CN108410877A - CRISPR-Cas9靶向敲除人细胞SANIL1基因及其特异性的sgRNA - Google Patents

CRISPR-Cas9靶向敲除人细胞SANIL1基因及其特异性的sgRNA Download PDF

Info

Publication number
CN108410877A
CN108410877A CN201810259431.8A CN201810259431A CN108410877A CN 108410877 A CN108410877 A CN 108410877A CN 201810259431 A CN201810259431 A CN 201810259431A CN 108410877 A CN108410877 A CN 108410877A
Authority
CN
China
Prior art keywords
sanil1
sgrna
genes
cell
cas9
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN201810259431.8A
Other languages
English (en)
Inventor
杨蕊菊
陆路
杨兴林
潘讴东
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yuan Biotechnology (shanghai) Ltd By Share Ltd
Original Assignee
Yuan Biotechnology (shanghai) Ltd By Share Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yuan Biotechnology (shanghai) Ltd By Share Ltd filed Critical Yuan Biotechnology (shanghai) Ltd By Share Ltd
Priority to CN201810259431.8A priority Critical patent/CN108410877A/zh
Publication of CN108410877A publication Critical patent/CN108410877A/zh
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Abstract

本发明公开了CRISPR/Cas9靶向敲除人细胞SANIL1基因及其特异性sgRNA。首先是获得特异性靶向SANIL1基因外显子的sgRNA，其碱基序列如SEQ ID NO.1所示；其次是构建SANIL1基因的sgRNA到病毒载体系统，该系统含有Cas9蛋白；最后将含有该sgRNA的CRISPR/Cas9病毒感染人细胞，获得SANIL1蛋白表达水平显著降低的细胞株。本发明具有操作步骤简单、sgRNA靶向性好、且对SANIL1基因切割效率高；此外，所构建的CRISPR/Cas9慢病毒系统具有敲除效率高的优势，并能特异性敲除SANIL1基因，得到敲除SANIL1基因的人细胞，从而为进一步研究人细胞中SANIL1的作用机制提供强有力的工具。

