CN105567688A - 一种可用于艾滋病基因治疗的CRISPR/SaCas9系统 - Google Patents

一种可用于艾滋病基因治疗的CRISPR/SaCas9系统 Download PDF

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CN105567688A
CN105567688A CN201610056141.4A CN201610056141A CN105567688A CN 105567688 A CN105567688 A CN 105567688A CN 201610056141 A CN201610056141 A CN 201610056141A CN 105567688 A CN105567688 A CN 105567688A
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sacas9
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郭德银
陈述亮
肖巧巧
刘哲鹏
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Abstract

本发明属于基因工程领域，更具体地说，涉及CRISPR/SaCas9特异性敲除人CCR5基因的方法以及用于特异性靶向CCR5基因的sgRNA。本发明提供了CRISPR/SaCas9特异性敲除人CCR5基因的方法以及用于特异性靶向CCR5基因的sgRNA以及涉及到的CRISPR/SaCas9重组慢病毒和腺相关病毒载体系统和应用。本发明所提供的CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体能够表达针对于人和恒河猴同一基因的CCR5靶点的SaCas9-RNA，使得应用范围更广，效率更高。该制备方法构建简单，靶向效率高，为HIV的基因治疗提供了一种新型的技术。

Description

一种可用于艾滋病基因治疗的CRISPR/SaCas9系统
技术领域
本发明属于基因工程领域，更具体地说涉及CRISPR/SaCas9特异性敲除人CCR5基因的方法以及用于特异性靶向CCR5基因的sgRNA。
背景技术
人类免疫缺陷病毒(humanimmunodeficiencyvirus，HIV)归属于逆转录病毒科慢病毒属中的人类慢病毒组，其感染引起的获得性免疫缺陷综合征(acquiredimmunodeficiencysyndrome，AIDS)是目前威胁全球人类健康的主要疾病之一。人类免疫缺陷病毒通常也俗称为“艾滋病毒”，截止到2014年底，艾滋病毒携带者多达3690万人。自1981年发现第一例由HIV引起的艾滋病的25年以来，尽管对艾滋病的临床治疗已有了很大进展，但是仍无有效治愈手段攻破此科学难题。
病毒特异的CD4+T细胞是针对HIV免疫应答的重要组成部分，也是HIV-1感染过程的首要靶点。自1984年发现HIV-1通过与CD4结合感染宿主细胞，随后研究又发现仅有CD4分子并不能介导HIV-1的侵入，同时还需要一种或几种辅助受体。1996年证实，趋化因子受体CXCR4和CCR5是HIV-1感染的辅助受体。其中，趋化因子CCR5，作为G蛋白偶联因子超家族(GPCR)成员的细胞膜蛋白，是HIV-1入侵机体细胞的主要辅助受体之一。一个接触HIV但未检测到HIV感染的个人，带有CCR5编码基因32个核苷酸缺失，表明了CCR5影响HIV病毒入侵细胞。因而以CCR5为靶点的HIV-1受体拮抗剂越来越受关注，主要有趋化因子衍生物、非肽类小分子化合物、单克隆抗体、肽类化合物等。这些抗病毒活性强、亲和力高的CCR5拮抗剂，已有一部分进入了临床试验阶段。
尽管辅助受体拮抗剂可有效预防HIV-1感染，遏制艾滋病的流行，但是此类抑制剂的发展也面临着挑战。长期使用一种抑制剂，最终会使HIV-1产生耐药性。随着耐药性的不断出现，使得现有的抗HIV-1药物难以达到理想的治疗效果。因此，从基因组层面失活CCR5具有重要的治疗价值，这样，特异性靶向CCR5引起了人们的广泛关注。
