 1360 Glu Leu Asp Tyr Leu Ile Ser Pro Val Ala Asp Val Asn Gly Asn Phe 1365 1370 1375 Phe Asp Ser Arg Gln Ala Pro Lys Asn Met Pro Gln Asp Ala Asp Ala 1380 1385 1390 Asn Gly Ala Tyr His Ile Gly Leu Lys Gly Leu Met Leu Leu Gly Arg 1395 1400 1405 Ile Lys Asn Asn Gln Glu Gly Lys Lys Leu Asn Leu Val Ile Lys Asn 1410 1415 1420 Glu Glu Tyr Phe Glu Phe Val Gln Asn Arg Asn Asn Gln Ala Ala Ala 1425 1430 1435 1440 Leu Glu Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys 1445 1450 1455 Lys Lys Ser Thr Pro Pro Pro Pro Pro Leu Arg Ser Gly Cys 1460 1465 1470 <210> 80 <211> 4413 <212> DNA <213> Artificial Sequence <220> <223> DSB-FnCpf1 <400> 80 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggctaa aaaagaaatg 120 gttgaatttg atgaagctat ccatggcgaa gacttggcta aatttattaa agaagcatct 180 gatcataaac tgaaaatttc cggttataat gaactgatta aagatattcg aattcgtgct 240 aaagatgaac ttggcgttga tggtaagatg tttaatcgtc tattagcttt gtatcataaa 300 gataaccgtg atgtgtttga agctgaaact gaagaggtag ttgaacttta tgacacagtt 360 ttctctaaag gatccgaatt cgagctccgt cgacaagctt gcggccgcat gtcaatttat 420 caagaatttg ttaataaata tagtttaagt aaaactctaa gatttgagtt aatcccacag 480 ggtaaaacac ttgaaaacat aaaagcaaga ggtttgattt tagatgatga gaaaagagct 540 aaagactaca aaaaggctaa acaaataatt gataaatatc atcagttttt tatagaggag 600 atattaagtt cggtttgtat tagcgaagat ttattacaaa actattctga tgtttatttt 660 aaacttaaaa agagtgatga tgataatcta caaaaagatt ttaaaagtgc aaaagatacg 720 ataaagaaac aaatatctga atatataaag gactcagaga aatttaagaa tttgtttaat 780 caaaacctta tcgatgctaa aaaagggcaa gagtcagatt taattctatg gctaaagcaa 840 tctaaggata atggtataga actatttaaa gccaatagtg atatcacaga tatagatgag 900 gcgttagaaa taatcaaatc ttttaaaggt tggacaactt attttaaggg ttttcatgaa 960 aatagaaaaa atgtttatag tagcaatgat attcctacat ctattattta taggatagta 1020 gatgataatt tgcctaaatt tctagaaaat aaagctaagt atgagagttt aaaagacaaa 1080 gctccagaag ctataaacta tgaacaaatt aaaaaagatt tggcagaaga gctaaccttt 1140 gatattgact acaaaacatc tgaagttaat caaagagttt tttcacttga tgaagttttt 1200 gagatagcaa actttaataa ttatctaaat caaagtggta ttactaaatt taatactatt 1260 attggtggta aatttgtaaa tggtgaaaat acaaagagaa aaggtataaa tgaatatata 1320 aatctatact cacagcaaat aaatgataaa acactcaaaa aatataaaat gagtgtttta 1380 tttaagcaaa ttttaagtga tacagaatct aaatcttttg taattgataa gttagaagat 1440 gatagtgatg tagttacaac gatgcaaagt ttttatgagc aaatagcagc ttttaaaaca 1500 gtagaagaaa aatctattaa agaaacacta tctttattat ttgatgattt aaaagctcaa 1560 aaacttgatt tgagtaaaat ttattttaaa aatgataaat ctcttactga tctatcacaa 1620 caagtttttg atgattatag tgttattggt acagcggtac tagaatatat aactcaacaa 1680 atagcaccta aaaatcttga taaccctagt aagaaagagc aagaattaat agccaaaaaa 1740 actgaaaaag caaaatactt atctctagaa actataaagc ttgccttaga agaatttaat 1800 aagcatagag atatagataa acagtgtagg tttgaagaaa tacttgcaaa ctttgcggct 1860 attccgatga tatttgatga aatagctcaa aacaaagaca atttggcaca gatatctatc 1920 aaatatcaaa atcaaggtaa aaaagaccta cttcaagcta gtgcggaaga tgatgttaaa 1980 gctatcaagg atcttttaga tcaaactaat aatctcttac ataaactaaa aatatttcat 2040 attagtcagt cagaagataa ggcaaatatt ttagacaagg atgagcattt ttatctagta 2100 tttgaggagt gctactttga gctagcgaat atagtgcctc tttataacaa aattagaaac 2160 tatataactc aaaagccata tagtgatgag aaatttaagc tcaattttga gaactcgact 2220 ttggctaatg gttgggataa aaataaagag cctgacaata cggcaatttt atttatcaaa 2280 gatgataaat attatctggg tgtgatgaat aagaaaaata acaaaatatt tgatgataaa 