CN108642053A - CRISPR-Cas9靶向敲除人肠癌细胞PPP1R1C基因及其特异性的sgRNA - Google Patents

CRISPR-Cas9靶向敲除人肠癌细胞PPP1R1C基因及其特异性的sgRNA Download PDF

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CN108642053A
CN108642053A CN201810396783.8A CN201810396783A CN108642053A CN 108642053 A CN108642053 A CN 108642053A CN 201810396783 A CN201810396783 A CN 201810396783A CN 108642053 A CN108642053 A CN 108642053A
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ppp1r1c
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杨佳丽
杨兴林
潘讴东
夏清梅
刘梅锐
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Yuan Biotechnology (shanghai) Ltd By Share Ltd
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Abstract

本发明公开了CRISPR/Cas9靶向敲除人肠癌细胞PPP1R1C基因及其特异性sgRNA序列。首先是获得特异性靶向PPP1R1C基因功能区的sgRNA，其碱基序列如SEQ ID NO.1所示；其次是构建PPP1R1C基因的sgRNA到慢病毒载体系统，该系统含有Cas9蛋白；最后将含有该sgRNA的CRISPR/Cas9慢病毒感染人肠癌HT‑29细胞，获得PPP1R1C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、对PPP1R1C基因切割效率高；且所构建的CRISPR/Cas9慢病毒系统具有敲除效率高并能特异性敲除PPP1R1C基因，得到敲除PPP1R1C基因的人肠癌细胞，从而为进一步研究肠癌细胞中PPP1R1C的作用机制提供强有力的工具。

Description

CRISPR-Cas9靶向敲除人肠癌细胞PPP1R1C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靶向敲除人肠癌细胞PPP1R1C基因及其特异性的gRNA。
背景技术
蛋白质磷酸酶1(PP1)具有丝氨酸/苏氨酸磷酸酶的活性，广泛参与细胞信号转导过程。蛋白激酶和蛋白磷酸酶能够使多种蛋白质磷酸化和去磷酸化，从而改变酶、受体和离子通道蛋白质电荷、溶解性和结合能力等，它们广泛参与细胞分裂、基因表达调控、神经信息传递、肌肉收缩和细胞信号转导等重要生命过程。蛋白磷酸酶PP1和PP2A活性受损会导致细胞功能障碍，如发生肿瘤、神经元分化异常等。蛋白磷酸酶1调节抑制亚基1C(proteinphosphatase 1regulatory inhibitor subunit 1C，PPP1R1C)又被称作IPP5，是一个新型的蛋白磷酸酶1(PP1)抑制因子，在其高度保守的N-端有一个PP1结合模体和一个PP1抑制模体，并且其本身受磷酸化作用调控。人PPP1R1C基因位于染色体2q31.3-q32.1，其表达的蛋白广泛分布于心脏、睾丸及肝脏等器官。
肠癌是指发生于肠道的癌病类疾病，是胃肠道中常见的恶性肿瘤，发病率仅次于胃癌和食管癌。目前，研究人员在肠癌的发生机制方面做了大量研究，发现了众多参与肠癌进展的途径和机制，如原癌基因激活、抑癌基因失活(点突变、重排、缺失)及染色体异常等，但目前肠癌发生与进展中所涉及的分子机制仍有许多尚未明确，需要进一步研究。
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PPP1R1C基因的人肠癌细胞，为研究肠癌细胞中PPP1R1C的作用机制提供有力工具。
参考文献：
1)Bensaid M,Gary-Bobo M,Esclangon A,Maffrand J P,Le Fur G,Oury-Donat Fand Soubrie P,2003.The cannabinoid CB1receptor antagonist SR141716increasesAcrp30mRNA expression in adipose tissue of obese fa/fa rats and in culturedadipocyte cells.Molecular Pharmacology,63(4):908～914
2)Benzinou M,Chevre J C,Ward K J,Lecoeur C,Dina C,Lobbens S,Durand E,Delplanque J,Horber F F,Heude B,Balkau B,Borch-Johnsen K,Jorgensen T,HansenT,Pedersen O,Meyre D and Froguel P,2008.Endocannabinoid receptor 1genevariations increase risk for obesity and modulate body mass index in Europeanpopulations.Human Molecular Genetics,17(13):1916～1921
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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PPP1R1C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PPP1R1C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PPP1R1C基因的人肠癌细胞HT-29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PPP1R1C基因的sgRNA序列，选自DNA序列如下的PPP1R1C sgRNA：
PPP1R1C sgRNA的序列如下：
PPP1R1C sgRNA oligo1：5’-CACCGTCAGGGGTTAAGCATCTGAAGCCC-3’；
PPP1R1CsgRNA oligo2：5’-AAACTTCAGATGCTTAACCCCTGACGGTGC-3’；
一种靶向敲除PPP1R1C基因的CRISPR/Cas9慢病毒系统，含有上述靶向敲除PPP1R1C基因的sgRNA的DNA序列。
所述的靶向敲除PPP1R1C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PPP1R1C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PPP1R1C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PPP1R1C基因的CRISPR/Cas9慢病毒系统在制备敲除PPP1R1C基因的细胞株中的应用。
一种敲除PPP1R1C基因的细胞株，是将所述的靶向敲除PPP1R1C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PPP1R1C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PPP1R1C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PPP1R1C基因的细胞株。
所述的目的细胞株优选为肿瘤细胞株。
所述的肿瘤细胞株优选为肠癌细胞株。
所述的肠癌细胞株优选为人结肠癌永生化细胞HT-29。
所述的敲除PPP1R1C基因的细胞株，是HT-29细胞中的PPP1R1C基因缺失或插入核苷酸得到的细胞株，为如下的细胞株中的任一株：
细胞株13#是：sgRNA的切割位点位于在野生型PPP1R1C基因上的sgRNA PAM区前3个碱基处，导致PPP1R1C基因的外显子增加1个碱基；细胞株14#：是sgRNA的切割位点位于在野生型PPP1R1C基因上的sgRNA PAM区前2个碱基处，导致PPP1R1C基因的外显子缺失14个碱基。。PPP1R1C基因的具体靶向敲除位点与野生型PPP1R1C基因序列的对比如图6和图7。
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PPP1R1C基因的sgRNA能特异靶向PPP1R1C基因，将其构建入CRISPR/Cas9慢病毒系统，该系统能特异靶向敲除PPP1R1C基因，得到敲除PPP1R1C基因的细胞株，从而有利于研究细胞株中的PPP1R1C的作用机制。
附图说明
图1为本发明实施例中使用的载体质粒Lenti-CRISPR v2的质粒图谱；
图2为本发明实施例中使用的表达载体质粒Lenti-CRISPRv2-hPPP1R1Csg的质粒图谱；
图3为本发明实施例中使用的包装质粒pLP-VSVG的质粒图谱；
图4为本发明实施例中使用的包装质粒psPAX2的质粒图谱；
图5为HT-29细胞病毒感染含有PPP1R1CsgRNA的活性鉴定测序图。
图6为敲除PPP1R1C基因的单克隆HT-29细胞株13#中PPP1R1C基因的测序图。
图7为敲除PPP1R1C基因的单克隆HT-29细胞株14#中PPP1R1C基因的测序图。
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PPP1R1C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PPP1R1C的第三外显子上设计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1 PPP1R1C靶向位点及sgRNA寡核苷酸序列表
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
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
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-hPPP1R1Csg。
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小时后，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 ID NO.4～7)PCR扩增靶向序列。用PPP1R1C-4-1和PPP1R1C-4-2进行PCR，得到930bp的片段a，以a为模板，用PPP1R1C-4-3和PPP1R1C-4-4进行PCR，得到404bp的片段b，该片段电泳后胶回收用于测序，测序引物为PPP1R1C-4-3。
表2引物序列表
引物名称 引物序列
PPP1R1C-4-1 AAGATAGCCTGCCTCTAATGG
PPP1R1C-4-2 CATTGTGTTCTGTTCTGAATACAG
PPP1R1C-4-3 CCCaagctt GGATTACATTGTTAAGATTCTTGG
PPP1R1C-4-4 CCGgaattc GTTCAGTCAAGGAGACTTCCAG
2.包装慢病毒
100mm皿种入4X106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：
包装质粒psPAX2 12μg；辅助质粒pLP-VSVG 10μg；表达质粒Lenti-CRISPRv2-hPPP1R1Csg 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HT-29-PPP1R1C。并使用有限稀释法挑选HT-29-PPP1R1C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PPP1R1C的单克隆细胞株基因组DNA为模板，分别设计针对PPP1R1C序列的引物，序列如SEQ ID NO.4～7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PPP1R1C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PPP1R1C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PPP1R1C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靶向敲除人肠癌细胞PPP1R1C基因及其特异性的sgRNA
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 1
tcaggggtta agcatctgaa 20
<210> 2
<211> 29
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 2
caccgtcagg ggttaagcat ctgaagccc 29
<210> 3
<211> 30
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 3
aaacttcaga tgcttaaccc ctgacggtgc 30
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 4
aagatagcct gcctctaatg g 21
<210> 5
<211> 24
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 5
cattgtgttc tgttctgaat acag 24
<210> 6
<211> 33
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 6
cccaagcttg gattacattg ttaagattct tgg 33
<210> 7
<211> 31
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 7
ccggaattcg ttcagtcaag gagacttcca g 31
<210> 8
<211> 14873
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 8
tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 60
caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 120
ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 180
gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc 240
tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 300
ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 360
ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga 420
aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca 480
cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct 540
agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat 600
gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat 660
gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag 720
gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta 780
gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag 840
acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac 900
agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt 960
gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca 1020
aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg 1080
ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc 1140
agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg 1200
caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg 1260
gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa 1320
ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag 1380
atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta 1440
atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg 1500
gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat 1560
ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta 1620
ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc 1680
ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac 1740
agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca 1800
aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg 1860
ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 1920
tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat 1980
taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct 2040
gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg 2100
tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg 2160
gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg 2220
tggaaaggac gaaacaccgg agacggttgt aaatgagcac acaaaataca catgctaaaa 2280
tattatattc tatgaccttt ataaaatcaa ccaaaatctt ctttttaata actttagtat 2340
caataattag aatttttatg ttcctttttg caaactttta ataaaaatga gcaaaataaa 2400
aaaacgctag ttttagtaac tcgcgttgtt ttcttcacct ttaataatag ctactccacc 2460
acttgttcct aagcggtcag ctcctgcttc aatcattttt tgagcatctt caaatgttct 2520
aactccacca gctgctttaa ctaaagcatt gtctttaaca actgacttca ttagtttaac 2580
atcttcaaat gttgcacctg attttgaaaa tcctgttgat gttttaacaa attctaatcc 2640
agcttcaaca gctatttcac aagctttcat gatttcttct tttgttaata aacaattttc 2700
cataatacat ttaacaacat gtgatccagc tgcttttttt acagctttca tgtcttctaa 2760
aactaattca taatttttgt cttttaatgc accaatattt aataccatat caatttctgt 2820
tgcaccatct ttaattgctt cagaaacttc gaatgctttt gtagctgttg tgcatgcacc 2880
tagaggaaaa cctacaacat ttgttattcc tacatttgtg ccttttaata attctttaca 2940
atagcttgtt caatatgaat taacacaaac tgttgcaaaa tcaaattcaa ttgcttcatc 3000
acataattgt ttaatttcag ctttcgtagc atcttgtttt aataatgtgt gatctatata 3060
tttgtttagt ttcatttttt ctcctatata ttcattttta attttaattc tttaataatt 3120
tcgtctactt taactttagc gttttgaaca gattcaccaa cacctataaa ataaattttt 3180
agtttaggtt cagttccact tgggcgaaca gcaaatcatg acttatcttc taaataaaat 3240
tttagtaagt cttgtcctgg catattatac attccatcga tgtagtcttc aacattaaca 3300
actttaagtc cagcaatttg agttaagggt gttgctctca atgatttcat taatggttca 3360
atttttaatt tcttttcttc tggtttaaaa ttcaagttta aagtgaaagt gtaatatgca 3420
cccatttctt taaataaatc ttctaaatag tctactaatg ttttattttg ttttttataa 3480
aatcaagcag cctctgctat taatatagaa gcttgtattc catctttatc tctagctgag 3540
tcatcaatta catatccata actttcttca taagcaaaaa caaaatttaa tccgttatct 3600
tcttctttag caatttctct acccattcat ttaaatccag ttaaagtttt tacaatatta 3660
actccatatt tttcatgagc gattctatca cccaaatcac ttgttacaaa acttgaatat 3720
agagccggat tttttggaat gctatttaag cgttttagat ttgataattt tcaatcaatt 3780
aaaattggtc ctgtttgatt tccatctaat cttacaaaat gaccatcatg ttttattgcc 3840
attccaaatc tgtcagcatc tgggtcattc ataataataa tatctgcatc atgtttaata 3900
ccatattcaa gcggtatttt tcatgcagga tcaaattctg gatttggatt tacaacattt 3960
ttaaatgttt catcttcaaa tgcatgctct tcaacctcaa taacgttata tcctgattca 4020
cgtaatattt ttggggtaaa tttagttcct gttccattaa ctgcgctaaa aataattttt 4080
aaatcttttt tagcttcttg ctcttttttg tacgtctctg ttttagagct agaaatagca 4140
agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 4200
tgaattcgct agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc 4260
agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg 4320
tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct 4380
ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt 4440
tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa 4500
gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga 4560
cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat 4620
caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg 4680
gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca 4740
agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga 4800
gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt 4860
ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt 4920
ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa 4980
gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa 5040
gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc 5100
cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga 5160
aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc 5220
cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa 5280
actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg 5340
cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag 5400
cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa 5460
gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct 5520
gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat 5580
tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat 5640
ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg 5700
gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct 5760
gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat 5820
cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc 5880
ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga 5940
caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc 6000
caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga 6060
gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga 6120
gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca 6180
gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt 6240
ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga 6300
caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct 6360
gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt 6420
cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag 6480
ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct 6540
gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac 6600
ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca 6660
cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt 6720
ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat 6780
ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg 6840
gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa 6900
cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta 6960
cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc 7020
tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa 7080
ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg 7140
gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc 7200
cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga 7260
aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta 7320
cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt 7380
gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca 7440
cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa 7500
gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc 7560
caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat 7620
gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat 7680
cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt 7740
gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg 7800
cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa 7860
gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt 7920
gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct 7980
gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga 8040
agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct 8100
gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg 8160
aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga 8220
gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa 8280
gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc 8340
cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag 8400
agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc 8460
cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct 8520
ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc 8580
tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa 8640
gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa 8700
acaagccgga gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct 8760
cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta 8820
ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca 8880
agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg 8940
cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga 9000
gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga 9060
aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt 9120
ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc 9180
ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt 9240
ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac 9300
ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga 9360
ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 9420
tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 9480
ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 9540
gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 9600
caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 9660
actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 9720
ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac 9780
ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 9840
tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 9900
gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac 9960
aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt 10020
cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag 10080
atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc 10140
ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga 10200
tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg 10260
atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 10320
ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 10380
atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 10440
gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc 10500
tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc 10560
attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 10620
agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 10680
tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 10740
ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 10800
ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 10860
aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 10920
ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg 10980
aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 11040
agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 11100
agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 11160
cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 11220
ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga 11280
ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt 11340
ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat 11400
aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca 11460
ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg 11520
acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg 11580
cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg 11640
acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc 11700
cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg 11760
ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg 11820
attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct 11880
ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta 11940
ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 12000
ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 12060
gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt 12120
gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 12180
cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 12240
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 12300
gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 12360
ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 12420
caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 12480
aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 12540
atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 12600
cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 12660
ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 12720
gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 12780
accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 12840
cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 12900
cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 12960
gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 13020
aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 13080
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 13140
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 13200
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 13260
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 13320
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 13380
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 13440
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 13500
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 13560
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 13620
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 13680
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 13740
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 13800