2008年，Sangamo成功的利用Engineeredzincfingernucleases(ZFNs)在CD4+T细胞上实现CCR5的敲除，如今这项名为SB-728-T的项目已被尝试应用于HIV感染人群的基因治疗，并已进入临床二期阶段。这一基因编辑产品通过敲除分离到的病人的CD4+T细胞的CCR5，然后自体回输到病人，可以用于HIV感染的治疗。越来越多的实验结果表明CCR5基因是一个可靠、高效和安全的治疗HIV的靶点，能够实现CCR5在基因组层面上的敲除也是可靠、高效和安全的治疗方法。然而，即使在经过真核抗性筛选之后，ZFN靶向敲除CCR5的效率也只有大约为30％。很明显，这个效率还是差强人意。
1987年，日本研究员在试验中发现大肠杆菌中存在串联间隔重复序列DNA，但是并不知道它们具体的功能。经过深入的研究发现，这是广泛存在于细菌和古生菌中的一种序列，最终在2002年将其正式命名为规律成簇间隔短回文重复系统CRISPR(ClusteredRegulatoryInterspacedShortPalindromicRepeats)。在2007年，研究者发现细菌可以利用CRISPR系统抵抗噬菌体的入侵，接着又有研究发现细菌的CRISPR系统能够阻止外源质粒的侵入，由此可见这种存在于大多数细菌和所有古生菌中的特殊的DNA重复序列在获得性免疫系统发挥着重要的作用。
CRISPR主要有3部分组成，其包括一个前导区(Leader)、多个短而保守的重复序列区(Repeat)和多个间隔区(Spacer)。前导区是位于CRISPR簇的上游，是富含AT长度300-500bp的区域，被大多数人认为是启动子序列；重复序列区是长度为21-48bp为的含有回文序列的碱基，其可形成发卡结构，而重复序列间又被26-72bp间隔区碱基序列隔开；间隔区主要有外源的DNA序列组成，其类似于记忆性的免疫，当含有相同序列的外源DNA再次入侵机体时，就可被自身机体识别，然后对外源DNA进行切割使之不能表达，以保护自身不被外源病毒或细菌所侵害，因此，正是这些间隔序列发挥着对靶基因的识别作用。研究人员对CRISPR进一步研究发现，其侧翼序列附近存在一个多态性家族基因，该基因编码的蛋白质存在能与核酸相互作用的功能域，此功能域具有整合酶、核酸酶、聚合酶等酶活性，与CRISPR区域共同起作用，被命名为CRISPR关联基因(CRISPR-associated)，缩写为Cas。Cas系统有typeI，II，III型，其中typeII中含有一个特殊的蛋白Cas9，Cas9靶向切割DNA是通过CRISPR系统中的两种小RNA—crRNA(CRISPRRNA)和tracrRNA(trans-activatigcrRNA)和靶序列互补识别的原理实现的。现在已经把两种小RNA融合成一条RNA链，简称sgRNA(singleguideRNA)。Cas9和CRISPR形成复合体识别特定的DNA序列，进行特定位点切割造成双链DNA断裂，在没有模板的条件下，发生非同源重组末端连接(Non-homologousendjoining,NHEJ)，造成移码突变，导致基因敲除(Wiedenheft,Sternbergetal.2012,Cong,Ranetal.2013,F.AnnRanetal.2015)。
这一技术由于能快速、简便、高效地靶向基因组任何基因，从而引起了广泛的关注，在2012年开始像爆炸一般流行开来。研究人员优先选择改造了来自化脓链球菌的Cas9酶(SpCas9)来改变高等生物的DNA，并已成为一系列高度通用的基因组修饰技术的基础。之前的一些研究利用CRISPR/SpCas9系统在CD4+T细胞或者CD34+干细胞对艾滋病毒的辅助受体CCR5进行基因修饰，在一定的程度上可以抑制HIV-1病毒的侵入，但是也存在着一定的脱靶率(Cradick,Fineetal.2013,Ye,Wangetal.2014)。
最近发现了一种新的CRISPR/Cas9系统，它是一种是来自金黄色葡糖球菌的Cas9酶(SaCas9)，其基因敲除效率与之前的CRISPR/SpCas9系统相当，但是其大小却减少了25％(F.AnnRanetal.2015)。腺相关病毒(AAV)是基因治疗的最佳载体，但是由于其负载能力有限，使其包装SpCas9酶和其他必须的基因元件进入单个病毒颗粒存在一定的难度，而SaCas9的出现为这一问题的解决带来了新的希望。