Description

CRISPR-Cas9靶向敲除人细胞SANIL1基因及其特异性的sgRNA
技术领域
本发明属于基因工程领域，更具体地说涉及CRISPR-Cas9靶向敲除人细胞SANIL1基因及其特异性的sgRNA。
背景技术
SANIL1(snail family transcriptional repressor 1)：是锌指转录抑制子，下调中胚层中的外胚层基因。由该基因编码的核蛋白在结构上和果蝇snail蛋白类似，而且同样也被认为在发育中的胚胎的中胚层形成是至关重要的。在2号染色体上，人们已经发现了至少2种类似的加工过的假基因的突变体。
Snail1蛋白是锌指蛋白Snail超家族的成员,具有强大的EMT诱导能力。借由其羧基末端的锌指结构与CDH1核心启动子区的E-box基序结合,Snail1蛋白能有效遏制后者的转录表达,造成E-cadherin表达缺失,诱导EMT发生。
CRISPR-Cas系统是最近几年兴起用于靶向基因特定DNA修饰的重要工具，具有快速、高效及特异性靶向等优势，已被应用于多种模式生物，包括人、小鼠、大鼠、斑马鱼、秀丽隐杆线虫、植物及细菌。CRISPR RNA(crRNA)与转录激活crRNA退火形成的复合物能特异识别基因组序列，引导Cas9核酸内切酶在目的片段生成DNA双链断裂。这个识别复合体可以通过融合crRNA与tracrRNA序列形成sgRNA进行简化。基因组的靶序列中有长约20bp的片段与crRNA或sgRNA互补配对；靶序列末端的三核苷酸区域PAM(5’-NGG-3’)为spCas9识别位点，是实现剪切功能的关键。sgRNA能否做到特异性、精确靶向目标基因是CRISPR/Cas9特异性敲除目标基因的先决条件，脱靶和错误靶向都会影响CRISPR/Cas9对目标基因的特异性敲除，因此，能够设计、制备出精确性和特异性靶向目标基因的sgRNA成为CRISPR/Cas9基因敲除的关键技术。
本发明利用CRISPR/Cas9慢病毒系统，获得敲除SANIL1基因的人细胞，为研究肠癌细胞中SANIL1的作用机制提供有力工具。
参考文献：
1.Doudna，J.A.；Charpentier，E.，Genome editing.The new frontier ofgenome engineering with CRISPR-Cas9.Science，2014，346，(6213)，1258096.
2.Fang，R.；Chang，F.；Sun，Z.-L.；Li，N.；Meng，Q.-Y.，New Method of GenomeEditing Derived From CRISPR/Cas9.Acta Agronomica Sinica，2013，40，(8)，691.
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种靶向敲除SANIL1基因的sgRNA序列；本发明的另一目的在于提供所述一种靶向敲除SANIL1基因的CRISPR/Cas9慢病毒系统的应用；本发明的再一目的在于提供靶向敲除SANIL1基因的人细胞的应用。
本发明的目的通过下述技术方案实现：一种靶向敲除SANIL1基因的sgRNA序列，选自DNA序列如下的SANIL1sgRNA：
SANIL1sgRNA的序列如下：
SANIL1sgRNA oligo1：5’-ACCGACGCCGCTGCGGAAACCTTC-3’；
SANIL1sgRNA oligo2：5’-AAACGAAGGTTTCCGCAGCGGCGT-3’；
SANIL1sgRNA oligo3：5’-ACCGCAACCTCTCCCCTGGCCCTG-3’；
SANIL1sgRNA oligo4：5’-AAACCAGGGCCAGGGGAGAGGTTG-3’；
SANIL1sgRNA oligo5：5’-ACCGTGCAGGCCCAAGCTGCCCGCC-3’；
SANIL1sgRNA oligo6：5’-AAACAAACGGCGGGCAGCTTGGGCCTGCA-3’；
一种靶向敲除SANIL1基因的CRISPR/Cas9慢病毒系统，含有上述靶向敲除SANIL1基因的sgRNA的DNA序列。
所述的靶向敲除SANIL1基因的CRISPR/Cas9慢病毒系统的构建，包括如下步骤：
(1)使用BsmBI酶切CRISPR/Cas9慢病毒载体Lenti-CRISPRv2，得到酶切后的CRISPR/Cas9慢病毒载体；
(2)将上述靶向敲除SANIL1基因的sgRNA的DNA序列磷酸化后与酶切后的CRISPR/Cas9慢病毒载体连接，得到靶向敲除SANIL1基因的CRISPR/Cas9慢病毒系统。
步骤(2)中所述的DNA序列是将寡核苷酸链1(oligo1)和寡核苷酸链2(oligo2)退火得到双链序列。
所述的靶向敲除SANIL1基因的CRISPR/Cas9慢病毒系统在制备敲除SANIL1基因的细胞株中的应用。
一种敲除SANIL1基因的细胞株，是将所述的靶向敲除SANIL1基因的CRISPR/Cas9慢病毒系统转染目的细胞株得到的。
所述的敲除SANIL1基因的细胞株，具体是通过如下步骤构建得到：
1)将所述的靶向敲除SANIL1基因的CRISPR/Cas9慢病毒系统通过包装细胞进行包装，得到慢病毒颗粒；
2)将慢病毒颗粒感染目的细胞株；
3)将感染后的细胞铺单克隆，得到敲除SANIL1基因的细胞株。
所述的目的细胞株优选为人细胞株。
所述的目的细胞株优选为肿瘤细胞株。
本发明相对于现有技术具有如下的优点及效果：
本发明提供SANIL1基因的sgRNA能特异靶向SANIL1基因，将其构建入CRISPR/Cas9慢病毒系统，该系统能特异靶向敲除SANIL1基因，得到敲除SANIL1基因的细胞株，从而有利于研究细胞株中的SANIL1的作用机制。
附图说明
附图1:pLenti-U6-gRNA v2.0-CMV-Puro-P2A-3Flag-spCas9质粒图谱；
附图2：pLenti-U6-SANIL1 gRNA 1-CMV-Puro-P2A-3Flag-spCas9质粒图谱；
附图3：pLenti-U6-SANIL1gRNA 2-CMV-Puro-P2A-3Flag-spCas9质粒图谱；
附图4：pLenti-U6-SANIL1 gRNA 3-CMV-Puro-P2A-3Flag-spCas9质粒图谱；
附图5：293T细胞转染含有SANIL1gRNA1的活性鉴定测序图；
附图6：293T细胞转染含有SANIL1gRNA2的活性鉴定测序图；
附图7：293T细胞转染含有SANIL1gRNA3的活性鉴定测序图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
1.使用CRISPR/Cas9技术构建敲除SANIL1质粒
1.1sgRNA寡核苷酸链合成
使用CRISPR在线设计工具(http://crispr.mit.edu/)根据评分系统，在SANIL1的外显子2上设计1个20bp的sgRNA，并通过BLAST验证无非特异性基因。编码链模板5′端添加ACCG，非编码链模板3′端添加AAAC，与BsmBI酶切后形成的粘性末端互补，设计1对CRISPR寡核苷酸链，见表1。
表1SANIL1靶向位点及sgRNA寡核苷酸序列
sgRNA名称 sgRNA寡核苷酸序列
SANIL1sgRNAoligo1 5’-ACCGACGCCGCTGCGGAAACCTTC-3’
SANIL1sgRNA oligo2 5’-AAACGAAGGTTTCCGCAGCGGCGT-3’
SANIL1sgRNAoligo3 5’-ACCGCAACCTCTCCCCTGGCCCTG-3’
SANIL1sgRNA oligo4 5’-AAACCAGGGCCAGGGGAGAGGTTG-3’
SANIL1sgRNAoligo5 5’-ACCGTGCAGGCCCAAGCTGCCCGCC-3’
SANIL1sgRNA oligo6 5’-AAACAAACGGCGGGCAGCTTGGGCCTGCA-3’
1.2载体构建
1.2.1使用BsmBI酶切2μg Lenti-CRISPRv2质粒(购于Addgene公司)，2h，37℃,
酶切体系为：
2μg(2μl) Lenti-CRISPRv2
1μl BsmBI(NEB)
5μl 10X Cutsmart
42μl ddH2O
50μl 合计
1.2.2使用捷瑞基因胶回收试剂盒纯化酶切质粒产物，按说明书进行操作。
1.2.3磷酸化并退火sgRNA oligos：
酶连体系为：
1μl Oligo1(100μM)
1μl Oligo2(100μM)
1μl 10X T4链接缓冲液(NEB)
6.5μl ddH2O
0.5μl T4PNK(NEB)
10μl 合计
PCR仪器退火程序：
37℃30min,95℃维持5min，每分钟降低5℃至25℃，4℃维持
1.2.4将退火形成的Oligo双链和酶切后的Lenti-CRISPRv2载体直接连接，室温下，10min。
1.2.5将连接后的质粒转化至感受态细胞DH5α中，均匀涂至LB固体培养基平板中，置于37℃培养箱中培养12-16小时，单个菌落即可出现。
1.3挑取单个菌落扩大培养并质粒小提。
1.4测序鉴定质粒构建成功，并命名为Lenti-CRISPRv2-hSANIL1sg。
1.5敲除效率验证。
用含10％胎牛血清的高糖DMEM培养基于5％CO2，37℃恒温培养293T细胞(购于美国ATCC细胞库)。取对数期细胞以1×105/孔接种到24孔板培养。待细胞融合度达到70％～80％时更换成Opti-MEM培养基，1小时后将相应的CRISPR/Cas9敲除质粒0.8μg经Lipo2000试剂，转染到293T细胞中，转染48小时候，荧光显微镜下观察转染效果，消化收集细胞，采用基因组DNA提取试剂盒提取细胞基因组DNA。
以各组细胞基因组DNA为模板，利用表2的引物(Seq No.8～11)PCR扩增靶向序列。用SANIL1-4-1和SANIL1-4-2进行PCR，得到751bp的片段a，以a为模板，用SANIL1-4-3和SANIL1-4-4进行PCR，得到388bp的片段b，该片段电泳后胶回收用于测序，测序引物为SANIL1-4-3。
表2引物情况表
引物名称 引物序列
SANIL1-4-1 GCTCAGAGAAGCCAGACAAC
SANIL1-4-2 CAGTGAGTCTGTCAGCCTTTG
SANIL1-4-3 TTGTCCCACGGCTCACTC
SANIL1-4-4 AATGGTCTTCATCAAAGTCC
2.包装慢病毒
100mm皿种入4×106个293TN细胞，培养24h(37℃,5％CO2)后，更换新鲜培养基，转染包装细胞：
a.混匀以下3个质粒：1)包装质粒psPAX2 12μg；辅助质粒pLP-VSVG 10μg；表达质粒Lenti-CRISPRv2-hSANIL1sg 22μg；加入CaCl2 250μl；加入ddH2O至总体积为500μl，37℃放置20min；
b. 2X BES 500μl，37℃放置20min；
c.把a中500μl三质粒混合液逐滴加入b中，并轻轻混匀；
d.小心的将c中混合液加入种有293TN的100mm皿中；培养12h后移除感染液，更换10ml新鲜培养基生产病毒液；培养48h后，收获病毒液至50ml离心管中，并重新向100mm皿中补加10ml培养基再次生产病毒；培养24h后，再次收获病毒液，室温离心，1000rpm，10min；使用0.22μm的滤器过滤病毒上清液；将病毒上清液装入超离管中，4℃，100000g，2h；弃去上清液后用Opti-MEM重悬过夜；再次使用0.22μm的滤器过滤重悬的病毒；分装后，-80℃保存。
3.病毒感染
1)将要感染的HT-29细胞(购于美国ATCC细胞库)接种到六孔板中，过夜，待细胞融合度约为50％时，移去培养基，更换2ml新鲜培养基；
2)每孔加入20μl病毒液，再加入12μl聚凝胺polybrene，至终浓度为6μg/ml；
3)37℃培养12h后，移除培养液，更换新鲜培养液后继续培养48h；
4.筛选稳定细胞株
感染结束48h后，向每孔加入嘌呤霉素(2.0μg/ml)，隔天换液，并保持培养基的嘌呤霉素浓度恒定，筛选阳性克隆细胞。所得细胞株命名为“胞名-SANIL1”。并使用有限稀释法挑选HT-29-SANIL1单克隆敲除细胞株。
5.稳定细胞株鉴定及基因打靶方式鉴定
以各组敲除SANIL1的单克隆细胞株基因组DNA为模板，分别设计针对SANIL1第二外显子的引物，序列如Seq No.8～11进行两轮巢式PCR扩增，对获得的第二轮PCR产物进行测序，如测序结果打靶位点附近出现双峰的情况，则可能为打靶成功。选择双峰的样品再次PCR，产物胶回收后TA克隆至T载体中，转化后挑取阳性克隆再次进行测序，如测序结果SANIL1基因靶位点附近发生非三倍数的碱基插入或碱基缺失，导致阅读框移码突变，则可判断为SANIL1基因敲除。鉴定出sgRNA是有活性的(图5)，并且找到2种类型的具有SANIL1基因缺失或插入突变的稳定细胞株(图6和图7)。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 和元生物技术(上海)股份有限公司
<120> CRISPR-Cas9特异性敲除人SNAIL1基因的方法及用于特异性靶向SNAIL1基因的sgRNA
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Aritifical synthesis)
<400> 1
<210> 2
<211> 21
<212> DNA
<213> 人工序列(Aritifical synthesis)
<400> 2
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Aritifical synthesis)
<400> 3
<210> 4
<211> 12584
<212> DNA
<213> 人工序列(Aritifical synthesis)
<400> 4
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga actacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgatacgcgt gcggccgccc ccttcaccga gggcctattt 2220
cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt 2280
aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt 2340
cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta 2400
acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg 2460
agacgatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg 2520
atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg 2580
cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg 2640
aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag 2700
aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct 2760
ggggaatata acgtctcagt ttcagagcta tgctggaaac agcatagcaa gttgaaataa 2820
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ggatccatta 2880
gacgcgtggg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 2940
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 3000
actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 3060
caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 3120
tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 3180
ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 3240
cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 3300
tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 3360
atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 3420
cagatcgcct gccatccacg ctgttttgac ctccatagaa gacaccgact ctactagagg 3480
atcgctagcg ctaccggact cagatctcga gctcaagctt cgaattcgcc accatgaccg 3540
agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc 3600
tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca 3660
tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca 3720
aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg 3780
aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc 3840
tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt 3900
ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg 3960
tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct 4020
ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg 4080
aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc ggctccggag 4140
ccacgaactt ctctctgtta aagcaagcag gcgacgtgga agaaaacccc ggtccggcta 4200
gcgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca 4260
aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc 4320
cagcagccga caagaagtac tccattgggc tcgatatcgg cacaaacagc gtcggctggg 4380
ccgtcattac ggacgagtac aaggtgccga gcaaaaaatt caaagttctg ggcaataccg 4440
atcgccacag cataaagaag aacctcattg gcgccctcct gttcgactcc ggggagaccg 4500
ccgaagccac gcggctcaaa agaacagcac ggcgcagata tacccgcaga aagaatcgga 4560
tctgctacct gcaggagatc tttagtaatg agatggctaa ggtggatgac tctttcttcc 4620
ataggctgga ggagtccttt ttggtggagg aggataaaaa gcacgagcgc cacccaatct 4680
ttggcaatat cgtggacgag gtggcgtacc atgaaaagta cccaaccata tatcatctga 4740
ggaagaagct tgtagacagt actgataagg ctgacttgcg gttgatctat ctcgcgctgg 4800
cgcatatgat caaatttcgg ggacacttcc tcatcgaggg ggacctgaac ccagacaaca 4860
gcgatgtgga caaactcttt atccaactgg ttcagactta caatcagctt ttcgaagaga 4920
acccgatcaa cgcatccgga gttgacgcca aagcaatcct gagcgctagg ctgtccaaat 4980
cccggcggct cgaaaacctc atcgcacagc tccctgggga gaagaagaac ggcctgtttg 5040
gtaatcttat cgccctgtca ctcgggctga cccccaactt taaatctaac ttcgacctgg 5100
ccgaagatgc caagcttcaa ctgagcaaag acacctacga tgatgatctc gacaatctgc 5160
tggcccagat cggcgaccag tacgcagacc tttttttggc ggcaaagaac ctgtcagacg 5220
ccattctgct gagtgatatt ctgcgagtga acacggagat caccaaagct ccgctgagcg 5280
ctagtatgat caagcgctat gatgagcacc accaagactt gactttgctg aaggcccttg 5340
tcagacagca actgcctgag aagtacaagg aaattttctt cgatcagtct aaaaatggct 5400
acgccggata cattgacggc ggagcaagcc aggaggaatt ttacaaattt attaagccca 5460
tcttggaaaa aatggacggc accgaggagc tgctggtaaa gcttaacaga gaagatctgt 5520
tgcgcaaaca gcgcactttc gacaatggaa gcatccccca ccagattcac ctgggcgaac 5580
tgcacgctat cctcaggcgg caagaggatt tctacccctt tttgaaagat aacagggaaa 5640
agattgagaa aatcctcaca tttcggatac cctactatgt aggccccctc gcccggggaa 5700
attccagatt cgcgtggatg actcgcaaat cagaagagac catcactccc tggaacttcg 5760
aggaagtcgt ggataagggg gcctctgccc agtccttcat cgaaaggatg actaactttg 5820
ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc tctgctgtac gagtacttca 5880
cagtttataa cgagctcacc aaggtcaaat acgtcacaga agggatgaga aagccagcat 5940
tcctgtctgg agagcagaag aaagctatcg tggacctcct cttcaagacg aaccggaaag 6000
ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat tgaatgtttc gactctgttg 6060