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 13860
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 13920
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 13980
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 14040
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 14100
cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 14160
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 14220
ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga 14280
tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct 14340
gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac 14400
aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct 14460
gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata 14520
gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact 14580
tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat 14640
gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta 14700
tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc 14760
tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg 14820
ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgg 14873
<210> 9
<211> 13012
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 9
tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 60
caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 120
ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 180
gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc 240
tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 300
ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 360
ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga 420
aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca 480
cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct 540
agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat 600
gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat 660
gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag 720
gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta 780
gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag 840
acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac 900
agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt 960
gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca 1020
aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg 1080
ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc 1140
agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg 1200
caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg 1260
gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa 1320
ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag 1380
atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta 1440
atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg 1500
gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat 1560
ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta 1620
ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc 1680
ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac 1740
agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca 1800
aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg 1860
ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 1920
tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat 1980
taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct 2040
gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg 2100
tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg 2160
gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg 2220
tggaaaggac gaaacacctc aggggttaag catctgaagt tttagagcta gaaatagcaa 2280
gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 2340
gaattcgcta gctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca 2400
gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt 2460
gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt 2520
tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt 2580
cgcaacgggt ttgccgccag aacacaggac cggttctaga gcgctgccac catggacaag 2640
aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac 2700
gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc 2760
aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg 2820
ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa 2880
gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag 2940
tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg 3000
gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg 3060
gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag 3120
ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag 3180
ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc 3240
agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa 3300
aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc 3360
ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa 3420
ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc 3480
gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc 3540
gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag 3600
agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg 3660
cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt 3720
gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg 3780
gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg 3840
accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg 3900
cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc 3960
ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc 4020
tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac 4080
aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc 4140
aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag 4200
ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag 4260
cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag 4320
ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg 4380
gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac 4440
aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg 4500
acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc 4560
gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc 4620
cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg 4680
aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc 4740
tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac 4800
attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg 4860
gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg 4920
gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg 4980
atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac 5040
acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac 5100
gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct 5160
cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac 5220
cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg 5280
cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc 5340
gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa 5400
acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac 5460
gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg 5520
tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac 5580
gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag 5640
ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc 5700
aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg 5760
aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc 5820
gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 5880
cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 5940
ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag 6000
aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg 6060
ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 6120
ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 6180
gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 6240
ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga 6300
aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 6360
aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 6420
cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 6480
gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 6540
gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc 6600
ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg 6660
gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 6720
cagctgggag gcgacaagcg acctgccgcc acaaagaagg ctggacaggc taagaagaag 6780
aaagattaca aagacgatga cgataaggga tccggcgcaa caaacttctc tctgctgaaa 6840
caagccggag atgtcgaaga gaatcctgga ccgaccgagt acaagcccac ggtgcgcctc 6900
gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac 6960
cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa 7020
gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc 7080
gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag 7140
atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa 7200
ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc 7260
tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg 7320
gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc 7380
tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc 7440
tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat 7500
tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 7560
ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 7620
tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 7680
caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 7740
accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 7800
ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 7860
tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc 7920
tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 7980
ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 8040
acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca 8100
aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc 8160
actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga 8220
tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct 8280
tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat 8340
ccctcagacc cttttagtca gtgtggaaaa tctctagcag ggcccgttta aacccgctga 8400
tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 8460
tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 8520
tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 8580
ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct 8640
gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca 8700
ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 8760
gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 8820
caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 8880
cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 8940
tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 9000
acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 9060
gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga 9120
atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 9180
gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca 9240
gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc 9300
ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 9360
tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag 9420
gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt 9480
tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata 9540
atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac 9600
cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga 9660
cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc 9720
ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga 9780
cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc 9840
ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc 9900
cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga 9960
ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg 10020
gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat 10080
tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 10140
tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 10200
tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 10260
aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 10320
ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 10380
ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 10440
cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 10500
tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 10560
aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 10620
aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 10680
tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 10740
ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 10800
cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 10860
ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 10920
ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 10980
gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 11040
agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 11100
cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 11160
aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 11220
aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 11280
ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 11340
aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 11400
ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 11460
agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 11520
cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 11580
ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 11640
gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 11700
cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 11760
cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 11820
ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 11880
catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 11940
tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 12000
ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 12060
catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 12120
cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 12180
cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 12240
acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 12300
ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 12360
tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat 12420
cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg 12480
ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca 12540
aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg 12600
cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag 12660
taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 12720
acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 12780
acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 12840
ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 12900
attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 12960
gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat gg 13012
<210> 10
<211> 6507
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 10
gcggccgctc tagagagctt ggcccattgc atacgttgta tccatatcat aatatgtaca 60
tttatattgg ctcatgtcca acattaccgc catgttgaca ttgattattg actagttatt 120
aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 180
aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 240
taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 300
agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 360
cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 420
tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 480
tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 540
gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 600
caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 660
aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac 720
gctgttttga cctccataga agacaccggg accgatccag cctccggtcg accgatcctg 780
agaacttcag ggtgagtttg gggacccttg attgttcttt ctttttcgct attgtaaaat 840
tcatgttata tggagggggc aaagttttca gggtgttgtt tagaatggga agatgtccct 900
tgtatcacca tggaccctca tgataatttt gtttctttca ctttctactc tgttgacaac 960
cattgtctcc tcttattttc ttttcatttt ctgtaacttt ttcgttaaac tttagcttgc 1020
atttgtaacg aatttttaaa ttcacttttg tttatttgtc agattgtaag tactttctct 1080
aatcactttt ttttcaaggc aatcagggta tattatattg tacttcagca cagttttaga 1140
gaacaattgt tataattaaa tgataaggta gaatatttct gcatataaat tctggctggc 1200
gtggaaatat tcttattggt agaaacaact acaccctggt catcatcctg cctttctctt 1260
tatggttaca atgatataca ctgtttgaga tgaggataaa atactctgag tccaaaccgg 1320
gcccctctgc taaccatgtt catgccttct tctctttcct acagctcctg ggcaacgtgc 1380
tggttgttgt gctgtctcat cattttggca aagaattcct cgacggatcc ctcgaggaat 1440
tctgacacta tgaagtgcct tttgtactta gcctttttat tcattggggt gaattgcaag 1500
ttcaccatag tttttccaca caaccaaaaa ggaaactgga aaaatgttcc ttctaattac 1560
cattattgcc cgtcaagctc agatttaaat tggcataatg acttaatagg cacagcctta 1620
caagtcaaaa tgcccaagag tcacaaggct attcaagcag acggttggat gtgtcatgct 1680
tccaaatggg tcactacttg tgatttccgc tggtatggac cgaagtatat aacacattcc 1740
atccgatcct tcactccatc tgtagaacaa tgcaaggaaa gcattgaaca aacgaaacaa 1800
ggaacttggc tgaatccagg cttccctcct caaagttgtg gatatgcaac tgtgacggat 1860
gccgaagcag tgattgtcca ggtgactcct caccatgtgc tggttgatga atacacagga 1920
gaatgggttg attcacagtt catcaacgga aaatgcagca attacatatg ccccactgtc 1980
cataactcta caacctggca ttctgactat aaggtcaaag ggctatgtga ttctaacctc 2040
atttccatgg acatcacctt cttctcagag gacggagagc tatcatccct gggaaaggag 2100
ggcacagggt tcagaagtaa ctactttgct tatgaaactg gaggcaaggc ctgcaaaatg 2160
caatactgca agcattgggg agtcagactc ccatcaggtg tctggttcga gatggctgat 2220
aaggatctct ttgctgcagc cagattccct gaatgcccag aagggtcaag tatctctgct 2280
ccatctcaga cctcagtgga tgtaagtcta attcaggacg ttgagaggat cttggattat 2340
tccctctgcc aagaaacctg gagcaaaatc agagcgggtc ttccaatctc tccagtggat 2400
ctcagctatc ttgctcctaa aaacccagga accggtcctg ctttcaccat aatcaatggt 2460
accctaaaat actttgagac cagatacatc agagtcgata ttgctgctcc aatcctctca 2520
agaatggtcg gaatgatcag tggaactacc acagaaaggg aactgtggga tgactgggca 2580
ccatatgaag acgtggaaat tggacccaat ggagttctga ggaccagttc aggatataag 2640
tttcctttat acatgattgg acatggtatg ttggactccg atcttcatct tagctcaaag 2700
gctcaggtgt tcgaacatcc tcacattcaa gacgctgctt cgcaacttcc tgatgatgag 2760
agtttatttt ttggtgatac tgggctatcc aaaaatccaa tcgagcttgt agaaggttgg 2820
ttcagtagtt ggaaaagctc tattgcctct tttttcttta tcatagggtt aatcattgga 2880
ctattcttgg ttctccgagt tggtatccat ctttgcatta aattaaagca caccaagaaa 2940
agacagattt atacagacat agagatgaac cgacttggaa agtaactcaa atcctgcaca 3000
acagattctt catgtttgga ccaaatcaac ttgtgatacc atgctcaaag aggcctcaat 3060
tatatttgag tttttaattt ttatgaaaaa aaaaaaaaaa aacggaattc ctcgagggat 3120
ccgtcgagga attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg 3180
ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg 3240
acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg 3300
caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc 3360
atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg 3420
ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg 3480
tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt 3540
ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat 3600
ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc 3660
tcgacggatc ggccgcaatt cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat 3720
ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 3780
taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 3840
aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 3900
attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 3960
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 4020
gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4080
ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 4140
agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 4200
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 4260
ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 4320
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 4380
ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 4440
gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 4500
aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 4560
aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 4620
ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 4680
gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 4740
gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 4800
tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 4860
ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 4920
ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 4980
atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 5040
ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 5100
tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 5160
attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 5220
tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 5280
ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 5340
gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 5400
gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 5460
gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 5520
aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 5580
taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 5640
tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 5700
tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 5760