2340 gctatcaaag aaaataaagg cgagggttat aaaaaaattg tttataaact tttacctggc 2400 gcaaataaaa tgttacctaa ggttttcttt tctgctaaat ctataaaatt ttataatcct 2460 agtgaagata tacttagaat aagaaatcat tccacacata caaaaaatgg tagtcctcaa 2520 aaaggatatg aaaaatttga gtttaatatt gaagattgcc gaaaatttat agatttttat 2580 aaacagtcta taagtaagca tccggagtgg aaagattttg gatttagatt ttctgatact 2640 caaagatata attctataga tgaattttat agagaagttg aaaatcaagg ctacaaacta 2700 acttttgaaa atatatcaga gagctatatt gatagcgtag ttaatcaggg taaattgtac 2760 ctattccaaa tctataataa agatttttca gcttatagca aagggcgacc aaatctacat 2820 actttatatt ggaaagcgct gtttgatgag agaaatcttc aagatgtggt ttataagcta 2880 aatggtgagg cagagctttt ttatcgtaaa caatcaatac ctaaaaaaat cactcaccca 2940 gctaaagagg caatagctaa taaaaacaaa gataatccta aaaaagagag tgtttttgaa 3000 tatgatttaa tcaaagataa acgctttact gaagataagt ttttctttca ctgtcctatt 3060 acaatcaatt ttaaatctag tggagctaat aagtttaatg atgaaatcaa tttattgcta 3120 aaagaaaaag caaatgatgt tcatatatta agtatagata gaggtgaaag acatttagct 3180 tactatactt tggtagatgg taaaggcaat atcatcaaac aagatacttt caacatcatt 3240 ggtaatgata gaatgaaaac aaactaccat gataagcttg ctgcaataga gaaagatagg 3300 gattcagcta ggaaagactg gaaaaagata aataacatca aagagatgaa agagggctat 3360 ctatctcagg tagttcatga aatagctaag ctagttatag agtataatgc tattgtggtt 3420 tttgaggatt taaattttgg atttaaaaga gggcgtttca aggtagagaa gcaggtctat 3480 caaaagttag aaaaaatgct aattgagaaa ctaaactatc tagttttcaa agataatgag 3540 tttgataaaa ctgggggagt gcttagagct tatcagctaa cagcaccttt tgagactttt 3600 aaaaagatgg gtaaacaaac aggtattatc tactatgtac cagctggttt tacttcaaaa 3660 atttgtcctg taactggttt tgtaaatcag ttatatccta agtatgaaag tgtcagcaaa 3720 tctcaagagt tctttagtaa gtttgacaag atttgttata accttgataa gggctatttt 3780 gagtttagtt ttgattataa aaactttggt gacaaggctg ccaaaggcaa gtggactata 3840 gctagctttg ggagtagatt gattaacttt agaaattcag ataaaaatca taattgggat 3900 actcgagaag tttatccaac taaagagttg gagaaattgc taaaagatta ttctatcgaa 3960 tatgggcatg gcgaatgtat caaagcagct atttgcggtg agagcgacaa aaagtttttt 4020 gctaagctaa ctagtgtcct aaatactatc ttacaaatgc gtaactcaaa aacaggtact 4080 gagttagatt atctaatttc accagtagca gatgtaaatg gcaatttctt tgattcgcga 4140 caggcgccaa aaaatatgcc tcaagatgct gatgccaatg gtgcttatca tattgggcta 4200 aaaggtctga tgctactagg taggatcaaa aataatcaag agggcaaaaa actcaatttg 4260 gttatcaaaa atgaagagta ttttgagttc gtgcagaata ggaataacca agcggccgca 4320 ctcgagaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtcgaca 4380 ccaccaccac caccactgag atccggctgc taa 4413 <210> 81 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Adapter primer sequences of CCR5 <400> 81 tcgtcggcag cgtcagatgt gtataagaga cagggagggc aactaaatac attct 55 <210> 82 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Adapter primer sequences of CCR5 <400> 82 gtctcgtggg ctcggagatg tgtataagag acaggaacac cagtgagtag agcgg 55 <210> 83 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Adapter primer sequences of DHCR7 <400> 83 tcgtcggcag cgtcagatgt gtataagaga cagggtttga gcaacagttc tcc 53 <210> 84 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Adapter primer sequences of DHCR7 <400> 84 gtctcgtggg ctcggagatg tgtataagag acagtgacga tgtccaccac ag 52 <210> 85 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Adapter primer sequences of CCR5 <400> 85 tcgtcggcag cgtcagatgt gtataagaga cagggtattt ctgttcagat cac 53 <210> 86 