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CN107164377A - 基于碱基编辑的基因敲除方法及其应用 - Google Patents

基于碱基编辑的基因敲除方法及其应用 Download PDF

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CN107164377A
CN107164377A CN201710437122.0A CN201710437122A CN107164377A CN 107164377 A CN107164377 A CN 107164377A CN 201710437122 A CN201710437122 A CN 201710437122A CN 107164377 A CN107164377 A CN 107164377A
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李广磊
尚小云
张琳琳
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Suzhou Mao Hang Bio Technology Co Ltd
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Abstract

基于碱基编辑的基因敲除方法及其应用。该基因敲除方法包括：选定待敲除基因的编码区的20bp–NGG目标序列，使其包含完整的目标密码子CAA、CAG或CGA；利用sgRNA序列来将BE3定位到目标序列以使目标密码子中的目标单碱基C变成T从而相应引入终止密码子TAA或TAG、TGA以实现基因敲除，其中目标单碱基C优选位于目标序列的第4‑8位，目标密码子与NGG间隔12‑14bp,并且目标密码子的上游紧邻碱基(H)不能为G；所述sgRNA序列为与目标序列互补对应的20bp序列。本发明的敲除方法更高效、更精确以及更少脱靶效应。

Description

基于碱基编辑的基因敲除方法及其应用
技术领域
本发明涉及基因敲除策略，更具体来说，涉及基于碱基编辑的基因敲除方法及其应用。
背景技术
传统的真核生物靶向基因操作是通过全能胚胎干细胞的同源重组和囊胚注射实现的。由于受到建立全能胚胎干细胞这一限制，主要是小鼠(也有大鼠的报道)可以通过全能胚胎干细胞的同源重组完成基因靶向改造[Capecchi,2005]。进行基因靶向操作的另一种途径是克隆，即体细胞的基因改造和核移植。不过，克隆技术存在一些缺陷[Carter etal.,2002；Zhu et al.,2004]。比如，1、终末分化的体细胞克隆后很难完全去分化成未分化细胞，影响胚胎发育，造成发育缺陷；2、所有的遗传物质仅为母源；3、成功率低等。传统的基因靶向操作技术制约了基因敲除。
可编程核酸内切酶技术包括锌指核酸酶(zinc-finger nucleases,ZFNs)技术和转录激活因子样效应物核酸酶(transcription activator-like effectors nucleases,TALENs)技术，以及规律成簇间隔短回文重复系统(clustered regularly interspacedshort palindromic repeat；CRISPR-associated，CRISPR/Cas9)[Kim and Kim,2014]。这类技术的发明和推广打破了全能胚胎干细胞的限制，使得不同物种的基因操作变得可能。特别是CRISPR/Cas9系统，由于简便、高效、价廉，出现之后立即席卷全球，成为了基因编辑领域最新，但发展最快、应用最广的技术，引发了基因编辑领域的革命。现在CRISPR/Cas9系统已经被成功地用于DNA敲除、DNA敲入、DNA替代、DNA修饰、RNA修饰、DNA标记、基因转录调节等[Hsu et al.,2014；Komor et al.,2017]。并已经成功应用于多个物种的基因编辑[Barrangou R&Doudna JA,2016；Komor et al.,2017]。
CRISPR/Cas9介导特异性基因编辑是利用sgRNA(single guided RNA)通过靶序列互补引导Cas9蛋白定位剪切双链DNA，造成双链DNA断裂(double-strand breaks,DSB)，在没有模板的条件下，发生非同源末端连接(non-homologous end joining,NHEJ)修复，造成移码突变(frameshift mutation)，导致基因敲除(knockout)；在有模板的条件下，通过同源重组进行修复(homology-directed repair,HDR)，实现基因敲入(knockin)[Hsu etal.,2014；Kim and Kim,2014；Komor et al.,2017]。由于HDR效率低(整合很少发生)，而且非同源性末端接合机制容易产生随机插入和删除(indel)，使得在断裂点附近可能随机引入新的碱基，从而导致不精确的基因编辑。此外，CRISPR/Cas9介导的基因编辑总有一些脱靶效应[Gorski et al.,2017]。
发明内容
本发明的目的之一是提供一种高效精准的基因敲除策略。
最新研究表明，基于CRISPR/Cas9技术所构建的Cas9融合蛋白可作为“碱基编辑器(Base editor，BE)”。这些融合蛋白包含dCas9或Cas9切口酶以及大鼠胞苷脱氨酶APOBEC1，它通过脱氨基作用将胞嘧啶(C)转化为尿嘧啶，而无需切割DNA。之后，通过DNA复制或修复，尿嘧啶被转化成胸腺嘧啶(T)。类似地，其也能将单碱基G转化成A。特别是Cas9切口酶与APOBEC1组成的BE3，其能够将碱基编辑效率显著提高到15～75％。由于不需切割DNA造成DSB，形成的indel低于1％，实现的基因编辑更精确[Komor et al.,2016]；而且，这种方式将脱靶效率降低到低于自然背景的10倍，实现的基因编辑更安全[Nishida et al.,2017]。BE3已经被成功地用于在体内碱基编辑，实现了小鼠的CT突变，效率达到44～57％[Kim etal.,2017]。
发明人基于上述BE介导单碱基突变，尤其是BE3介导的单碱基编辑的精确性和特异性，巧妙设计了一种基因敲除策略：通过CT突变引入终止密码子，例如将CAA、CAG、CGA突变成终止密码子TAA、TAG、TGA，或通过GA突变将TGG突变成终止密码子TAA、TGA、TAG，终止编码基因的翻译，从而实现基因敲除。
根据本发明的第一方面，提供了一种基因敲除方法，其包括：
选定待敲除基因的编码区(CDS区)的20bp–NGG目标序列(PAM序列)，使其包含完整的目标密码子CAA、CAG或CGA；
利用sgRNA序列来将BE3定位到目标序列以使目标密码子中的目标单碱基C变成T从而相应引入终止密码子TAA或TAG、TGA以实现基因敲除，
其中目标单碱基C位于目标序列的(左端)第1-8位，优选为第4-8位，目标密码子与NGG间隔12-14bp,优选为14bp，并且目标密码子的上游紧邻碱基(H)不能为G；
所述sgRNA序列为与目标序列互补对应的20bp序列。
可替代地，在上述方法中，也可选定待敲除基因的编码区的CCN-20bp目标序列(PAM序列)，使其包含完整的目标密码子TGG，目标密码子的下游紧邻碱基(D)不能为C；相应地，目标单碱基G位于目标序列的(右端)第1-8位，优选为第4-8位，目标密码子与CCN间隔12-14bp，优选为14bp。
根据本发明，BE3可以选自：rAPOBEC1-SaCas9-NLS-UGI-NLS；3xUGI-rAPOBEC1-SaCas9-NLS-UGI-NLS；rAPOBEC1-SpCas9-NLS-UGI-NLS；3xUGI-rAPOBEC1-SpCas9-NLS-UGI-NLS，优选采用后两种。
根据本发明的方法可以用于敲除如下八个靶基因：人PD1、LAG3、TIGIT、VISTA、2B4、CD160以及小鼠TIM3和LAG3，与其相应的sgRNA序列分别与序列一至八所示目标基因序列互补。
根据本发明的第二方面，提供了上述方法在细胞系HEK293T进行人PD1、LAG3、TIGIT、VISTA、2B4、CD160基因敲除的应用。
根据本发明的第三方面，提供了上述方法在人T细胞进行人PD1、LAG3、TIGIT、VISTA、2B4、CD160基因敲除的应用。
根据本发明的第四方面，提供了根据上述应用而获得的分离的T细胞或细胞系或它们的次代培养物。
根据本发明的第五方面，提供了一种用于基因敲除的试剂盒，包括(与待敲除基因相应的)上述sgRNA、BE3以及相应的扩增试剂。
本发明利用CRISPR/Cas9基础上发展的碱基编辑技术，通过精准的CT或GA单碱基突变创造终止密码子，从而建立了比CRISPR/Cas9更高效、更精确以及更少脱靶效应的基因敲除策略。
附图说明
图1为根据本发明的利用CT突变实现目标基因被敲除的示意图；
图2-5为不同BE3的结构示意图。
具体实施方式
首先，构建不同的BE3，如图2-5所示，将不同的Cas9 nickase与胞苷脱氨酶(APOBEC1)融合后形成如下四种BE3：
(1)rAPOBEC1-SaCas9-NLS-UGI-NLS，图2，序列1；
(2)3xUGI-rAPOBEC1-SaCas9-NLS-UGI-NLS，图3，序列2；
(3)rAPOBEC1-SpCas9-NLS-UGI-NLS，图4，序列3；
(4)3xUGI-rAPOBEC1-SpCas9-NLS-UGI-NLS，图5，序列4。
在下面进行基因敲除时，可以采用上述任一种BE3，优选为(3)或(4)。
接下来进行sgRNA的设计。碱基定点编辑是利用sgRNA将BE3定位到或靶向特异靶位点，靶基因特异性sgRNA的选择和设计是本发明的关键之处。本发明如下选择设计sgRNA：
选定待敲除基因的编码区的20bp–NGG目标序列(PAM序列)，使其包含完整的目标密码子CAA、CAG或CGA；
目标单碱基C优选位于目标序列的(左端)第4-8位，目标密码子与NGG优选间隔14bp,并且目标密码子的上游紧邻碱基(H)不能为G；
制备与目标序列互补对应的20bp sgRNA序列。
在替代方案中，则选定待敲除基因的编码区的CCN-20bp目标序列(PAM序列)，使其包含完整的目标密码子TGG，目标密码子的下游紧邻碱基(D)不能为C；相应地，目标单碱基G优选位于目标序列的(右端)第4-8位，目标密码子优选与CCN间隔14bp。
针对八个靶基因——人PD1、LAG3、TIGIT、VISTA、2B4、CD160以及小鼠TIM3和LAG3，本发明选定下述目标基因序列来设计相应的sgRNA(粗体下划表示PAM；斜体下划表示候选突变编码子)：
一.hPD-1
Sg-1:
Sg-2:
Sg-3:
二.hLAG3
Sg-1:
Sg-2:
Sg-3:
三.hTIGIT
Sg-1:
四.hVISTA
Sg-1:
五.2B4
Sg-1:
六.hCD160
Sg-1:
七.mTIM3
Sg-1:
八.mLAG3
Sg-1:
针对上述选定的目标基因序列，人PD1(3条)、LAG3(3条)、TIGIT、VISTA、2B4、CD160以及鼠TIM3、LAG3，构建相应的sgRNA表达载体，将不同的sgRNA分别导入pGL3-U6-sgRNA。
实施例1
在细胞株上进行BE3介导的碱基编辑，引入终止密码子，实现基因敲除。按常规操作，进行细胞株的基因敲除(通过电转或脂质体转染)，以脂质体转染为例。
(1)以HEK293T细胞为例，本发明进行真核生物细胞的培养与转染：HEK293T细胞接种培养于添加10％FBS的DMEM高糖培养液中(HyClone,SH30022.01B),其中含penicillin(100U/ml)和streptomycin(100μg/ml)。
(2)在转染前分至6孔板中，待密度达到70％-80％时进行转染。
(3)转染以脂质体转染为例。按照LipofectamineTM 2000 Transfection Reagent(Invitrogen,11668-019)的操作手册，以SpCas9 nickase为例，将2μg BE3质粒与2μgpGL3-U6-sgRNA质粒混匀,共转染至每孔细胞中，6-8小时后换液，72小时后收取细胞。
(4)基因型分析
A、收取部分细胞在裂解液(10μM Tris-HCl,0.4M NaCl,2μM EDTA,1％SDS)中用100μg/ml蛋白酶K裂解消化后,酚-氯仿抽提后溶解到50μl去离子水中。
B、使用一对引物N-For和N-Rev进行PCR扩增，用AxyPrep PCR cleanup纯化获得PCR回收产物，取200ng统一稀释到20μl进行变性、退火，程序如：95℃，5min；95-85℃at-2℃/s；85-25℃at-0.1℃/s；hold at 4℃。
C、获得的PCR回收产物用rTaq进行加A反应。加A反应体系为：
700-800ng PCR回收产物
5μl 10X Buffer(Mg2+PLUS)
4μl dNTP
0.5μl rTaq(TAKARA,R001AM)
补水至50μl体系。
37℃温育30分钟后，取1μl产物与pMD19-T vector(TAKARA,3271)连接并转化DH5感受态细胞(TransGen,CD201)。
D、挑取单克隆，用通用引物M13-F测序各靶基因突变，测序结果如下(粗体下划表示PAM；斜体表示突变编码子；斜体下划表示突变碱基)：
1.hPD-1
Sg-1:
Mut:
Sg-2:
Mut:
Sg-3:
Mut:
2.hLAG3
Sg-1:
Mut:
Sg-2:
Mut:
Sg-3:
Mut:
3.hTIGIT
Sg-1：
Mut:
4.hVISTA
Sg-1:
Mut:
5.2B4
Sg-1:
Mut:
6.hCD160
Sg-1:
Mut:
结果表明：靶基因发生了sgRNA靶向的碱基突变，引入了终止密码子，PD1、LAG3、TIGIT、VISTA、2B4、CD160等基因敲除成功。
实施例2
在原代细胞上进行BE3介导的碱基编辑，引入终止密码子，实现基因敲除。
按常规操作，以人的T细胞进原代细胞的基因敲除(通过电转或脂质体转染)，以电转为例。
(1)PBMC细胞的分离纯化：
A、用抗凝管采集外周血，边采集边摇晃使外周血与抗凝剂充分混合；
B、外周血细胞与淋巴细胞分离液等体积混合，离心，吸取离心后的白膜层细胞；
C、将得到的白膜层细胞与PBS或者无血清细胞培养基1640混合后离心，沉淀即为所述PBMC细胞。
重复三遍。
(2)CD3阳性细胞的富集
A、调整PBMC细胞浓度至50x106cell/ml。
B、按每1ml加入CD3+enriched antibodies cocktail 50μl，混匀后室温静置5分钟。
C、按每1ml加入magnet 150μl，混匀后室温静置10分钟，
D、将离心管置于磁力架上静置5分钟，吸取上层细胞悬液至新的15ml离心管中。
E、重复该操作一次。
F、室温离心300*g，10分钟，收集细胞。
G、细胞计数。
(3)CD3阳性细胞的电转
A、配置电转体系
向1.5ml离心管中分别加入8μg BE3质粒与8μg pGL3-U6-sgRNA质粒，并按照LonzaAmaxa电转试剂盒说明书要求，加入82μl电转缓冲液和18μl supplement1,混匀。
B、收取20X106个细胞到15ml离心管中，300g离心10分钟，弃掉上清。
C、以A中配好的质粒电转缓冲液混合物重悬细胞，并转移至电转杯中。
D、使用仪器Lonza 2B,U-014程序进行电转。
E、电转后的细胞迅速转移至提前预热的添加有10％FBS的AIM-V培养基中，37度5％二氧化碳培养箱中培养2小时。
F、电转后的细胞全换液，以1X106个/ml的密度重悬细胞，培养过夜。
(4)T细胞的激活培养
A、电转培养24小时后，向培养基中加入100U/ml IL-2，并按照1:1的比例加入CD3/CD28dynabeads，激活T细胞。
B、每两天对细胞半换液，或者是补加IL-2，细胞密度始终维持在1X106个/ml。
C、激活5天后，将T细胞收集到15ml离心管中，并将离心管置于磁力架中，慢慢将上清转移到另外一个干净的15ml离心管中，重复此步骤一次。
D、室温离心300*g，10分钟，弃上清，使用10％FBS,300U/ml IL-2AIM-V培养基重悬细胞，密度控制在1X106个/ml。
E、每两天对细胞半换液，或者是补加IL-2，并计数，细胞密度始终维持在1X106个/ml。
(5)基因型分析
A、收取部分细胞在裂解液(10μM Tris-HCl,0.4M NaCl,2μM EDTA,1％SDS)中用100μg/ml蛋白酶K裂解消化后,酚-氯仿抽提后溶解到50μl去离子水中。
B、使用一对引物N-For和N-Rev进行PCR扩增，用AxyPrep PCR cleanup纯化获得PCR回收产物，取200ng统一稀释到20μl进行变性、退火，程序如：95℃，5min；95-85℃at-2℃/s；85-25℃at-0.1℃/s；hold at 4℃。
C、获得的PCR回收产物用rTaq进行加A反应。加A反应体系为：
700-800ng PCR回收产物
5μl 10X Buffer(Mg2+PLUS)
4μl dNTP
0.5μl rTaq(TAKARA,R001AM)
补水至50μl体系。
37℃温育30分钟后，取1μl产物与pMD19-T vector(TAKARA,3271)连接并转化DH5感受态细胞(TransGen,CD201)。
D、挑取单克隆，用通用引物M13-F测序T细胞各靶基因突变，测序结果如下(粗体下划表示PAM；斜体表示候选突变编码子；斜体下划表示突变碱基)：
1.hPD-1
Sg-1:
Mut:
Sg-2:
Mut:
Sg-3:
Mut:
2.hLAG3
Sg-1:
Mut:
Sg-2:
Mut:
Sg-3:
Mut:
3.hTIGIT
Sg-1:
Mut:
4.hVISTA
Sg-1:
Mut:
5.2B4
Sg-1:
Mut:
6.hCD160
Sg-1:
Mut:
结果表明：靶基因发生了sgRNA靶向的碱基突变，引入了终止密码子，PD1、LAG3、TIGIT、VISTA、2B4、CD160等基因敲除成功。
实施例3
构建BE3介导的基因敲除小鼠
按常规操作进行小鼠的胚胎收集、显微注射、胚胎培养和胚胎移植等。以TIM3和LAG3基因为例构建敲除小鼠。
(1)显微注射：受精卵分别注射BE3mRNA和TIM3特异性sgRNA(对应于上述序列七)，或BE3mRNA和LAG3特异性sgRNA(对应于上述序列八)。常规进行胚胎移植；
(2)基因型分析：常规小鼠剪尾提取基因组DNA，分别PCR扩增编码区域，Sanger测序，测序结果如下(粗体下划表示PAM；斜体表示突变编码子；斜体下划表示突变碱基)：
7.mTIM3
Sg-1:
Mut:
8.mLAG3
Sg-1:
Mut:
上述结果确证TIM3和LAG3的CT突变和终止密码子的引入。构建TIM3和LAG3敲除小鼠成功。
　　序 列 表
<110> 黄行许
<120> 基于碱基编辑的基因敲除方法及其应用
<160> 4
<210> 1
<211> 7686
<212> DNA
<213> 人工序列
<400> 1
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 420
actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480
gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540
gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600
aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660
acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720
tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780
ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 840
actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900
gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960
atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020
ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080
gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140
agtaagcgga actatatcct cgggctggct attggcatca catctgtcgg ctatggtata 1200
atagactatg aaacaaggga cgtgattgac gcaggtgtga ggctgttcaa ggaggcaaac 1260
gtcgagaaca acgaaggtcg gagaagcaag aggggtgccc ggaggctgaa gaggaggaga 1320
aggcacagaa tacagcgggt caagaaactc ctgttcgact ataacctgct gaccgatcat 1380
tccgaactgt caggcatcaa tccttacgaa gccagagtca agggtctgtc tcaaaaactc 1440
tctgaggaag agttttccgc agccctgctg cacctggcta agaggagagg agtccacaac 1500
gtcaatgagg ttgaggagga tacagggaac gaactgtcta caaaggaaca gatcagccgg 1560
aatagcaagg ccctggaaga gaagtacgtt gctgaactgc agctggaaag gctcaagaaa 1620
gatggagagg ttcggggttc catcaacagg ttcaagacat ctgactatgt gaaggaagcc 1680
aagcaactgc tcaaggtgca gaaggcctac catcagctcg accagagctt cattgatact 1740
tacatagacc tgctggagac taggagaact tactacgaag ggcctggcga gggcagccct 1800
ttcggctgga aagatatcaa ggagtggtac gagatgctca tggggcattg cacctacttc 1860
cccgaagaac tgaggtcagt caagtacgcc tacaacgcag acctgtacaa cgccctgaat 1920
gatctcaaca atctcgtcat aactcgggat gaaaacgaga agctggaata ttatgagaag 1980
ttccagatta ttgaaaatgt gttcaaacag aagaagaaac ctaccctgaa acaaattgcc 2040
aaagagatcc tggtgaatga ggaggatatc aagggatatc gggttacttc taccggcaaa 2100
ccagagttca caaatctgaa agtttaccat gacatcaaag atattaccgc aagaaaggag 2160
atcatcgaga acgctgagct cctggaccag atcgctaaga ttctcactat ctatcagtcc 2220
agcgaggata ttcaggaaga gctgaccaac ctgaactcag agctgactca ggaagaaatc 2280
gaacaaatct ccaatctgaa aggatacact ggtacccata atctctcact caaggctatc 2340
aatctgatcc tggatgaact gtggcatact aacgacaatc agatcgccat cttcaatcgg 2400
ctcaaactgg tgcccaaaaa agtggacctg agccaacaga aagagattcc tacaaccctg 2460
gtggacgatt tcattctgag cccagtggtt aagcggagct tcatccaatc catcaaggtg 2520
atcaacgcta tcatcaagaa gtatggcctg cctaatgaca taatcattga actcgcaagg 2580
gaaaagaata gcaaagatgc ccagaagatg ataaacgaga tgcagaaacg gaacagacag 2640
actaacgaaa gaatcgagga aataatacgg accactggta aggagaacgc taagtatctg 2700
atcgagaaaa tcaagctgca cgatatgcag gaaggcaagt gcctgtattc tctggaggct 2760
atacccctgg aggatctgct caataatcct ttcaattacg aggtggatca catcatacca 2820
agatccgtga gctttgacaa tagctttaat aataaggtgc tcgtgaagca ggaggaaaac 2880
tcaaagaaag gcaacaggac cccattccag tacctgtcca gctctgacag caagattagc 2940
tacgaaacct tcaagaaaca catcctgaat ctggccaagg gcaagggaag aataagcaaa 3000
acaaagaaag agtatctcct ggaggaaagg gacatcaaca ggttttcagt gcagaaagat 3060
tttatcaatc ggaatctcgt tgacacaaga tatgctacca gagggctcat gaatctgctc 3120
aggtcatact ttagggtgaa caacctggat gtgaaggtca aatccataaa tggagggttc 3180
acttcctttc tcaggagaaa atggaagttt aagaaagaga gaaataaggg ttacaaacat 3240
cacgccgagg acgcactgat cattgccaac gctgacttta tctttaagga atggaagaag 3300
ctggacaaag caaagaaggt gatggagaat cagatgtttg aggaaaagca ggccgagtct 3360
atgcctgaga ttgaaacaga gcaggaatac aaagagatct tcattactcc acatcagatt 3420
aagcacatca aggactttaa ggactataaa tactcacata gggtggataa gaaacctaat 3480