aaatcagcgg agtggaggat cgcttcaacg catccctggg aacgtatcac gatctcctga 6120
aaatcattaa agacaaggac ttcctggaca atgaggagaa cgaggacatt cttgaggaca 6180
ttgtcctcac ccttacgttg tttgaagata gggagatgat tgaagaacgc ttgaaaactt 6240
acgctcatct cttcgacgac aaagtcatga aacagctcaa gaggcgccga tatacaggat 6300
gggggcggct gtcaagaaaa ctgatcaatg gcatccgaga caagcagagt ggaaagacaa 6360
tcctggattt tcttaagtcc gatggatttg ccaaccggaa cttcatgcag ttgatccatg 6420
atgactctct cacctttaag gaggacatcc agaaagcaca agtttctggc cagggggaca 6480
gtcttcacga gcacatcgct aatcttgcag gtagcccagc tatcaaaaag ggaatactgc 6540
agaccgttaa ggtcgtggat gaactcgtca aagtaatggg aaggcataag cccgagaata 6600
tcgttatcga gatggcccga gagaaccaaa ctacccagaa gggacagaag aacagtaggg 6660
aaaggatgaa gaggattgaa gagggtataa aagaactggg gtcccaaatc cttaaggaac 6720
acccagttga aaacacccag cttcagaatg agaagctcta cctgtactac ctgcagaacg 6780
gcagggacat gtacgtggat caggaactgg acatcaatcg gctctccgac tacgacgtgg 6840
atcatatcgt gccccagtct tttctcaaag atgattctat tgataataaa gtgttgacaa 6900
gatccgataa aaatagaggg aagagtgata acgtcccctc agaagaagtt gtcaagaaaa 6960
tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat cacacaacgg aagttcgata 7020
atctgactaa ggctgaacga ggtggcctgt ctgagttgga taaagccggc ttcatcaaaa 7080
ggcagcttgt tgagacacgc cagatcacca agcacgtggc ccaaattctc gattcacgca 7140
tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga ggtgaaagtt attactctga 7200
agtctaagct ggtctcagat ttcagaaagg actttcagtt ttataaggtg agagagatca 7260
acaattacca ccatgcgcat gatgcctacc tgaatgcagt ggtaggcact gcacttatca 7320
aaaaatatcc caagcttgaa tctgaatttg tttacggaga ctataaagtg tacgatgtta 7380
ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc caccgctaag tacttctttt 7440
acagcaatat tatgaatttt ttcaagaccg agattacact ggccaatgga gagattcgga 7500
agcgaccact tatcgaaaca aacggagaaa caggagaaat cgtgtgggac aagggtaggg 7560
atttcgcgac agtccggaag gtcctgtcca tgccgcaggt gaacatcgtt aaaaagaccg 7620
aagtacagac cggaggcttc tccaaggaaa gtatcctccc gaaaaggaac agcgacaagc 7680
tgatcgcacg caaaaaagat tgggacccca agaaatacgg cggattcgat tctcctacag 7740
tcgcttacag tgtactggtt gtggccaaag tggagaaagg gaagtctaaa aaactcaaaa 7800
gcgtcaagga actgctgggc atcacaatca tggagcgatc aagcttcgaa aaaaacccca 7860
tcgactttct ggaggcgaaa ggatataaag aggtcaaaaa agacctcatc attaagcttc 7920
ccaagtactc tctctttgag cttgaaaacg gccggaaacg aatgctcgct agtgcgggcg 7980
agctgcagaa aggtaacgag ctggcactgc cctctaaata cgttaatttc ttgtatctgg 8040
ccagccacta tgaaaagctc aaagggtctc ccgaagataa tgagcagaag cagctgttcg 8100
tggaacaaca caaacactac cttgatgaga tcatcgagca aataagcgag ttctccaaaa 8160
gagtgatcct cgccgacgct aacctcgata aggtgctttc tgcttacaat aagcacaggg 8220
ataagcccat cagggagcag gcagaaaaca ttatccactt gtttactctg accaacttgg 8280
gcgcgcctgc agccttcaag tacttcgaca ccaccataga cagaaagcgg tacacctcta 8340
caaaggaggt cctggacgcc acactgattc atcagtcaat tacggggctc tatgaaacaa 8400
gaatcgacct ctctcagctc ggtggagaca agcgtcctgc tgctactaag aaagctggtc 8460
aagctaagaa aaagaaatga gtcgactcta gaccgcgtct ggaacaatca acctctggat 8520
tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 8580
ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 8640
tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 8700
caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 8760
accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 8820
ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 8880
tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc 8940
tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 9000
ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 9060
acgagtcgga tctccctttg ggccgcctcc ccgcctggaa ttaattctgc agtcgagacc 9120
tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct 9180
ggctagaagc acaagaggag gaggaggtgg gtttttccag tcacacctca ggtaccttta 9240
agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagagggga 9300
ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac 9360
acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt cagatatcca 9420
ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt agaagaggcc 9480
aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat ggatgacccg 9540
gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca cgtggcccga 9600
gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 9660
gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg agccctcaga 9720
tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag accagatctg 9780
agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 9840
ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 9900
cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat 9960
tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggcctt gacattgcta 10020
gcgttttacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg 10080
tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 10140
aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 10200
ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 10260
gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 10320
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 10380
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 10440
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 10500
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 10560
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 10620
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 10680
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 10740
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 10800
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 10860
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 10920
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 10980
aacaaaccac cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 11040
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 11100
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 11160
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 11220
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 11280
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 11340
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 11400
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 11460
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 11520
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 11580
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 11640
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 11700
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 11760
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 11820
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 11880
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 11940
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 12000
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 12060
cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 12120
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 12180
ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatcaacttg 12240
tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 12300
gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 12360
gtctggatca actggataac tcaagctaac caaaatcatc ccaaacttcc caccccatac 12420
cctattacca ctgccaatta cctagtggtt tcatttactc taaacctgtg attcctctga 12480
attattttca ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagtttt 12540
taaagaaatt gtatttgtta aatatgtact acaaacttag tagt 12584
<210> 5
<211> 12283
<212> DNA
<213> 人工序列(Aritifical synthesis)
<400> 5
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga actacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgatacgcgt gcggccgccc ccttcaccga gggcctattt 2220
cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt 2280
aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt 2340
cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta 2400
acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgat 2460
gtccccgctg accctcggtt tcagagctat gctggaaaca gcatagcaag ttgaaataag 2520
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg gatccattag 2580
acgcgtggga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 2640
acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 2700
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 2760
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 2820
ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 2880
tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 2940
ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 3000
ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 3060
tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 3120
agatcgcctg ccatccacgc tgttttgacc tccatagaag acaccgactc tactagagga 3180
tcgctagcgc taccggactc agatctcgag ctcaagcttc gaattcgcca ccatgaccga 3240
gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct 3300
cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat 3360
cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa 3420
ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga 3480
agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct 3540
ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg 3600
gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt 3660
cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc 3720
cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga 3780
ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gctccggagc 3840
cacgaacttc tctctgttaa agcaagcagg cgacgtggaa gaaaaccccg gtccggctag 3900
cgccaccatg gactataagg accacgacgg agactacaag gatcatgata ttgattacaa 3960
agacgatgac gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc 4020
agcagccgac aagaagtact ccattgggct cgatatcggc acaaacagcg tcggctgggc 4080
cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg gcaataccga 4140
tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg gggagaccgc 4200
cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa agaatcggat 4260
ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact ctttcttcca 4320
taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc acccaatctt 4380
tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat atcatctgag 4440
gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc 4500
gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc cagacaacag 4560
cgatgtggac aaactcttta tccaactggt tcagacttac aatcagcttt tcgaagagaa 4620
cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc tgtccaaatc 4680
ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg gcctgtttgg 4740
taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact tcgacctggc 4800
cgaagatgcc aagcttcaac tgagcaaaga cacctacgat gatgatctcg acaatctgct 4860
ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc tgtcagacgc 4920
cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc cgctgagcgc 4980
tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga aggcccttgt 5040
cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta aaaatggcta 5100
cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta ttaagcccat 5160
cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag aagatctgtt 5220
gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc tgggcgaact 5280
gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata acagggaaaa 5340
gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg cccggggaaa 5400
ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct ggaacttcga 5460
ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga ctaactttga 5520
taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg agtacttcac 5580
agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa agccagcatt 5640
cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga accggaaagt 5700
taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtttcg actctgttga 5760
aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg atctcctgaa 5820
aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc ttgaggacat 5880
tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct tgaaaactta 5940
cgctcatctc ttcgacgaca aagtcatgaa acagctcaag aggcgccgat atacaggatg 6000
ggggcggctg tcaagaaaac tgatcaatgg catccgagac aagcagagtg gaaagacaat 6060
cctggatttt cttaagtccg atggatttgc caaccggaac ttcatgcagt tgatccatga 6120
tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc agggggacag 6180
tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg gaatactgca 6240
gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc ccgagaatat 6300
cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga acagtaggga 6360
aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc ttaaggaaca 6420
cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc tgcagaacgg 6480
cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact acgacgtgga 6540
tcatatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag tgttgacaag 6600
atccgataaa aatagaggga agagtgataa cgtcccctca gaagaagttg tcaagaaaat 6660
gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga agttcgataa 6720
tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct tcatcaaaag 6780
gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg attcacgcat 6840
gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta ttactctgaa 6900
gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga gagagatcaa 6960
caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg cacttatcaa 7020
aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt acgatgttag 7080
gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt acttctttta 7140
cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag agattcggaa 7200
gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca agggtaggga 7260
tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta aaaagaccga 7320
agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct 7380
gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt ctcctacagt 7440
cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag 7500
cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa aaaaccccat 7560
cgactttctg gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca ttaagcttcc 7620
caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga 7680
gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct tgtatctggc 7740
cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc agctgttcgt 7800
ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgagt tctccaaaag 7860
agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata agcacaggga 7920
taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga ccaacttggg 7980
cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt acacctctac 8040
aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct atgaaacaag 8100
aatcgacctc tctcagctcg gtggagacaa gcgtcctgct gctactaaga aagctggtca 8160
agctaagaaa aagaaatgag tcgactctag accgcgtctg gaacaatcaa cctctggatt 8220
acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 8280
gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 8340
cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 8400
aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 8460
ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 8520
tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 8580
ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct 8640
ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 8700
cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 8760
cgagtcggat ctccctttgg gccgcctccc cgcctggaat taattctgca gtcgagacct 8820
agaaaaacat ggagcaatca caagtagcaa tacagcagct accaatgctg attgtgcctg 8880
gctagaagca caagaggagg aggaggtggg tttttccagt cacacctcag gtacctttaa 8940
gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aagaggggac 9000
tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 9060
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 9120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 9180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 9240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 9300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 9360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 9420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 9480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 9540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 9600
agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt 9660
cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggccttg acattgctag 9720
cgttttaccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt 9780
gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 9840
agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 9900
tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 9960
aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 10020
cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 10080
atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 10140
taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 10200
aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 10260
tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 10320
gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 10380
cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 10440
cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 10500
atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 10560
tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 10620
ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 10680
acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 10740
aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 10800
ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 10860
aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 10920
ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 10980
agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 11040
cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 11100
ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 11160
gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 11220
cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 11280
cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 11340
ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 11400
catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 11460
tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 11520
ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 11580
catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 11640
cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 11700
cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 11760
acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 11820
ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 11880
tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcaacttgt 11940
ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 12000
catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 12060
tctggatcaa ctggataact caagctaacc aaaatcatcc caaacttccc accccatacc 12120
ctattaccac tgccaattac ctagtggttt catttactct aaacctgtga ttcctctgaa 12180
ttattttcat tttaaagaaa ttgtatttgt taaatatgta ctacaaactt agtagttttt 12240
aaagaaattg tatttgttaa atatgtacta caaacttagt agt 12283
<210> 6
<211> 12284
<212> DNA
<213> 人工序列(Aritifical synthesis)
<400> 6
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga actacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgatacgcgt gcggccgccc ccttcaccga gggcctattt 2220
cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt 2280
aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt 2340
cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta 2400
acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgca 2460
ccaagagtcc ggctgctcgt ttcagagcta tgctggaaac agcatagcaa gttgaaataa 2520
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ggatccatta 2580
gacgcgtggg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 2640
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 2700
actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 2760
caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 2820
tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 2880
ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 2940
cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 3000
tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 3060
atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 3120
cagatcgcct gccatccacg ctgttttgac ctccatagaa gacaccgact ctactagagg 3180
atcgctagcg ctaccggact cagatctcga gctcaagctt cgaattcgcc accatgaccg 3240
agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc 3300
tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca 3360
tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca 3420
aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg 3480
aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc 3540
tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt 3600
ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg 3660
tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct 3720
ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg 3780
aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc ggctccggag 3840
ccacgaactt ctctctgtta aagcaagcag gcgacgtgga agaaaacccc ggtccggcta 3900
gcgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca 3960
aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc 4020
cagcagccga caagaagtac tccattgggc tcgatatcgg cacaaacagc gtcggctggg 4080
ccgtcattac ggacgagtac aaggtgccga gcaaaaaatt caaagttctg ggcaataccg 4140