SaCas9与SpCas9存在着不同之处，SaCas9主要识别5’-NNGRRT-3’特征区域的NGGRRT位点，然后对与sgRNA互补的DNA序列进行剪切，被剪切后断裂的DNA双链通过非同源末端直接连接(non-homologousendjoining，NHEJ)或者同源重组(homology-directedrepair，HDR)的修补方法进行修复，修复后的DNA会由于某些碱基的随机插入、缺失、突变等使基因的序列发生改变从而抑制了基因的表达，由此在DNA水平上对基因进行定向敲除。而SpCas9的识别位点为PAM序列5’-NGG-3’，然后再对靶位点进行剪切。
新发现的SaCas9与之前的SpCas9相比具有很多优点：1)SaCas9在哺乳动物体内具有很高的剪切效率，其所带有的sgRNA的长度范围为21-23个碱基，其本身的大小比SpCas9小25％，更有利于AAV载体携带其它相关基因进入单个病毒颗粒，经验证其与SpCas9可以达到相当的效率。2)在SaCas9系统中，把其sgRNA的第一个碱基替换成鸟嘌呤，使得SaCas9酶的活性大大提高，从而提高了其在机体内的剪切效率。3)SaCas9在靶向精确性上效率更高。
目前还没有针对HIV-1的辅助受体CCR5进行特异性敲除的CRISPR/SaCas9系统。
参考文献：
1.Yuan,J.,J.Wang,K.Crain,C.Fearns,K.A.Kim,K.L.Hua,P.D.Gregory,M.C.HolmesandB.E.Torbett(2012)."Zinc-fingernucleaseeditingofhumancxcr4promotesHIV-1CD4(+)Tcellresistanceandenrichment."MolTher20(4):849-859
2.Ye,L.,J.Wang,A.I.Beyer,F.Teque,T.J.Cradick,Z.Qi,J.C.Chang,G.Bao,M.O.Muench,J.Yu,J.A.LevyandY.W.Kan(2014)."Seamlessmodificationofwild-typeinducedpluripotentstemcellstothenaturalCCR5Delta32mutationconfersresistancetoHIVinfection."ProcNatlAcadSciUSA
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发明内容
CAS9靶向切割DNA是通过sgRNA和靶序列互补识别的原理实现的。因此，sgRNA能否做到特异性、精确靶向目标基因是CRISPR/Cas9能否特异性敲除目标基因的先决条件。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。
本发明设计出了针对HIV-1的辅助受体CCR5的CRISPR/SaCas9系统，以阻止CCR5的表达，抑制病毒的侵入，实现细胞内清除HIV病毒的目的。本发明筛选了CCR5基因的多个靶位点并且把这些靶位点构建到了CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体系统中，以得到长期抑制作用。
为了达到上述目的，本申请的技术方案如下：
一、靶向CCR5基因的sgRNA的设计：
在本发明选择了HIV-1的辅助受体蛋白即宿主蛋白CCR5(NCBI基因号：NM_000579)作为靶标，依据SaCas9编辑规则(F.AnnRanetal.2015)筛选出了多个在恒河猴和人的基因都保守的靶位点(13个靶位点见附表1，其中10个靶位点在人与恒河猴的基因位点上保守，有一个碱基不一样的有3个靶位点，即在附表1中分别是7、9和11号以粗体显示的靶点，序列中括号内是恒河猴基因组上对应的碱基)。
所述sgRNA在CCR5基因上的靶序列符合5’-G(21N)NNGRRT-3’(下划线代表PAM)或者5’-ARRCNN(21N)C-3’(下划线代表PAM)的序列排列规则；由此获得了在CRISPR/SaCas9特异性敲除人CCR5基因中用于特异性靶向CCR5基因的sgRNA,如附表1所示。