agagagctga tcaacgatac actctactca acaaggaaag acgacaaagg aaacaccctg 3540
attgttaata atctcaatgg gctgtatgac aaagataatg acaagctgaa gaaactcatc 3600
aacaagtccc cagaaaagct gctgatgtat caccacgatc cccaaacata tcagaagctg 3660
aagctgatta tggagcagta tggtgatgag aagaaccctc tgtacaagta ctatgaagag 3720
acagggaact acctcactaa gtacagcaag aaagacaacg gacccgttat caagaagatc 3780
aagtactacg gcaataagct gaacgcccac ctggatatca cagatgacta tccaaactct 3840
aggaacaaag tggtgaaact gtccctgaag ccatacagat ttgatgtgta tctggataac 3900
ggagtctata agttcgtcac agtcaagaac ctggacgtca tcaagaagga gaattactat 3960
gaagtgaaca gcaaatgcta cgaggaagcc aagaagctca agaagatttc taaccaggca 4020
gagtttatcg cctctttcta caataacgat ctgatcaaga tcaacggaga actgtacaga 4080
gtgatcggcg tgaataatga cctcctgaat aggatcgagg ttaacatgat cgatatcaca 4140
tatcgggagt acctggagaa tatgaatgac aagaggcctc ccagaattat caagactatt 4200
gccagcaaaa cccaatctat aaaaaagtac tcaacagata tcctggggaa cctgtatgag 4260
gtgaagtcaa agaagcatcc ccagattatc aagaaaggcg gcagccccaa gaagaagagg 4320
aaggtgagca gcgactacaa ggaccacgac ggcgactaca aggaccacga catcgactac 4380
aaggacgacg acgacaagTC Tggtggttct actaatctgt cagatattat tgaaaaggag 4440
accggtaagc aactggttat ccaggaatcc atcctcatgc tcccagagga ggtggaagaa 4500
gtcattggga acaagccgga aagcgatata ctcgtgcaca ccgcctacga cgagagcacc 4560
gacgagaatg tcatgcttct gactagcgac gcccctgaat acaagccttg ggctctggtc 4620
atacaggata gcaacggtga gaacaagatt aagatgctct ctggtggttc tcccaagaag 4680
aagaggaaag tctaaccggt catcatcacc atcaccattg agtttaaacc cgctgatcag 4740
cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 4800
tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 4860
attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 4920
aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg 4980
cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg gcgtaatcat 5040
ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 5100
ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc acattaattg 5160
cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 5220
tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 5280
ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 5340
taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 5400
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 5460
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 5520
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 5580
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 5640
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 5700
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 5760
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5820
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 5880
gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5940
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 6000
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 6060
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 6120
ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 6180
atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 6240
tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 6300
gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 6360
ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 6420
caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 6480
cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 6540
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 6600
cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 6660
agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 6720
tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 6780
agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 6840
atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 6900
ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 6960
cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 7020
caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 7080
attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 7140
agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg 7200
acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca atctgctctg 7260
atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 7320
gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 7380
tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 7440
attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 7500
atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 7560
acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 7620
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 7680
tgtatc 7686
<210> 2
<211> 8670
<212> DNA
<213> 人工序列
<400> 2
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccgg gcccatgact 420
aatctgtcag atattattga aaaggagacc ggtaagcaac tggttatcca ggaatccatc 480
ctcatgctcc cagaggaggt ggaagaagtc attgggaaca agccggaaag cgatatactc 540
gtgcacaccg cctacgacga gagcaccgac gagaatgtca tgcttctgac tagcgacgcc 600
cctgaataca agccttgggc tctggtcata caggatagca acggtgagaa caagattaag 660
atgctcccca agaagaagag gaaagtcgag ggcagaggaa gtctgctaac atgcggtgac 720
gtcgaggaga atcctggccc aaccaacctg tccgatatca ttgagaaaga gaccggcaaa 780
cagctggtga tccaggagag catcctgatg ctgcccgaag aggtggagga agtgatcggc 840
aacaagcccg agtccgacat cctggtgcac acagcctatg atgaatccac cgacgagaac 900
gtgatgctgc tgacctccga tgctcccgag tataaaccct gggcactggt gatccaggac 960
tctaatggag agaacaagat caagatgctg cccaagaaga agaggaaagt cgctactaac 1020
ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctac aaacctcagt 1080
gacattatcg agaaggaaac aggaaaacag ctcgtcattc aagaatctat tcttatgttg 1140
cctgaggaag tcgaagaggt tattggcaat aaacctgaat ctgatattct tgtccatacc 1200
gcttacgatg agtccacaga tgaaaatgtt atgctgctca catctgacgc accagagtac 1260
aaaccatggg cgctcgttat tcaagattcc aacggcgaaa acaaaatcaa aatgcttccc 1320
aagaagaaga ggaaagtcga aggacggggc tccctcctga cctgtggcga tgtggaagag 1380
aaccccggcc ccatgagctc agagactggc ccagtggctg tggaccccac attgagacgg 1440
cggatcgagc cccatgagtt tgaggtattc ttcgatccga gagagctccg caaggagacc 1500