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Adapter primer sequences of CCR5 <400> 86 gtctcgtggg ctcggagatg tgtataagag acaggcccat caattataga aagcc 55 <210> 87 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Adapter primer sequences of DNMT1 <400> 87 tcgtcggcag cgtcagatgt gtataagaga cagctgcaca cagcaggcct ttg 53 <210> 88 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Adapter primer sequences of DNMT1 <400> 88 gtctcgtggg ctcggagatg tgtataagag acagcccaat aagtggcaga gtgc 54 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Spacer sequence of CCR5 <400> 89 tgacatcaat tattatacat 20 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Spacer sequence of ADCY5 <400> 90 tgacatcaat tattatagat 20 <210> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Spacer sequence of KCNJ6 <400> 91 tgacatcact tattatgcat 20 <210> 92 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Spacer sequence of CNTNAP2 <400> 92 tgacataaat tattctacat 20 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Spacer sequence of Chr. 5N/A <400> 93 tgaaatcaat tatcatagat 20 <210> 94 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> ssODN donor sequences for CCR5 <400> 94 tggaacaaga tggattatca agtgtcaagt ccaatctatg acatcaatta ttat 54 <210> 95 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> ssODN donor sequences for CCR5 <400> 95 acatatgcat cggagccctg ccaaaaaatc aatgtgaagc aaatcgcagc cc 52 <210> 96 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> ssODN donor sequences for DHCR7 <400> 96 atgggcccca gtgtgactgc ctgcatccgt cctcgcaggg aggtggactg gt 52 <210> 97 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> ssODN donor sequences for DHCR7 <400> 97 tttgaattcc actggcgagc gtcatcttcc tactgctgtt cgcccccttc atcg 54 <210> 98 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> ssODN donor sequences for CCR5 <400> 98 aattctctga ggctttcttt taaatataca taaggaactt tcggagtgaa ggg 53 <210> 99 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> ssODN donor sequences for CCR5 <400> 99 agagtttcat atggtcaata acttgatgca tgtgaagggg agataaaaag gtt 53 <210> 100 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> ssODN donor sequences for DNMT1 <400> 100 tggccctggg gccgtttccc tcactcctgc tcggtgaatt tggctcagca ggc 53 <210> 101 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> ssODN donor sequences for DNMT1 <400> 101 acctgccgaa ttctcagctg ctcacttgag cctctgggtc tagaaccctc tgg 53 <210> 102 <211> 3358 <212> DNA <213> Artificial Sequence <220> <223> pGEM _447 <400> 102 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggccgcgggt ttgtcgctgg 60 aagaggataa attcgaactc tttcaacaac ggtttggcga gctggttacc gagtggctgg 120 acccttcgct attgcaaggc gaagtggtgt cagacggccc gttaagcccg gccgaaatga 180 ccatggaagt ggcgcagctg ctgcgcgatg ctggcccgtg ggggcagatg ttcccggagc 240 cgctgtttga tggtcatttc cgtctgctgc aacagcggct ggtgggcgaa cgtcatttga 300 aagtcatggt cgaaccggtc ggcggcggtc cgctgctgga tggtattgct tttaatgtcg 360 ataccgccct ctggccggat aacggcgtgc gcgaagtgca actggcttaa tcactagtgc 420 ggccgcctgc aggtcgacca tatgggagag ctcccaacgc gttggatgca tagcttgagt 480 attctatagt gtcacctaaa tagcttggcg taatcatggt catagctgtt tcctgtgtga 540 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 600 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 660 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 720 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 