atcgccacag cataaagaag aacctcattg gcgccctcct gttcgactcc ggggagaccg 4200
ccgaagccac gcggctcaaa agaacagcac ggcgcagata tacccgcaga aagaatcgga 4260
tctgctacct gcaggagatc tttagtaatg agatggctaa ggtggatgac tctttcttcc 4320
ataggctgga ggagtccttt ttggtggagg aggataaaaa gcacgagcgc cacccaatct 4380
ttggcaatat cgtggacgag gtggcgtacc atgaaaagta cccaaccata tatcatctga 4440
ggaagaagct tgtagacagt actgataagg ctgacttgcg gttgatctat ctcgcgctgg 4500
cgcatatgat caaatttcgg ggacacttcc tcatcgaggg ggacctgaac ccagacaaca 4560
gcgatgtgga caaactcttt atccaactgg ttcagactta caatcagctt ttcgaagaga 4620
acccgatcaa cgcatccgga gttgacgcca aagcaatcct gagcgctagg ctgtccaaat 4680
cccggcggct cgaaaacctc atcgcacagc tccctgggga gaagaagaac ggcctgtttg 4740
gtaatcttat cgccctgtca ctcgggctga cccccaactt taaatctaac ttcgacctgg 4800
ccgaagatgc caagcttcaa ctgagcaaag acacctacga tgatgatctc gacaatctgc 4860
tggcccagat cggcgaccag tacgcagacc tttttttggc ggcaaagaac ctgtcagacg 4920
ccattctgct gagtgatatt ctgcgagtga acacggagat caccaaagct ccgctgagcg 4980
ctagtatgat caagcgctat gatgagcacc accaagactt gactttgctg aaggcccttg 5040
tcagacagca actgcctgag aagtacaagg aaattttctt cgatcagtct aaaaatggct 5100
acgccggata cattgacggc ggagcaagcc aggaggaatt ttacaaattt attaagccca 5160
tcttggaaaa aatggacggc accgaggagc tgctggtaaa gcttaacaga gaagatctgt 5220
tgcgcaaaca gcgcactttc gacaatggaa gcatccccca ccagattcac ctgggcgaac 5280
tgcacgctat cctcaggcgg caagaggatt tctacccctt tttgaaagat aacagggaaa 5340
agattgagaa aatcctcaca tttcggatac cctactatgt aggccccctc gcccggggaa 5400
attccagatt cgcgtggatg actcgcaaat cagaagagac catcactccc tggaacttcg 5460
aggaagtcgt ggataagggg gcctctgccc agtccttcat cgaaaggatg actaactttg 5520
ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc tctgctgtac gagtacttca 5580
cagtttataa cgagctcacc aaggtcaaat acgtcacaga agggatgaga aagccagcat 5640
tcctgtctgg agagcagaag aaagctatcg tggacctcct cttcaagacg aaccggaaag 5700
ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat tgaatgtttc gactctgttg 5760
aaatcagcgg agtggaggat cgcttcaacg catccctggg aacgtatcac gatctcctga 5820
aaatcattaa agacaaggac ttcctggaca atgaggagaa cgaggacatt cttgaggaca 5880
ttgtcctcac ccttacgttg tttgaagata gggagatgat tgaagaacgc ttgaaaactt 5940
acgctcatct cttcgacgac aaagtcatga aacagctcaa gaggcgccga tatacaggat 6000
gggggcggct gtcaagaaaa ctgatcaatg gcatccgaga caagcagagt ggaaagacaa 6060
tcctggattt tcttaagtcc gatggatttg ccaaccggaa cttcatgcag ttgatccatg 6120
atgactctct cacctttaag gaggacatcc agaaagcaca agtttctggc cagggggaca 6180
gtcttcacga gcacatcgct aatcttgcag gtagcccagc tatcaaaaag ggaatactgc 6240
agaccgttaa ggtcgtggat gaactcgtca aagtaatggg aaggcataag cccgagaata 6300
tcgttatcga gatggcccga gagaaccaaa ctacccagaa gggacagaag aacagtaggg 6360
aaaggatgaa gaggattgaa gagggtataa aagaactggg gtcccaaatc cttaaggaac 6420
acccagttga aaacacccag cttcagaatg agaagctcta cctgtactac ctgcagaacg 6480
gcagggacat gtacgtggat caggaactgg acatcaatcg gctctccgac tacgacgtgg 6540
atcatatcgt gccccagtct tttctcaaag atgattctat tgataataaa gtgttgacaa 6600
gatccgataa aaatagaggg aagagtgata acgtcccctc agaagaagtt gtcaagaaaa 6660
tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat cacacaacgg aagttcgata 6720
atctgactaa ggctgaacga ggtggcctgt ctgagttgga taaagccggc ttcatcaaaa 6780
ggcagcttgt tgagacacgc cagatcacca agcacgtggc ccaaattctc gattcacgca 6840
tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga ggtgaaagtt attactctga 6900
agtctaagct ggtctcagat ttcagaaagg actttcagtt ttataaggtg agagagatca 6960
acaattacca ccatgcgcat gatgcctacc tgaatgcagt ggtaggcact gcacttatca 7020
aaaaatatcc caagcttgaa tctgaatttg tttacggaga ctataaagtg tacgatgtta 7080
ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc caccgctaag tacttctttt 7140
acagcaatat tatgaatttt ttcaagaccg agattacact ggccaatgga gagattcgga 7200
agcgaccact tatcgaaaca aacggagaaa caggagaaat cgtgtgggac aagggtaggg 7260
atttcgcgac agtccggaag gtcctgtcca tgccgcaggt gaacatcgtt aaaaagaccg 7320
aagtacagac cggaggcttc tccaaggaaa gtatcctccc gaaaaggaac agcgacaagc 7380
tgatcgcacg caaaaaagat tgggacccca agaaatacgg cggattcgat tctcctacag 7440
tcgcttacag tgtactggtt gtggccaaag tggagaaagg gaagtctaaa aaactcaaaa 7500
gcgtcaagga actgctgggc atcacaatca tggagcgatc aagcttcgaa aaaaacccca 7560
tcgactttct ggaggcgaaa ggatataaag aggtcaaaaa agacctcatc attaagcttc 7620
ccaagtactc tctctttgag cttgaaaacg gccggaaacg aatgctcgct agtgcgggcg 7680
agctgcagaa aggtaacgag ctggcactgc cctctaaata cgttaatttc ttgtatctgg 7740
ccagccacta tgaaaagctc aaagggtctc ccgaagataa tgagcagaag cagctgttcg 7800
tggaacaaca caaacactac cttgatgaga tcatcgagca aataagcgag ttctccaaaa 7860
gagtgatcct cgccgacgct aacctcgata aggtgctttc tgcttacaat aagcacaggg 7920
ataagcccat cagggagcag gcagaaaaca ttatccactt gtttactctg accaacttgg 7980
gcgcgcctgc agccttcaag tacttcgaca ccaccataga cagaaagcgg tacacctcta 8040
caaaggaggt cctggacgcc acactgattc atcagtcaat tacggggctc tatgaaacaa 8100
gaatcgacct ctctcagctc ggtggagaca agcgtcctgc tgctactaag aaagctggtc 8160
aagctaagaa aaagaaatga gtcgactcta gaccgcgtct ggaacaatca acctctggat 8220
tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 8280
ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 8340
tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 8400
caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 8460
accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 8520
ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 8580
tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc 8640
tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 8700
ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 8760
acgagtcgga tctccctttg ggccgcctcc ccgcctggaa ttaattctgc agtcgagacc 8820
tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct 8880
ggctagaagc acaagaggag gaggaggtgg gtttttccag tcacacctca ggtaccttta 8940
agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagagggga 9000
ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac 9060
acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt cagatatcca 9120
ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt agaagaggcc 9180
aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat ggatgacccg 9240
gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca cgtggcccga 9300
gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 9360
gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg agccctcaga 9420
tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag accagatctg 9480
agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 9540
ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 9600
cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat 9660
tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggcctt gacattgcta 9720
gcgttttacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg 9780
tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 9840
aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 9900
ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 9960
gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 10020
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 10080
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 10140
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 10200
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 10260
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 10320
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 10380
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 10440
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 10500
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 10560
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 10620
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 10680
aacaaaccac cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 10740
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 10800
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 10860
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 10920
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 10980
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 11040
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 11100
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 11160
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 11220
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 11280
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 11340
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 11400
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 11460
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 11520
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 11580
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 11640
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 11700
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 11760
cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 11820
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 11880
ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatcaacttg 11940
tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 12000
gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 12060
gtctggatca actggataac tcaagctaac caaaatcatc ccaaacttcc caccccatac 12120
cctattacca ctgccaatta cctagtggtt tcatttactc taaacctgtg attcctctga 12180
attattttca ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagtttt 12240
taaagaaatt gtatttgtta aatatgtact acaaacttag tagt 12284
<210> 7
<211> 12284
<212> DNA
<213> 人工序列(Aritifical synthesis)
<400> 7
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga actacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgatacgcgt gcggccgccc ccttcaccga gggcctattt 2220
cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt 2280
aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt 2340
cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta 2400
acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgat 2460
cctgagcagc cggactctgt ttcagagcta tgctggaaac agcatagcaa gttgaaataa 2520
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ggatccatta 2580
gacgcgtggg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 2640
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 2700
actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 2760
caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 2820
tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 2880
ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 2940
cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 3000
tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 3060
atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 3120
cagatcgcct gccatccacg ctgttttgac ctccatagaa gacaccgact ctactagagg 3180
atcgctagcg ctaccggact cagatctcga gctcaagctt cgaattcgcc accatgaccg 3240
agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc 3300
tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca 3360
tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca 3420
aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg 3480
aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc 3540
tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt 3600
ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg 3660
tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct 3720
ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg 3780
aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc ggctccggag 3840
ccacgaactt ctctctgtta aagcaagcag gcgacgtgga agaaaacccc ggtccggcta 3900
gcgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca 3960
aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc 4020
cagcagccga caagaagtac tccattgggc tcgatatcgg cacaaacagc gtcggctggg 4080
ccgtcattac ggacgagtac aaggtgccga gcaaaaaatt caaagttctg ggcaataccg 4140
atcgccacag cataaagaag aacctcattg gcgccctcct gttcgactcc ggggagaccg 4200
ccgaagccac gcggctcaaa agaacagcac ggcgcagata tacccgcaga aagaatcgga 4260
tctgctacct gcaggagatc tttagtaatg agatggctaa ggtggatgac tctttcttcc 4320
ataggctgga ggagtccttt ttggtggagg aggataaaaa gcacgagcgc cacccaatct 4380
ttggcaatat cgtggacgag gtggcgtacc atgaaaagta cccaaccata tatcatctga 4440
ggaagaagct tgtagacagt actgataagg ctgacttgcg gttgatctat ctcgcgctgg 4500
cgcatatgat caaatttcgg ggacacttcc tcatcgaggg ggacctgaac ccagacaaca 4560
gcgatgtgga caaactcttt atccaactgg ttcagactta caatcagctt ttcgaagaga 4620
acccgatcaa cgcatccgga gttgacgcca aagcaatcct gagcgctagg ctgtccaaat 4680
cccggcggct cgaaaacctc atcgcacagc tccctgggga gaagaagaac ggcctgtttg 4740
gtaatcttat cgccctgtca ctcgggctga cccccaactt taaatctaac ttcgacctgg 4800
ccgaagatgc caagcttcaa ctgagcaaag acacctacga tgatgatctc gacaatctgc 4860
tggcccagat cggcgaccag tacgcagacc tttttttggc ggcaaagaac ctgtcagacg 4920
ccattctgct gagtgatatt ctgcgagtga acacggagat caccaaagct ccgctgagcg 4980
ctagtatgat caagcgctat gatgagcacc accaagactt gactttgctg aaggcccttg 5040
tcagacagca actgcctgag aagtacaagg aaattttctt cgatcagtct aaaaatggct 5100
acgccggata cattgacggc ggagcaagcc aggaggaatt ttacaaattt attaagccca 5160
tcttggaaaa aatggacggc accgaggagc tgctggtaaa gcttaacaga gaagatctgt 5220
tgcgcaaaca gcgcactttc gacaatggaa gcatccccca ccagattcac ctgggcgaac 5280
tgcacgctat cctcaggcgg caagaggatt tctacccctt tttgaaagat aacagggaaa 5340
agattgagaa aatcctcaca tttcggatac cctactatgt aggccccctc gcccggggaa 5400
attccagatt cgcgtggatg actcgcaaat cagaagagac catcactccc tggaacttcg 5460
aggaagtcgt ggataagggg gcctctgccc agtccttcat cgaaaggatg actaactttg 5520
ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc tctgctgtac gagtacttca 5580
cagtttataa cgagctcacc aaggtcaaat acgtcacaga agggatgaga aagccagcat 5640
tcctgtctgg agagcagaag aaagctatcg tggacctcct cttcaagacg aaccggaaag 5700
ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat tgaatgtttc gactctgttg 5760
aaatcagcgg agtggaggat cgcttcaacg catccctggg aacgtatcac gatctcctga 5820
aaatcattaa agacaaggac ttcctggaca atgaggagaa cgaggacatt cttgaggaca 5880
ttgtcctcac ccttacgttg tttgaagata gggagatgat tgaagaacgc ttgaaaactt 5940
acgctcatct cttcgacgac aaagtcatga aacagctcaa gaggcgccga tatacaggat 6000
gggggcggct gtcaagaaaa ctgatcaatg gcatccgaga caagcagagt ggaaagacaa 6060
tcctggattt tcttaagtcc gatggatttg ccaaccggaa cttcatgcag ttgatccatg 6120
atgactctct cacctttaag gaggacatcc agaaagcaca agtttctggc cagggggaca 6180
gtcttcacga gcacatcgct aatcttgcag gtagcccagc tatcaaaaag ggaatactgc 6240
agaccgttaa ggtcgtggat gaactcgtca aagtaatggg aaggcataag cccgagaata 6300
tcgttatcga gatggcccga gagaaccaaa ctacccagaa gggacagaag aacagtaggg 6360
aaaggatgaa gaggattgaa gagggtataa aagaactggg gtcccaaatc cttaaggaac 6420
acccagttga aaacacccag cttcagaatg agaagctcta cctgtactac ctgcagaacg 6480
gcagggacat gtacgtggat caggaactgg acatcaatcg gctctccgac tacgacgtgg 6540
atcatatcgt gccccagtct tttctcaaag atgattctat tgataataaa gtgttgacaa 6600
gatccgataa aaatagaggg aagagtgata acgtcccctc agaagaagtt gtcaagaaaa 6660
tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat cacacaacgg aagttcgata 6720
atctgactaa ggctgaacga ggtggcctgt ctgagttgga taaagccggc ttcatcaaaa 6780
ggcagcttgt tgagacacgc cagatcacca agcacgtggc ccaaattctc gattcacgca 6840
tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga ggtgaaagtt attactctga 6900
agtctaagct ggtctcagat ttcagaaagg actttcagtt ttataaggtg agagagatca 6960
acaattacca ccatgcgcat gatgcctacc tgaatgcagt ggtaggcact gcacttatca 7020
aaaaatatcc caagcttgaa tctgaatttg tttacggaga ctataaagtg tacgatgtta 7080
ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc caccgctaag tacttctttt 7140
acagcaatat tatgaatttt ttcaagaccg agattacact ggccaatgga gagattcgga 7200
agcgaccact tatcgaaaca aacggagaaa caggagaaat cgtgtgggac aagggtaggg 7260
atttcgcgac agtccggaag gtcctgtcca tgccgcaggt gaacatcgtt aaaaagaccg 7320
aagtacagac cggaggcttc tccaaggaaa gtatcctccc gaaaaggaac agcgacaagc 7380
tgatcgcacg caaaaaagat tgggacccca agaaatacgg cggattcgat tctcctacag 7440
tcgcttacag tgtactggtt gtggccaaag tggagaaagg gaagtctaaa aaactcaaaa 7500
gcgtcaagga actgctgggc atcacaatca tggagcgatc aagcttcgaa aaaaacccca 7560
tcgactttct ggaggcgaaa ggatataaag aggtcaaaaa agacctcatc attaagcttc 7620
ccaagtactc tctctttgag cttgaaaacg gccggaaacg aatgctcgct agtgcgggcg 7680
agctgcagaa aggtaacgag ctggcactgc cctctaaata cgttaatttc ttgtatctgg 7740
ccagccacta tgaaaagctc aaagggtctc ccgaagataa tgagcagaag cagctgttcg 7800
tggaacaaca caaacactac cttgatgaga tcatcgagca aataagcgag ttctccaaaa 7860
gagtgatcct cgccgacgct aacctcgata aggtgctttc tgcttacaat aagcacaggg 7920
ataagcccat cagggagcag gcagaaaaca ttatccactt gtttactctg accaacttgg 7980
gcgcgcctgc agccttcaag tacttcgaca ccaccataga cagaaagcgg tacacctcta 8040
caaaggaggt cctggacgcc acactgattc atcagtcaat tacggggctc tatgaaacaa 8100
gaatcgacct ctctcagctc ggtggagaca agcgtcctgc tgctactaag aaagctggtc 8160
aagctaagaa aaagaaatga gtcgactcta gaccgcgtct ggaacaatca acctctggat 8220
tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 8280
ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 8340
tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 8400
caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 8460
accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 8520
ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 8580
tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc 8640
tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 8700
ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 8760
acgagtcgga tctccctttg ggccgcctcc ccgcctggaa ttaattctgc agtcgagacc 8820
tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct 8880
ggctagaagc acaagaggag gaggaggtgg gtttttccag tcacacctca ggtaccttta 8940
agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagagggga 9000
ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac 9060
acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt cagatatcca 9120
ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt agaagaggcc 9180
aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat ggatgacccg 9240
gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca cgtggcccga 9300
gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 9360
gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg agccctcaga 9420
tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag accagatctg 9480
agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 9540
ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 9600
cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat 9660
tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggcctt gacattgcta 9720
gcgttttacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg 9780
tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 9840
aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 9900
ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 9960
gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 10020
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 10080
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 10140
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 10200
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 10260
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 10320
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 10380
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 10440
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 10500
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 10560
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 10620
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 10680
aacaaaccac cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 10740
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 10800
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 10860
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 10920
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 10980
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 11040
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 11100
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 11160
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 11220
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 11280
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 11340
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 11400
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 11460
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 11520
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 11580
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 11640
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 11700
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 11760
cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 11820
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 11880
ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatcaacttg 11940
tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 12000
gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 12060
gtctggatca actggataac tcaagctaac caaaatcatc ccaaacttcc caccccatac 12120
cctattacca ctgccaatta cctagtggtt tcatttactc taaacctgtg attcctctga 12180
attattttca ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagtttt 12240
taaagaaatt gtatttgtta aatatgtact acaaacttag tagt 12284
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Aritifical synthesis)
<400> 8
<210> 9
<211> 21
<212> DNA
<213> 人工序列(Aritifical synthesis)
<400> 9
<210> 10
<211> 18
<212> DNA
<213> 人工序列(Aritifical synthesis)
<400> 10
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Aritifical synthesis)
<400> 11