tgcctgcttt acgaaattaa ttgggggggc cggcactcca tttggcgaca tacatcacag 1560
aacactaaca agcacgtcga agtcaacttc atcgagaagt tcacgacaga aagatatttc 1620
tgtccgaaca caaggtgcag cattacctgg tttctcagct ggagcccatg cggcgaatgt 1680
agtagggcca tcactgaatt cctgtcaagg tatccccacg tcactctgtt tatttacatc 1740
gcaaggctgt accaccacgc tgacccccgc aatcgacaag gcctgcggga tttgatctct 1800
tcaggtgtga ctatccaaat tatgactgag caggagtcag gatactgctg gagaaacttt 1860
gtgaattata gcccgagtaa tgaagcccac tggcctaggt atccccatct gtgggtacga 1920
ctgtacgttc ttgaactgta ctgcatcata ctgggcctgc ctccttgtct caacattctg 1980
agaaggaagc agccacagct gacattcttt accatcgctc ttcagtcttg tcattaccag 2040
cgactgcccc cacacattct ctgggccacc gggttgaaaa gcggcagcga gactcccggg 2100
acctcagagt ccgccacacc cgaaagtaag cggaactata tcctcgggct ggctattggc 2160
atcacatctg tcggctatgg tataatagac tatgaaacaa gggacgtgat tgacgcaggt 2220
gtgaggctgt tcaaggaggc aaacgtcgag aacaacgaag gtcggagaag caagaggggt 2280
gcccggaggc tgaagaggag gagaaggcac agaatacagc gggtcaagaa actcctgttc 2340
gactataacc tgctgaccga tcattccgaa ctgtcaggca tcaatcctta cgaagccaga 2400
gtcaagggtc tgtctcaaaa actctctgag gaagagtttt ccgcagccct gctgcacctg 2460
gctaagagga gaggagtcca caacgtcaat gaggttgagg aggatacagg gaacgaactg 2520
tctacaaagg aacagatcag ccggaatagc aaggccctgg aagagaagta cgttgctgaa 2580
ctgcagctgg aaaggctcaa gaaagatgga gaggttcggg gttccatcaa caggttcaag 2640
acatctgact atgtgaagga agccaagcaa ctgctcaagg tgcagaaggc ctaccatcag 2700
ctcgaccaga gcttcattga tacttacata gacctgctgg agactaggag aacttactac 2760
gaagggcctg gcgagggcag ccctttcggc tggaaagata tcaaggagtg gtacgagatg 2820
ctcatggggc attgcaccta cttccccgaa gaactgaggt cagtcaagta cgcctacaac 2880
gcagacctgt acaacgccct gaatgatctc aacaatctcg tcataactcg ggatgaaaac 2940
gagaagctgg aatattatga gaagttccag attattgaaa atgtgttcaa acagaagaag 3000
aaacctaccc tgaaacaaat tgccaaagag atcctggtga atgaggagga tatcaaggga 3060
tatcgggtta cttctaccgg caaaccagag ttcacaaatc tgaaagttta ccatgacatc 3120
aaagatatta ccgcaagaaa ggagatcatc gagaacgctg agctcctgga ccagatcgct 3180
aagattctca ctatctatca gtccagcgag gatattcagg aagagctgac caacctgaac 3240
tcagagctga ctcaggaaga aatcgaacaa atctccaatc tgaaaggata cactggtacc 3300
cataatctct cactcaaggc tatcaatctg atcctggatg aactgtggca tactaacgac 3360
aatcagatcg ccatcttcaa tcggctcaaa ctggtgccca aaaaagtgga cctgagccaa 3420
cagaaagaga ttcctacaac cctggtggac gatttcattc tgagcccagt ggttaagcgg 3480
agcttcatcc aatccatcaa ggtgatcaac gctatcatca agaagtatgg cctgcctaat 3540
gacataatca ttgaactcgc aagggaaaag aatagcaaag atgcccagaa gatgataaac 3600
gagatgcaga aacggaacag acagactaac gaaagaatcg aggaaataat acggaccact 3660
ggtaaggaga acgctaagta tctgatcgag aaaatcaagc tgcacgatat gcaggaaggc 3720
aagtgcctgt attctctgga ggctataccc ctggaggatc tgctcaataa tcctttcaat 3780
tacgaggtgg atcacatcat accaagatcc gtgagctttg acaatagctt taataataag 3840
gtgctcgtga agcaggagga aaactcaaag aaaggcaaca ggaccccatt ccagtacctg 3900
tccagctctg acagcaagat tagctacgaa accttcaaga aacacatcct gaatctggcc 3960
aagggcaagg gaagaataag caaaacaaag aaagagtatc tcctggagga aagggacatc 4020
aacaggtttt cagtgcagaa agattttatc aatcggaatc tcgttgacac aagatatgct 4080
accagagggc tcatgaatct gctcaggtca tactttaggg tgaacaacct ggatgtgaag 4140
gtcaaatcca taaatggagg gttcacttcc tttctcagga gaaaatggaa gtttaagaaa 4200
gagagaaata agggttacaa acatcacgcc gaggacgcac tgatcattgc caacgctgac 4260
tttatcttta aggaatggaa gaagctggac aaagcaaaga aggtgatgga gaatcagatg 4320
tttgaggaaa agcaggccga gtctatgcct gagattgaaa cagagcagga atacaaagag 4380
atcttcatta ctccacatca gattaagcac atcaaggact ttaaggacta taaatactca 4440
catagggtgg ataagaaacc taatagagag ctgatcaacg atacactcta ctcaacaagg 4500
aaagacgaca aaggaaacac cctgattgtt aataatctca atgggctgta tgacaaagat 4560
aatgacaagc tgaagaaact catcaacaag tccccagaaa agctgctgat gtatcaccac 4620
gatccccaaa catatcagaa gctgaagctg attatggagc agtatggtga tgagaagaac 4680
cctctgtaca agtactatga agagacaggg aactacctca ctaagtacag caagaaagac 4740
aacggacccg ttatcaagaa gatcaagtac tacggcaata agctgaacgc ccacctggat 4800
atcacagatg actatccaaa ctctaggaac aaagtggtga aactgtccct gaagccatac 4860
agatttgatg tgtatctgga taacggagtc tataagttcg tcacagtcaa gaacctggac 4920
gtcatcaaga aggagaatta ctatgaagtg aacagcaaat gctacgagga agccaagaag 4980
ctcaagaaga tttctaacca ggcagagttt atcgcctctt tctacaataa cgatctgatc 5040
aagatcaacg gagaactgta cagagtgatc ggcgtgaata atgacctcct gaataggatc 5100
gaggttaaca tgatcgatat cacatatcgg gagtacctgg agaatatgaa tgacaagagg 5160
cctcccagaa ttatcaagac tattgccagc aaaacccaat ctataaaaaa gtactcaaca 5220
gatatcctgg ggaacctgta tgaggtgaag tcaaagaagc atccccagat tatcaagaaa 5280
ggcggcagcc ccaagaagaa gaggaaggtg agcagcgact acaaggacca cgacggcgac 5340
tacaaggacc acgacatcga ctacaaggac gacgacgaca agTCTggtgg ttctactaat 5400
ctgtcagata ttattgaaaa ggagaccggt aagcaactgg ttatccagga atccatcctc 5460
atgctcccag aggaggtgga agaagtcatt gggaacaagc cggaaagcga tatactcgtg 5520
cacaccgcct acgacgagag caccgacgag aatgtcatgc ttctgactag cgacgcccct 5580
gaatacaagc cttgggctct ggtcatacag gatagcaacg gtgagaacaa gattaagatg 5640
ctctctggtg gttctcccaa gaagaagagg aaagtctaac cggtcatcat caccatcacc 5700
attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 5760
tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 5820
aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 5880
gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 5940
cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata ccgtcgacct 6000
ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 6060
tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag ggtgcctaat 6120
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 6180
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 6240
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 6300
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 6360
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 6420
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 6480
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 6540
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 6600