二、构建sgRNA的寡核苷酸双链：
1、根据选择的sgRNA，通过化学合成法合成寡核苷酸链，格式如下：
有义链：5’-G(21N)NNGRRT-3’或者5’-ARRCNN(21N)C-3’(引物不含PAM)
PAMPAM
反义链：与有义链互补(引物不含PAM)，分别在有义链引物的5’端加上CACC,在反义链引物5’端加上AAAC以形成BsmBI酶切位点和BsaI酶切位点的粘性末端；(注：21N代表靶位点的21个碱基序列)
2、把有义链引物和反义链引物经过退火，形成带有BsmBI酶切位点和BsaI酶切位点的粘性末端小片段；
三、sgRNA寡核苷酸构建到慢病毒载体lentiCRISPR/SaCas9和腺相关病毒载体AAVCRISPR/SaCas9
1、选择慢病毒载体lentiCRISPR/SaCas9和腺相关病毒载体AAVCRISPR/SaCas9
本发明中选择的慢病毒载体为lentiCRISPR/SaCas9，该载体由本实验室构建，具体是将Addgene公司AAV-CRISPR-SaCas9(PX601)载体上的SaCas9基因通过PCR扩增后转移到lentiCRISPR-V2载体上，取代原来的SpCas9。将PX601载体上的SaCas9对应的crRNA通过重叠PCR扩增后转移到lentiCRISPR-V2载体上，取代原来的SpCas9对应的crRNA。具体是1)先用引物1F:5’-cggggtaccgagggcctatttccc-3’(SEQIDNO.8)和1R:5’-acagagacgctcgagtacaaccgtctccggtgtttcgtc-3’(SEQIDNO.9)以lenti-crispr-v2为模版扩增含U6promoter序列的片段1(大小为286bp)；2)用引物2F:5’-ctcgagCGTCTCTgttttagtactctggaaacagaa-3’(SEQIDNO.10)和2R:5’-ctgagaattcaaaaatctcgccaacaagttg-3’(SEQIDNO.11)以PX601为模版PCR扩增SaCas9对应的crRNA序列的片段2(大小为104bp)；3)最后，用片段1和2作为模版，用引物1F和2R扩增286bp+104bp＝390bp片段，用KpnI+EcoRI酶切片段后连入KpnI+EcoRI酶切后的lenti-crisrp-v2载体中(切除一个2214bp片段)。以上载体称为lenti-U6-BsmBI-SacrRNA(先PCR鉴定，再用XhoI鉴定)。用AgeI和BamHI酶切lenti-U6-BsmBI-SacrRNA载体，用引物3F:5’-acaggaccggttctagagcgctgccaccatggccccaaagaagaagcgg-3’(SEQIDNO.12)和3R:5’-cgcggatccctcgagcttatcgtcatcgtctttgtaatcctttttcttttttgcctggc-3’(SEQIDNO.13)以PX601为模板，PCR扩增Sa-Cas9，用AgeI和BamHI酶切PCR片段后连入lenti-U6-BsmBI-SacrRNA载体。以上载体称为lenti-U6-BsmBI-SacrRNA-pEF-NLS-SaCas9-NLS-Flag-2A-puro(简称lenti-SaCas9)(SEQIDNO.14)此载体含有一个人的U6启动子，该启动子控制着sgRNA的表达，用BsmBI核酸内切酶对此载体进行消化，然后再插入长为21bp的sgRNA片段。在酶切位点BsmBI后有长为81bp的crRNA核酸序列为sgRNA的骨架序列。其后的EFS的启动子位点控制人类编码最优的SaCas9的表达(因为SpCas9和SaCas9最开始是在低等的原核生物中发现的蛋白，其编码氨基酸的密码子适合在原核生物中表达，如果要在人类细胞中表达SpCas9和SaCas9蛋白，那么就必须根据密码子摆动性原理重新设计适合在人类细胞中表达蛋白的氨基酸对应的密码子，此称为人类编码优化)。同时还含有自切割多肽P2A和带有筛选标记的基因Puromycin的编码序列以便于筛选有效的细胞从而提高靶向效率。