Claims (6)

1.一种CRISPR/Cas9 靶向敲除SANIL1基因及其特异性的sgRNA，其特征在于，首先设计获得特异性靶向SANIL1 基因外显子的sgRNA；其次构建SANIL1基因的sgRNA到病毒载体系统，然后将此质粒或者病毒载体系统转染或者感染人细胞，得到的SANIL1基因敲除细胞株。
2.根据权利要求1所述的特异性靶向SANIL1 基因外显子的sgRNA，其DNA 序列如SEQID NO.1、2、3所示，所述sgRNA 在SANIL1基因上的靶序列是唯一的。
3.根据权利要求2所述的特异性靶向SANIL1 基因外显子的sgRNA，其引物包括
针对SANIL1基因的靶点位于外显子上的引物对：
SANIL1sgRNAoligo1：如SEQ ID NO.2所示；
SANIL1sgRNAoligo2：如SEQ ID NO.3所示。
4.根据权利要求1所述的构建SANIL1基因的sgRNA到病毒载体系统，是一种靶向敲除SANIL1 基因的CRISPR/Cas9 重组表达病毒载体Lenti-CRISPRv2-hSANIL1sg，其特征在于：含有特异性靶向SANIL1 基因显子的sgRNA 和Cas9 蛋白的重组表达载体。
5.根据权利要求4所述的重组表达载体的骨架载体的序列如序列表中SEQ ID NO ：4所示；
根据权利要求1所述的CRISPR/Cas9 靶向敲除人SANIL1基因及其特异性的sgRNA，其特征在于包括如下步骤：
(1) 提供sgRNA序列，所述sgRNA 在SANIL1基因上的靶序列符合5’-N(21)G 的序列排列规则，所述sgRNA 在SANIL1基因上的靶序列位于基因的外显子，所述sgRNA 在SANIL1 基因上的靶序列位于不同的各种剪切形式的共有外显子上，所述sgRNA 在SANIL1基因上的靶序列是唯一的，并且所述sgRNA 在SANIL1 上的靶位点序列如序列表SEQ ID NO.1、2、3序列所示任意一条，在所述sgRNA 在SANIL1 上的靶位点序列的5’- 端加上ACCG序列 合成得到正向寡核苷酸即Forward oligo ；获得sgRNA 在SANIL1 上的靶位点序列的互补链，并且在互补链的5’ - 端加上AAAC序列， 合成得到反向寡核苷酸即Reverse oligo ；将合成的1对互补的sgRNA 寡聚核苷酸的Forward oligo 和Reverse oligo 成对变性、退火，退火之后形成可以连入U6 真核表达载体的双链sgRNA寡聚核苷酸；
(2) 线性化序列如序列表SEQ ID NO.4 所示的Lenti-CRISPRv2质粒；将退火的双链sgRNA 寡聚核苷酸与线性化的携带Cas9 基因的表达载体Lenti-CRISPRv2连接获得携带含相应靶序列的sgRNA 寡聚核苷酸和Cas9 基因的表达载体Lenti-CRISPRv2-hSANIL1sg 质粒，转化感受态细菌并涂Amp+ 平板，挑取单克隆并用通用引物U6 通过测序鉴定出阳性克隆，并对所述阳性克隆摇菌、提取pLenti-U6-SANIL1 gRNA v2.0-CMV-Puro-P2A-3Flag-spCas9质粒；
（3）用所述携带有sgRNA 寡聚核苷酸和Cas9 基因的序列为SEQ ID NO.9的表达载体Lenti-CRISPRv2-hSANIL1sg 质粒、序列为SEQ ID NO.10和SEQ ID NO.11 的pLP-VSVG(AddGene 8454) and psPAX2 (AddGene 12260)包装质粒和包装细胞系，包装细胞系为293TN 细胞；包装出同时携带靶向SANIL1基因的sgRNA 和Cas9 的假型病毒；
（4）使用所述假型病毒感染目的细胞，加puromycin抗性筛选后并进一步培养；
（5）铺单克隆细胞，然后收集单克隆细胞，抽提基因组DNA,以其基因组DNA 为模板扩增包含所述靶序列的基因片段，TA 克隆测序确认SANIL1 基因已经被敲除并获得基因敲除的细胞。
6.根据权利要求1所述的敲除SANIL1基因的细胞株，为人细胞；其特征在于：是人细胞中的SANIL1基因缺失或插入核苷酸得到的细胞株。
CN201810259431.8A 2018-03-27 2018-03-27 CRISPR-Cas9靶向敲除人细胞SANIL1基因及其特异性的sgRNA Withdrawn CN108410877A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810259431.8A CN108410877A (zh) 2018-03-27 2018-03-27 CRISPR-Cas9靶向敲除人细胞SANIL1基因及其特异性的sgRNA