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 6660
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 6720
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 6780
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 6840
ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 6900
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 6960
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 7020
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 7080
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 7140
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 7200
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 7260
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 7320
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 7380
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 7440
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 7500
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 7560
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 7620
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 7680
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 7740
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 7800
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 7860
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 7920
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 7980
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 8040
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 8100
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 8160
cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt 8220
cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt 8280
gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct 8340
tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg 8400
tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta 8460
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 8520
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 8580
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 8640
ctgcccactt ggcagtacat caagtgtatc 8670
<210> 3
<211> 8532
<212> DNA
<213> 人工序列
<400> 3
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 420
actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480
gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540
gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600
aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660
acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720
tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780
ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 840
actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900
gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960
atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020
ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080
gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140
agtgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 1200
ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 1260
cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 1320
gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 1380
tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 1440
ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 1500
aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 1560
aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 1620
atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 1680
gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 1740
ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 1800
cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 1860
cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 1920
gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 1980
caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 2040
ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 2100
atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 2160
cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 2220
ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 2280
gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 2340
aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 2400
gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 2460
gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 2520
cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 2580
gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 2640
aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 2700
tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 2760
agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 2820
gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 2880
tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 2940
attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 3000
ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 3060
cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 3120
cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 3180
gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 3240
tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 3300
cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 3360
gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 3420
atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 3480
atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 3540
gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 3600
gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 3660
attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 3720
gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 3780
aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 3840
actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 3900
ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 3960
acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 4020
aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 4080
taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 4140
tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 4200
atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 4260
aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 4320
cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 4380
gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 4440
cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 4500
gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 4560
tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 4620
aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 4680
ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 4740
tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 4800
caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 4860
cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 4920
cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 4980
atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 5040
cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 5100
ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 5160
gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 5220
gatttgtcac agcttggggg tgactctggt ggttctacta atctgtcaga tattattgaa 5280
aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5340
gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5400
agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5460
ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5520
aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 5580
gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5640
cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5700
catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5760
agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 5820
ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 5880
aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 5940
tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 6000
taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6060
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6120
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6180
aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6240
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6300
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6360
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6420
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6480
ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 6540
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6600
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6660
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6720
acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6780
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6840
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6900
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6960
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7020
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7080
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7140
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7200
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7260
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7320
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7380
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7440
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7500
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7560
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7620
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7680
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7740
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7800
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7860
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7920
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7980
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8040
acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 8100
gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 8160
agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 8220
agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 8280
gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8340
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8400
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8460
ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8520
atcaagtgta tc 8532
<210> 4
<211> 9516
<212> DNA
<213> 人工序列
<400> 4
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccgg gcccatgact 420
aatctgtcag atattattga aaaggagacc ggtaagcaac tggttatcca ggaatccatc 480
ctcatgctcc cagaggaggt ggaagaagtc attgggaaca agccggaaag cgatatactc 540
gtgcacaccg cctacgacga gagcaccgac gagaatgtca tgcttctgac tagcgacgcc 600
cctgaataca agccttgggc tctggtcata caggatagca acggtgagaa caagattaag 660
atgctcccca agaagaagag gaaagtcgag ggcagaggaa gtctgctaac atgcggtgac 720
gtcgaggaga atcctggccc aaccaacctg tccgatatca ttgagaaaga gaccggcaaa 780
cagctggtga tccaggagag catcctgatg ctgcccgaag aggtggagga agtgatcggc 840
aacaagcccg agtccgacat cctggtgcac acagcctatg atgaatccac cgacgagaac 900
gtgatgctgc tgacctccga tgctcccgag tataaaccct gggcactggt gatccaggac 960
tctaatggag agaacaagat caagatgctg cccaagaaga agaggaaagt cgctactaac 1020
ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctac aaacctcagt 1080
gacattatcg agaaggaaac aggaaaacag ctcgtcattc aagaatctat tcttatgttg 1140
cctgaggaag tcgaagaggt tattggcaat aaacctgaat ctgatattct tgtccatacc 1200
gcttacgatg agtccacaga tgaaaatgtt atgctgctca catctgacgc accagagtac 1260
aaaccatggg cgctcgttat tcaagattcc aacggcgaaa acaaaatcaa aatgcttccc 1320
aagaagaaga ggaaagtcga aggacggggc tccctcctga cctgtggcga tgtggaagag 1380
aaccccggcc ccatgagctc agagactggc ccagtggctg tggaccccac attgagacgg 1440
cggatcgagc cccatgagtt tgaggtattc ttcgatccga gagagctccg caaggagacc 1500
tgcctgcttt acgaaattaa ttgggggggc cggcactcca tttggcgaca tacatcacag 1560
aacactaaca agcacgtcga agtcaacttc atcgagaagt tcacgacaga aagatatttc 1620