780 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 840 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 900 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 960 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 1020 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 1080 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 1140 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 1200 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 1260 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 1320 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 1380 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 1440 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 1500 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 1560 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 1620 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 1680 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 1740 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 1800 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 1860 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 1920 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 1980 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 2040 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 2100 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 2160 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 2220 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 2280 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 2340 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 2400 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 2460 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 2520 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 2580 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 2640 ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt gaaataccgc acagatgcgt 2700 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta 2760 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 2820 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 2880 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 2940 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta 3000 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 3060 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 3120 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc 3180 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 3240 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 3300 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactata 3358

Claims (22)

  1. 크리스퍼 연관 단백질 및 엑소뉴클레아제인 RecJ를 포함하는 크리스퍼 플러스 단백질.
  2. 제1항에 있어서,
    상기 크리스퍼 플러스 단백질은 하기 구조식 1 내지 4에서 선택되는 어느 하나의 구조를 갖는 것인, 크리스퍼 플러스 단백질로서,
    <구조식 1>
    크리스퍼 연관 단백질-RecJ
    <구조식 2>
    크리스퍼 연관 단백질-링커 1-RecJ
    <구조식 3>
    RecJ-크리스퍼 연관 단백질
    <구조식 4>
    RecJ-링커 1-크리스퍼 연관 단백질
    상기 링커 1은 1 내지 20개의 아미노산으로 구성된 것일 수 있다.
  3. 제2항에 있어서,
    상기 링커 1은 서열번호 17, 18 , 19, 20, 21, 22, 23 또는 24의 아미노산 서열을 가지는 것인, 크리스퍼 플러스 단백질.
  4. 제1항에 있어서,
    추가적으로 DNA 결합 단백질(DNA binding protein, DBP)를 더 포함하는 것인, 크리스퍼 플러스 단백질.
  5. 제4항에 있어서,
    상기 DBP는 SSB(single stranded binding protein) 또는 DSB(double stranded binding protein)인 것인, 크리스퍼 플러스 단백질.
  6. 제1항에 있어서,
    상기 RecJ는 크리스퍼 연관 단백질의 N 말단 또는 C 말단에 결합되는 것인, 크리스퍼 플러스 단백질.