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810259431.8A CN108410877A (zh) 2018-03-27 2018-03-27 CRISPR-Cas9靶向敲除人细胞SANIL1基因及其特异性的sgRNA

Publications (1)

Publication Number Publication Date
CN108410877A true CN108410877A (zh) 2018-08-17

Family

ID=63133616

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810259431.8A Withdrawn CN108410877A (zh) 2018-03-27 2018-03-27 CRISPR-Cas9靶向敲除人细胞SANIL1基因及其特异性的sgRNA

Country Status (1)

Country Link
CN (1) CN108410877A (zh)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
CN113046330A (zh) * 2021-03-23 2021-06-29 中吉智药(南京)生物技术有限公司 携带红系基因编辑系统的慢病毒及药物
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105518137A (zh) * 2015-06-11 2016-04-20 深圳市第二人民医院 CRISPR-Cas9特异性敲除猪SALL1基因的方法及用于特异性靶向SALL1基因的sgRNA
CN105886498A (zh) * 2015-05-13 2016-08-24 沈志荣 CRISPR-Cas9特异性敲除人PCSK9基因的方法以及用于特异性靶向PCSK9基因的sgRNA

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105886498A (zh) * 2015-05-13 2016-08-24 沈志荣 CRISPR-Cas9特异性敲除人PCSK9基因的方法以及用于特异性靶向PCSK9基因的sgRNA
CN105518137A (zh) * 2015-06-11 2016-04-20 深圳市第二人民医院 CRISPR-Cas9特异性敲除猪SALL1基因的方法及用于特异性靶向SALL1基因的sgRNA

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DING ET AL: "Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome Editing", 《CIRCULATION RESEARCH》 *
XM 015139585.2: "PREDICTED: Macaca mulatta proprotein convertase subtilisin/kexin type 9 (PCSK9), transcript variant X3, Mrna", 《NCBI DATABASE》 *

Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
CN113046330B (zh) * 2021-03-23 2023-03-21 中吉智药(南京)生物技术有限公司 携带红系基因编辑系统的慢病毒及药物
CN113046330A (zh) * 2021-03-23 2021-06-29 中吉智药(南京)生物技术有限公司 携带红系基因编辑系统的慢病毒及药物

Similar Documents

Publication Publication Date Title
CN108410877A (zh) CRISPR-Cas9靶向敲除人细胞SANIL1基因及其特异性的sgRNA
CN107893075A (zh) CRISPR‑Cas9靶向敲除人肠癌细胞RITA基因及其特异性的sgRNA
CN108642053A (zh) CRISPR-Cas9靶向敲除人肠癌细胞PPP1R1C基因及其特异性的sgRNA
AU2018229561B2 (en) Recombinant adenoviruses and use thereof
CN108588071A (zh) CRISPR-Cas9靶向敲除人肠癌细胞CNR1基因及其特异性的sgRNA
KR102370675B1 (ko) 표적 핵산의 변형을 위한 개선된 방법
ES2805045T3 (es) Vectores lentivirales
AU2014273089B2 (en) A LAGLIDADG homing endonuclease cleaving the C-C Chemokine Receptor Type-5 (CCR5) gene and uses thereof
CN109563505A (zh) 用于真核细胞的组装系统
CN108559745A (zh) 基于CRISPR-Cas9技术提高B16F10细胞转染效率的方法
KR20180107155A (ko) Cpf1 또는 csm1을 사용하여 게놈을 변형하기 위한 조성물 및 방법
CA2441937A1 (en) Chromosome-based platforms
KR20130132405A (ko) 형질전환 빈도를 증가시키기 위해 변형된 아그로박테리움 균주
KR20210116712A (ko) 말라리아 백신
CN114181957B (zh) 一种基于病毒加帽酶的稳定t7表达系统及其在真核生物中表达蛋白质的方法
KR102614328B1 (ko) T-세포 수용체 합성 및 tcr-제시 세포에 대한 안정적인 게놈 통합을 위한 2-부분 디바이스
KR20210118402A (ko) 위스콧-알드리치(Wiskott-Aldrich) 증후군에 대한 조혈 줄기 세포-유전자 치료요법
KR20220034212A (ko) 재조합으로 변형된 아데노 연관 바이러스 헬퍼 벡터 및 재조합으로 변형된 아데노 연관 바이러스의 패키징 효율을 개선하기 위한 그의 용도
US20030180740A1 (en) Differential expression screening method
CN109762846B (zh) 利用碱基编辑修复与克拉伯病相关的galcc1586t突变的试剂和方法
CN113846124A (zh) 一种用于糖代谢相关疾病基因治疗的核酸构建体
CN114645066B (zh) 一种用于艾滋病基因治疗的核酸构建体
CN109666673B (zh) 利用碱基编辑修复与胆固醇酯贮积症相关的e8sjm-1g&gt;a突变的试剂和方法
CN110540962B (zh) 一种制备人定型内胚层细胞的方法
US20240082327A1 (en) Retroviral vectors

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication
WW01 Invention patent application withdrawn after publication

Application publication date: 20180817