tgtccgaaca caaggtgcag cattacctgg tttctcagct ggagcccatg cggcgaatgt 1680
agtagggcca tcactgaatt cctgtcaagg tatccccacg tcactctgtt tatttacatc 1740
gcaaggctgt accaccacgc tgacccccgc aatcgacaag gcctgcggga tttgatctct 1800
tcaggtgtga ctatccaaat tatgactgag caggagtcag gatactgctg gagaaacttt 1860
gtgaattata gcccgagtaa tgaagcccac tggcctaggt atccccatct gtgggtacga 1920
ctgtacgttc ttgaactgta ctgcatcata ctgggcctgc ctccttgtct caacattctg 1980
agaaggaagc agccacagct gacattcttt accatcgctc ttcagtcttg tcattaccag 2040
cgactgcccc cacacattct ctgggccacc gggttgaaaa gcggcagcga gactcccggg 2100
acctcagagt ccgccacacc cgaaagtgat aaaaagtatt ctattggttt agccatcggc 2160
actaattccg ttggatgggc tgtcataacc gatgaataca aagtaccttc aaagaaattt 2220
aaggtgttgg ggaacacaga ccgtcattcg attaaaaaga atcttatcgg tgccctccta 2280
ttcgatagtg gcgaaacggc agaggcgact cgcctgaaac gaaccgctcg gagaaggtat 2340
acacgtcgca agaaccgaat atgttactta caagaaattt ttagcaatga gatggccaaa 2400
gttgacgatt ctttctttca ccgtttggaa gagtccttcc ttgtcgaaga ggacaagaaa 2460
catgaacggc accccatctt tggaaacata gtagatgagg tggcatatca tgaaaagtac 2520
ccaacgattt atcacctcag aaaaaagcta gttgactcaa ctgataaagc ggacctgagg 2580
ttaatctact tggctcttgc ccatatgata aagttccgtg ggcactttct cattgagggt 2640
gatctaaatc cggacaactc ggatgtcgac aaactgttca tccagttagt acaaacctat 2700
aatcagttgt ttgaagagaa ccctataaat gcaagtggcg tggatgcgaa ggctattctt 2760
agcgcccgcc tctctaaatc ccgacggcta gaaaacctga tcgcacaatt acccggagag 2820
aagaaaaatg ggttgttcgg taaccttata gcgctctcac taggcctgac accaaatttt 2880
aagtcgaact tcgacttagc tgaagatgcc aaattgcagc ttagtaagga cacgtacgat 2940
gacgatctcg acaatctact ggcacaaatt ggagatcagt atgcggactt atttttggct 3000
gccaaaaacc ttagcgatgc aatcctccta tctgacatac tgagagttaa tactgagatt 3060
accaaggcgc cgttatccgc ttcaatgatc aaaaggtacg atgaacatca ccaagacttg 3120
acacttctca aggccctagt ccgtcagcaa ctgcctgaga aatataagga aatattcttt 3180
gatcagtcga aaaacgggta cgcaggttat attgacggcg gagcgagtca agaggaattc 3240
tacaagttta tcaaacccat attagagaag atggatggga cggaagagtt gcttgtaaaa 3300
ctcaatcgcg aagatctact gcgaaagcag cggactttcg acaacggtag cattccacat 3360
caaatccact taggcgaatt gcatgctata cttagaaggc aggaggattt ttatccgttc 3420
ctcaaagaca atcgtgaaaa gattgagaaa atcctaacct ttcgcatacc ttactatgtg 3480
ggacccctgg cccgagggaa ctctcggttc gcatggatga caagaaagtc cgaagaaacg 3540
attactccat ggaattttga ggaagttgtc gataaaggtg cgtcagctca atcgttcatc 3600
gagaggatga ccaactttga caagaattta ccgaacgaaa aagtattgcc taagcacagt 3660
ttactttacg agtatttcac agtgtacaat gaactcacga aagttaagta tgtcactgag 3720
ggcatgcgta aacccgcctt tctaagcgga gaacagaaga aagcaatagt agatctgtta 3780
ttcaagacca accgcaaagt gacagttaag caattgaaag aggactactt taagaaaatt 3840
gaatgcttcg attctgtcga gatctccggg gtagaagatc gatttaatgc gtcacttggt 3900
acgtatcatg acctcctaaa gataattaaa gataaggact tcctggataa cgaagagaat 3960
gaagatatct tagaagatat agtgttgact cttaccctct ttgaagatcg ggaaatgatt 4020
gaggaaagac taaaaacata cgctcacctg ttcgacgata aggttatgaa acagttaaag 4080
aggcgtcgct atacgggctg gggacgattg tcgcggaaac ttatcaacgg gataagagac 4140
aagcaaagtg gtaaaactat tctcgatttt ctaaagagcg acggcttcgc caataggaac 4200
tttatgcagc tgatccatga tgactcttta accttcaaag aggatataca aaaggcacag 4260
gtttccggac aaggggactc attgcacgaa catattgcga atcttgctgg ttcgccagcc 4320
atcaaaaagg gcatactcca gacagtcaaa gtagtggatg agctagttaa ggtcatggga 4380
cgtcacaaac cggaaaacat tgtaatcgag atggcacgcg aaaatcaaac gactcagaag 4440
gggcaaaaaa acagtcgaga gcggatgaag agaatagaag agggtattaa agaactgggc 4500
agccagatct taaaggagca tcctgtggaa aatacccaat tgcagaacga gaaactttac 4560
ctctattacc tacaaaatgg aagggacatg tatgttgatc aggaactgga cataaaccgt 4620
ttatctgatt acgacgtcga tcacattgta ccccaatcct ttttgaagga cgattcaatc 4680
gacaataaag tgcttacacg ctcggataag aaccgaggga aaagtgacaa tgttccaagc 4740
gaggaagtcg taaagaaaat gaagaactat tggcggcagc tcctaaatgc gaaactgata 4800
acgcaaagaa agttcgataa cttaactaaa gctgagaggg gtggcttgtc tgaacttgac 4860
aaggccggat ttattaaacg tcagctcgtg gaaacccgcc aaatcacaaa gcatgttgca 4920
cagatactag attcccgaat gaatacgaaa tacgacgaga acgataagct gattcgggaa 4980
gtcaaagtaa tcactttaaa gtcaaaattg gtgtcggact tcagaaagga ttttcaattc 5040
tataaagtta gggagataaa taactaccac catgcgcacg acgcttatct taatgccgtc 5100
gtagggaccg cactcattaa gaaatacccg aagctagaaa gtgagtttgt gtatggtgat 5160
tacaaagttt atgacgtccg taagatgatc gcgaaaagcg aacaggagat aggcaaggct 5220
acagccaaat acttctttta ttctaacatt atgaatttct ttaagacgga aatcactctg 5280
gcaaacggag agatacgcaa acgaccttta attgaaacca atggggagac aggtgaaatc 5340
gtatgggata agggccggga cttcgcgacg gtgagaaaag ttttgtccat gccccaagtc 5400
aacatagtaa agaaaactga ggtgcagacc ggagggtttt caaaggaatc gattcttcca 5460
aaaaggaata gtgataagct catcgctcgt aaaaaggact gggacccgaa aaagtacggt 5520
ggcttcgata gccctacagt tgcctattct gtcctagtag tggcaaaagt tgagaaggga 5580
aaatccaaga aactgaagtc agtcaaagaa ttattgggga taacgattat ggagcgctcg 5640
tcttttgaaa agaaccccat cgacttcctt gaggcgaaag gttacaagga agtaaaaaag 5700
gatctcataa ttaaactacc aaagtatagt ctgtttgagt tagaaaatgg ccgaaaacgg 5760
atgttggcta gcgccggaga gcttcaaaag gggaacgaac tcgcactacc gtctaaatac 5820
gtgaatttcc tgtatttagc gtcccattac gagaagttga aaggttcacc tgaagataac 5880
gaacagaagc aactttttgt tgagcagcac aaacattatc tcgacgaaat catagagcaa 5940
atttcggaat tcagtaagag agtcatccta gctgatgcca atctggacaa agtattaagc 6000
gcatacaaca agcacaggga taaacccata cgtgagcagg cggaaaatat tatccatttg 6060
tttactctta ccaacctcgg cgctccagcc gcattcaagt attttgacac aacgatagat 6120
cgcaaacgat acacttctac caaggaggtg ctagacgcga cactgattca ccaatccatc 6180
acgggattat atgaaactcg gatagatttg tcacagcttg ggggtgacTC Tggtggttct 6240
actaatctgt cagatattat tgaaaaggag accggtaagc aactggttat ccaggaatcc 6300
atcctcatgc tcccagagga ggtggaagaa gtcattggga acaagccgga aagcgatata 6360
ctcgtgcaca ccgcctacga cgagagcacc gacgagaatg tcatgcttct gactagcgac 6420
gcccctgaat acaagccttg ggctctggtc atacaggata gcaacggtga gaacaagatt 6480
aagatgctct ctggtggttc tcccaagaag aagaggaaag tctaaccggt catcatcacc 6540