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 5820
tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa 5880
atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 5940
aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 6000
tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 6060
cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa 6120
atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 6180
cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 6240
tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc 6300
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 6360
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 6420
tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat 6480
agggcgaatt ggagctccac cgcggtg 6507
<210> 11
<211> 10703
<212> DNA
<213> 人工序列(Artificial synthesis)
<400> 11
gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240
atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420
atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480
gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540
gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600
tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660
gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720
ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780
gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840
ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900
tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960
cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020
ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080
tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140
cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200
gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260
ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320
gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380
gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440
tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500
cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560
acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620
gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680
acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcgggccg gccgcgttga 1740
cgcgcacggc aagaggcgag gggcggcgac tggtgagaga tgggtgcgag agcgtcagta 1800
ttaagcgggg gagaattaga tcgatgggaa aaaattcggt taaggccagg gggaaagaaa 1860
aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat 1920
cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc 1980
cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt 2040
gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag 2100
caaaacaaaa gtaagaaaaa agcacagcaa gcagcagctg acacaggaca cagcaatcag 2160
gtcagccaaa attaccctat agtgcagaac atccaggggc aaatggtaca tcaggccata 2220
tcacctagaa ctttaaatgc atgggtaaaa gtagtagaag agaaggcttt cagcccagaa 2280
gtgataccca tgttttcagc attatcagaa ggagccaccc cacaagattt aaacaccatg 2340
ctaaacacag tggggggaca tcaagcagcc atgcaaatgt taaaagagac catcaatgag 2400
gaagctgcag aatgggatag agtgcatcca gtgcatgcag ggcctattgc accaggccag 2460
atgagagaac caaggggaag tgacatagca ggaactacta gtacccttca ggaacaaata 2520
ggatggatga cacataatcc acctatccca gtaggagaaa tctataaaag atggataatc 2580
ctgggattaa ataaaatagt aagaatgtat agccctacca gcattctgga cataagacaa 2640
ggaccaaagg aaccctttag agactatgta gaccgattct ataaaactct aagagccgag 2700
caagcttcac aagaggtaaa aaattggatg acagaaacct tgttggtcca aaatgcgaac 2760
ccagattgta agactatttt aaaagcattg ggaccaggag cgacactaga agaaatgatg 2820
acagcatgtc agggagtggg gggacccggc cataaagcaa gagttttggc tgaagcaatg 2880
agccaagtaa caaatccagc taccataatg atacagaaag gcaattttag gaaccaaaga 2940
aagactgtta agtgtttcaa ttgtggcaaa gaagggcaca tagccaaaaa ttgcagggcc 3000
cctaggaaaa agggctgttg gaaatgtgga aaggaaggac accaaatgaa agattgtact 3060
gagagacagg ctaatttttt agggaagatc tggccttccc acaagggaag gccagggaat 3120
tttcttcaga gcagaccaga gccaacagcc ccaccagaag agagcttcag gtttggggaa 3180
gagacaacaa ctccctctca gaagcaggag ccgatagaca aggaactgta tcctttagct 3240
tccctcagat cactctttgg cagcgacccc tcgtcacaat aaagataggg gggcaattaa 3300
aggaagctct attagataca ggagcagatg atacagtatt agaagaaatg aatttgccag 3360
gaagatggaa accaaaaatg atagggggaa ttggaggttt tatcaaagta ggacagtatg 3420
atcagatact catagaaatc tgcggacata aagctatagg tacagtatta gtaggaccta 3480
cacctgtcaa cataattgga agaaatctgt tgactcagat tggctgcact ttaaattttc 3540
ccattagtcc tattgagact gtaccagtaa aattaaagcc aggaatggat ggcccaaaag 3600
ttaaacaatg gccattgaca gaagaaaaaa taaaagcatt agtagaaatt tgtacagaaa 3660
tggaaaagga aggaaaaatt tcaaaaattg ggcctgaaaa tccatacaat actccagtat 3720
ttgccataaa gaaaaaagac agtactaaat ggagaaaatt agtagatttc agagaactta 3780
ataagagaac tcaagatttc tgggaagttc aattaggaat accacatcct gcagggttaa 3840
aacagaaaaa atcagtaaca gtactggatg tgggcgatgc atatttttca gttcccttag 3900
ataaagactt caggaagtat actgcattta ccatacctag tataaacaat gagacaccag 3960
ggattagata tcagtacaat gtgcttccac agggatggaa aggatcacca gcaatattcc 4020
agtgtagcat gacaaaaatc ttagagcctt ttagaaaaca aaatccagac atagtcatct 4080
atcaatacat ggatgatttg tatgtaggat ctgacttaga aatagggcag catagaacaa 4140
aaatagagga actgagacaa catctgttga ggtggggatt taccacacca gacaaaaaac 4200
atcagaaaga acctccattc ctttggatgg gttatgaact ccatcctgat aaatggacag 4260
tacagcctat agtgctgcca gaaaaggaca gctggactgt caatgacata cagaaattag 4320
tgggaaaatt gaattgggca agtcagattt atgcagggat taaagtaagg caattatgta 4380
aacttcttag gggaaccaaa gcactaacag aagtagtacc actaacagaa gaagcagagc 4440
tagaactggc agaaaacagg gagattctaa aagaaccggt acatggagtg tattatgacc 4500
catcaaaaga cttaatagca gaaatacaga agcaggggca aggccaatgg acatatcaaa 4560
tttatcaaga gccatttaaa aatctgaaaa caggaaaata tgcaagaatg aagggtgccc 4620
acactaatga tgtgaaacaa ttaacagagg cagtacaaaa aatagccaca gaaagcatag 4680
taatatgggg aaagactcct aaatttaaat tacccataca aaaggaaaca tgggaagcat 4740
ggtggacaga gtattggcaa gccacctgga ttcctgagtg ggagtttgtc aatacccctc 4800
ccttagtgaa gttatggtac cagttagaga aagaacccat aataggagca gaaactttct 4860
atgtagatgg ggcagccaat agggaaacta aattaggaaa agcaggatat gtaactgaca 4920
gaggaagaca aaaagttgtc cccctaacgg acacaacaaa tcagaagact gagttacaag 4980
caattcatct agctttgcag gattcgggat tagaagtaaa catagtgaca gactcacaat 5040
atgcattggg aatcattcaa gcacaaccag ataagagtga atcagagtta gtcagtcaaa 5100
taatagagca gttaataaaa aaggaaaaag tctacctggc atgggtacca gcacacaaag 5160
gaattggagg aaatgaacaa gtagatgggt tggtcagtgc tggaatcagg aaagtactat 5220
ttttagatgg aatagataag gcccaagaag aacatgagaa atatcacagt aattggagag 5280
caatggctag tgattttaac ctaccacctg tagtagcaaa agaaatagta gccagctgtg 5340
ataaatgtca gctaaaaggg gaagccatgc atggacaagt agactgtagc ccaggaatat 5400
ggcagctaga ttgtacacat ttagaaggaa aagttatctt ggtagcagtt catgtagcca 5460
gtggatatat agaagcagaa gtaattccag cagagacagg gcaagaaaca gcatacttcc 5520
tcttaaaatt agcaggaaga tggccagtaa aaacagtaca tacagacaat ggcagcaatt 5580
tcaccagtac tacagttaag gccgcctgtt ggtgggcggg gatcaagcag gaatttggca 5640
ttccctacaa tccccaaagt caaggagtaa tagaatctat gaataaagaa ttaaagaaaa 5700
ttataggaca ggtaagagat caggctgaac atcttaagac agcagtacaa atggcagtat 5760
tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag 5820
tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc 5880
aaaattttcg ggtttattac agggacagca gagatccagt ttggaaagga ccagcaaagc 5940
tcctctggaa aggtgaaggg gcagtagtaa tacaagataa tagtgacata aaagtagtgc 6000
caagaagaaa agcaaagatc atcagggatt atggaaaaca gatggcaggt gatgattgtg 6060
tggcaagtag acaggatgag gattaacaca tggaattctg caacaactgc tgtttatcca 6120
tttcagaatt gggtgtcgac atagcagaat aggcgttact cgacagagga gagcaagaaa 6180
tggagccagt agatcctaga ctagagccct ggaagcatcc aggaagtcag cctaaaactg 6240
cttgtaccaa ttgctattgt aaaaagtgtt gctttcattg ccaagtttgt ttcatgacaa 6300
aagccttagg catctcctat ggcaggaaga agcggagaca gcgacgaaga gctcatcaga 6360
acagtcagac tcatcaagct tctctatcaa agcagtaagt agtacatgta atgcaaccta 6420
taatagtagc aatagtagca ttagtagtag caataataat agcaatagtt gtgtggtcca 6480
tagtaatcat agaatatagg aaaatggccg ctgatcttca gacctggagg aggagatatg 6540
agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 6600
gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 6660
ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcctcaatg 6720
acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 6780
ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 6840
ctccaagcaa gaatcctagc tgtggaaaga tacctaaagg atcaacagct cctagggatt 6900
tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 6960
aataaatctc tggaacagat ctggaatcac acgacctgga tggagtggga cagagaaatt 7020
aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 7080
aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 7140
acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 7200
agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 7260
tcgtttcaga cccacctccc aatcccgagg ggacccgaca ggcccgaagg aatagaagaa 7320
gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc cttggcactt 7380
atctgggacg atctgcggag cctgtgcctc ttcagctacc accgcttgag agacttactc 7440
ttgattgtaa cgaggattgt ggaacttctg ggacgcaggg ggtgggaagc cctcaaatat 7500
tggtggaatc tcctacaata ttggagtcag gagctaaaga atagtgctgt tagcttgctc 7560
aatgccacag ccatagcagt agctgagggg acagataggg ttatagaagt agtacaagga 7620
gcttgtagag ctattcgcca catacctaga agaataagac agggcttgga aaggattttg 7680
ctataagctc gaaacaaccg gtacctctag aactatagct agcagatctt tttccctctg 7740
ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga 7800
aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat 7860
atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat 7920
atgccatatg ctggctgcca tgaacaaagg tggctataaa gaggtcatca gtatatgaaa 7980
cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt 8040
tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt 8100
ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc 8160
ttatgaagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc 8220
tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 8280
taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 8340
cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc 8400
atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 8460
ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct 8520
gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta 8580
acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 8640
ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 8700
atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 8760
tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 8820
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 8880
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 8940
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 9000
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 9060
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 9120
cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta 9180
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 9240
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 9300
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 9360
gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 9420
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 9480
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 9540
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 9600
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 9660
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 9720
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 9780
actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 9840
aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 9900
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 9960
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 10020
cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 10080
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 10140
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 10200
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 10260
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 10320
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 10380
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 10440
gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 10500
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 10560
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 10620
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 10680
atttccccga aaagtgccac ctg 10703

Claims (7)

1.一种CRISPR/Cas9靶向敲除人肠癌细胞PPP1R1C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PPP1R1C基因功能区的sgRNA；其次构建PPP1R1C基因的sgRNA到慢病毒载体系统；然后将此慢病毒载体系统感染人肠癌HT-29细胞，得到的PPP1R1C基因敲除细胞株。
2.根据权利要求1所述的特异性靶向PPP1R1C基因功能区的sgRNA，其DNA序列如SEQ IDNO.1所示，所述sgRNA在PPP1R1C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PPP1R1C基因功能区的sgRNA，其引物包括：
针对PPP1R1C基因的靶点的引物对：
PPP1R1CsgRNAoligo1：如SEQ ID NO.2所示；
PPP1R1CsgRNAoligo2：如SEQ ID NO.3所示。
4.根据权利要求1所述的构建PPP1R1C基因的sgRNA到慢病毒载体系统，是一种靶向敲除PPP1R1C基因的CRISPR/Cas9重组表达慢病毒载体Lenti-CRISPRv2-hPPP1R1Csg，其特征在于：含有特异性靶向PPP1R1C基因的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.8所示。
6.根据权利要求1所述的CRISPR/Cas9靶向敲除人肠癌细胞PPP1R1C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PPP1R1C基因上的靶序列符合5’-N(19)G的序列排列规则，所述sgRNA在PPP1R1C基因上的靶序列位于基因的外显子，所述sgRNA在PPP1R1C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PPP1R1C基因上的靶序列是唯一的，并且所述sgRNA在PPP1R1C上的靶位点序列如序列表SEQ ID NO.1序列所示，在所述sgRNA在PPP1R1C上的靶位点序列的5’-端加上CACCG序列合成得到正向寡核苷酸即Forward oligo；获得sgRNA在PPP1R1C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、3’-端加上C，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.8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-hPPP1R1Csg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-hPPP1R1Csg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PPP1R1C基因的sgRNA和Cas9的假型慢病毒；
(4)使用所述假型慢病毒感染目的细胞，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PPP1R1C基因已经被敲除并获得基因敲除的细胞。
7.根据权利要求1所述的敲除PPP1R1C基因的细胞株，为人HT-29细胞；其特征在于：是HT-29细胞中的PPP1R1C基因缺失或插入核苷酸得到的细胞株，为如下的细胞株13#和14#中的任一株：细胞株13#是：sgRNA的切割位点位于在野生型PPP1R1C基因上的sgRNA PAM区前3个碱基处，导致PPP1R1C基因的外显子增加1个碱基；细胞株14#：是sgRNA的切割位点位于在野生型PPP1R1C基因上的sgRNA PAM区前2个碱基处，导致PPP1R1C基因的外显子缺失14个碱基。
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