  7. 제4항에 있어서,
    상기 크리스퍼 플러스 단백질는 하기 구조식 5 내지 7에서 선택되는 어느 하나의 구조를 갖는 것인, 크리스퍼 플러스 단백질로서,
    <구조식 5>
    DBP-크리스퍼 연관 단백질-RecJ
    <구조식 6>
    DBP-링커 2-크리스퍼 연관 단백질-RecJ
    <구조식 7>
    DBP-링커 2-크리스퍼 연관 단백질-링커 1-RecJ
    이때, DBP는 SSB 또는 DSB이며,
    상기 링커 1은 1 내지 20개의 아미노산으로 구성된 것일 수 있으며,
    상기 링커 2는 1 내지 20개의 아미노산으로 구성된 것일 수 있다.
  8. 제1항에 있어서,
    추가적으로 핵 위치화 시그널( nuclear localization signal)을 더 포함하는 것인, 크리스퍼 플러스 단백질.
  9. 제8항에 있어서,
    상기 핵 위치화 시그널은 융합 단백질의 C 말단에 위치하는 것인, 크리스퍼 플러스 단백질.
  10. 제1항에 있어서,
    추가적으로 His tag을 포함하는 것인, 크리스퍼 플러스 단백질.
  11. 제1항에 있어서,
    상기 크리스퍼 연관 단백질은 Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 및 Csf4로 구성된 군으로부터 선택되는 어느 하나인 것인, 크리스퍼 플러스 단백질.
  12. 제1항에 있어서,
    상기 RecJ는 서열번호 1, 3, 5, 7, 9, 11, 13 또는 15로 이루어진 군에서 선택되는 어느 하나의 아미노산 서열을 가지는 것인, 크리스퍼 플러스 단백질.
  13. 제1항 내지 제12항에 있어서,
    상기 크리스퍼 플러스 단백질은 서열번호 31, 41, 49, 53, 57, 71 또는 77로 구성된 군에서 선택되는 어느 하나의 아미노산 서열을 가지는 것인, 크리스퍼 플러스 단백질.
  14. 제12항의 크리스퍼 플러스 단백질을 코딩하는 폴리뉴클레오티드.
  15. 제14항에 있어서,
    상기 폴리뉴클레오티드는 서열번호 32, 42, 50, 54, 58, 72 또는 78로 구성된 군에서 선택되는 어느 하나의 핵산 서열을 가지는 것인, 폴리뉴클레오티드.
  16. 제14항의 폴리뉴클레오티드가 적재된 벡터.
  17. 제16항에 있어서,
    상기 벡터는 플라스미드인, 벡터.
  18. 제16항의 벡터가 도입된 숙주 세포.
  19. 제1항 내지 제12항의 크리스퍼 플러스 단백질 및 crRNA가 결합된 RNP 복합체.
  20. 제19항의 RNP 복합체를 포함하는 유전자 편집용 조성물.
  21. 제1항 내지 제12항의 크리스퍼 플러스 단백질 및 crRNA를 유효성분으로 포함하는 유전자 편집용 키트.
  22. 제19항에 있어서,
    상기 crRNA는 표적 유전자에 상보적으로 결합 가능한 10 내지 30개의 뉴클레오티드로 구성된, 유전자 편집용 키트.
KR1020180167870A 2018-12-21 2018-12-21 크리스퍼 연관 단백질 및 엑소뉴클레아제를 포함하는 변형된 크리스퍼 연관 단백질 Ceased KR20200078200A (ko)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118531031A (zh) * 2024-06-12 2024-08-23 河南农业职业学院 单链DNA结合蛋白介导的体外CRISPR/Cas9基因编辑方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
B. Zetsche, et al., Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system, Cell, 2015, 163, 759-771.

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118531031A (zh) * 2024-06-12 2024-08-23 河南农业职业学院 单链DNA结合蛋白介导的体外CRISPR/Cas9基因编辑方法及其应用

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