atcaccattg agtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat 6600
ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 6660
tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg 6720
ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg 6780
gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tcgataccgt 6840
cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 6900
atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctagggtg 6960
cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 7020
gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 7080
gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 7140
ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 7200
acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 7260
cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 7320
caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 7380
gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 7440
tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 7500
aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 7560
ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 7620
cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 7680
tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 7740
tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 7800
ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 7860
aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 7920
aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 7980
aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 8040
gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 8100
gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 8160
caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 8220
ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 8280
attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 8340
ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 8400
gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 8460
ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 8520
tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 8580
gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 8640
cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 8700
gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 8760
tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 8820
ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 8880
gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 8940
tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 9000
catttccccg aaaagtgcca cctgacgtcg acggatcggg agatcgatct cccgatcccc 9060
tagggtcgac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc 9120
ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc 9180
aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc 9240
gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat 9300
caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg 9360
taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 9420
atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 9480
ggtaaactgc ccacttggca gtacatcaag tgtatc 9516

Claims (7)

1.一种基因敲除方法，包括：
选定待敲除基因的编码区的20bp–NGG目标序列，使其包含完整的目标密码子CAA、CAG或CGA；
利用sgRNA序列来将BE3定位到目标序列以使目标密码子中的目标单碱基C变成T从而相应引入终止密码子TAA或TAG、TGA以实现基因敲除，
其中目标单碱基C位于目标序列的第1-8位，目标密码子与NGG间隔12-14bp,并且目标密码子的上游紧邻碱基不能为G；
所述sgRNA序列为与目标序列互补对应的20bp序列。
2.根据权利要求1所述的方法，其中BE3选自：rAPOBEC1-SaCas9-NLS-UGI-NLS；3xUGI-rAPOBEC1-SaCas9-NLS-UGI-NLS；rAPOBEC1-SpCas9-NLS-UGI-NLS；3xUGI-rAPOBEC1-SpCas9-NLS-UGI-NLS。
3.根据权利要求1所述的方法，用于敲除如下八个靶基因：人PD1、LAG3、TIGIT、VISTA、2B4、CD160以及小鼠TIM3和LAG3，与其相应的sgRNA序列分别与序列一至八所示目标基因序列互补。
4.权利要求1所述方法在细胞系HEK293T进行人PD1、LAG3、TIGIT、VISTA、2B4、CD160基因敲除的应用。
5.权利要求1所述方法在人T细胞进行人PD1、LAG3、TIGIT、VISTA、2B4、CD160基因敲除的应用。
6.根据权利要求4或5的应用而获得的分离的T细胞或细胞系或它们的次代培养物。
7.一种用于基因敲除的试剂盒，包括权利要求1或2中所述的sgRNA、BE3以及扩增试剂。
CN201710437122.0A 2017-06-12 2017-06-12 基于碱基编辑的基因敲除方法及其应用 Pending CN107164377A (zh)

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Cited By (13)

* Cited by examiner, † Cited by third party
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CN108504676A (zh) * 2018-02-05 2018-09-07 上海科技大学 一种pnCasSA-BEC质粒及其应用
CN108753823A (zh) * 2018-06-20 2018-11-06 李广磊 利用碱基编辑技术实现基因敲除的方法及其应用
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WO2019222971A1 (zh) * 2018-05-24 2019-11-28 深圳市博奥康生物科技有限公司 CRISPR/Cas9靶向敲除人VSTM3基因及其特异性gRNA
WO2020000439A1 (zh) * 2018-06-29 2020-01-02 深圳市博奥康生物科技有限公司 一种应用CRISPR/Cas9系统对小鼠CD366基因进行敲除的方法
CN112779288A (zh) * 2018-09-25 2021-05-11 肇庆华夏凯奇生物技术有限公司 基因敲除及试剂盒
WO2021155607A1 (zh) * 2020-02-07 2021-08-12 辉大(上海)生物科技有限公司 经改造的胞嘧啶碱基编辑器及其应用
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WO2018228183A1 (zh) * 2017-06-12 2018-12-20 苏州茂行生物科技有限公司 基于碱基编辑的基因敲除方法及其应用
CN108504676B (zh) * 2018-02-05 2021-12-10 上海科技大学 一种pnCasSA-BEC质粒及其应用
CN108504676A (zh) * 2018-02-05 2018-09-07 上海科技大学 一种pnCasSA-BEC质粒及其应用
WO2019222971A1 (zh) * 2018-05-24 2019-11-28 深圳市博奥康生物科技有限公司 CRISPR/Cas9靶向敲除人VSTM3基因及其特异性gRNA
CN108753823B (zh) * 2018-06-20 2022-09-23 李广磊 利用碱基编辑技术实现基因敲除的方法及其应用
CN108753823A (zh) * 2018-06-20 2018-11-06 李广磊 利用碱基编辑技术实现基因敲除的方法及其应用
WO2020000439A1 (zh) * 2018-06-29 2020-01-02 深圳市博奥康生物科技有限公司 一种应用CRISPR/Cas9系统对小鼠CD366基因进行敲除的方法
CN109022477A (zh) * 2018-07-12 2018-12-18 上海科技大学 一种pnCasPA-BEC质粒及其应用
CN108949825A (zh) * 2018-07-30 2018-12-07 苏州茂行生物科技有限公司 一种靶向her2的car-t细胞的制备方法及应用
CN112779288A (zh) * 2018-09-25 2021-05-11 肇庆华夏凯奇生物技术有限公司 基因敲除及试剂盒
CN109706185A (zh) * 2019-02-01 2019-05-03 国家卫生计生委科学技术研究所 基于碱基编辑系统突变起始密码子实现基因敲除的方法及应用
CN110042124A (zh) * 2019-04-25 2019-07-23 国家卫生健康委科学技术研究所 基因组碱基编辑增加人红细胞中胎儿血红蛋白水平的试剂盒及应用
WO2021155607A1 (zh) * 2020-02-07 2021-08-12 辉大(上海)生物科技有限公司 经改造的胞嘧啶碱基编辑器及其应用
WO2022152266A1 (zh) * 2021-01-18 2022-07-21 上海邦耀生物科技有限公司 用于基因编辑的组合物及其应用
CN115820728A (zh) * 2022-07-11 2023-03-21 上海贝斯昂科生物科技有限公司 一种基因编辑的方法和用途
WO2024012300A1 (zh) * 2022-07-11 2024-01-18 上海贝斯昂科生物科技有限公司 一种基因编辑的方法和用途
CN115820728B (zh) * 2022-07-11 2025-07-04 上海贝斯昂科生物科技有限公司 一种基因编辑的方法和用途

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