本发明中选择的腺相关病毒载体为PX601，此载体同样含有一个U6启动子，控制着sgRNA的表达，同时含有一个TBG启动子来调控SaCas9的表达，用BsaI核酸内切酶切割载体后，同样插入和慢病毒载体一样的长为21bp的sgRNA片段。
2、将退火形成的小片段连入由BsmBI酶和BsaI酶进行酶切的载体lentiCRISPR和AAVCRISPR；
3、将连接产物转化大肠杆菌Stbl3感受态细胞中(Stbl3菌株购自Invitrogen公司)；
4、挑取单克隆，经初步鉴定后把准阳性克隆送公司测序确定。
四、lentiCRISPR/SaCas9重组慢病毒载体和AAVCRISPR/SaCas9重组腺相关病毒载体的有效性验证
申请人将所构建的CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体转染进入TZM-bl细胞(购买自ATCC)，72h后收取细胞样品提取基因组，然后用PCR方法扩增CCR5基因，把扩增后回收的片段用T7EndonucleaseI(购买自BioLabs)进行酶切鉴定。所得的结果中由申请人所设计的重组慢病毒载体针对CCR5的4个靶位点有一定程度的突变，而重组腺相关病毒载体针对CCR5的6个靶位点有不同程度的突变，然后申请人把这些PCR产物分别连入T-easy载体(购买自Promega)后进行基因测序，发现重组慢病毒载体中的4个靶位点和重组腺相关病毒载体中的2个靶位点具有不同程度的基因突变、插入或者缺失，甚至移码突变。具体结果分析如下表1和表2所示：
表1、慢病毒重组载体的4个靶位点及其突变率
CXCR5基因靶点 基因突变率
靶点2GCCTATAAAATAGAGCCCTGTC 50％
靶点6GGCTGTGTTTGCGTCTCTCCC 60％
靶点8GTACAGTCAGTATCAATTCTGG 10％
靶点11GCTTCTCATTTCGACACCGAAG 50％
表2、腺相关病毒重组载体的4个靶位点：
CXCR5基因靶点 基因突变率
靶点2GCCTATAAAATAGAGCCCTGTC 0
靶点3GCTATTTTATAGGCTTCTTCTC 0
靶点5GGTGGTGACAAGTGTGATCAC 0
靶点8GCTTCTCATTTCGACACCGAAG 60％
靶点11GCTTCTCATTTCGACACCGAAG 50％
靶点12GTCCTTCTCCTGAACACCTTCC 0
五、CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体阻止HIV侵入有效性的验证。
为了提高CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体的基因敲除效率，为后续改造人和恒河猴的造血干细胞做准备，申请人把CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体转入到HEK-293T细胞中包装成慢病毒和腺相关病毒。其中慢病毒的包装体系包括：psPAX2包装质粒(购自Addgene)，pMD2.G包膜质粒(购自Addgene)，以及不同靶点的重组lentiCRISPR载体；腺相关病毒的包装体系包括：AAV6包装质粒，pDF辅助质粒和不同靶点的AAVCRISPR载体。
为了进一步检测CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体的基因敲除效率，申请人用2号，6号，8号和11号靶点lentiCRISPR载体(对应的序列如SEQIDNO.15-18所示)包装的慢病毒和8号，11号靶点AAVCRISPR载体包装的腺相关病毒，建立了基于TZM-bl细胞的CCR5基因缺失细胞系。然后用APC标记的CCR5抗体(购自BioLegend公司)对以上细胞系进行了流式检测以测定CCR5膜蛋白表达水平，同时也运用单荧光素酶报告基因对改造后的细胞中HIV的感染复制水平进行了检测，结果发现CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体改造后的细胞中HIV水平显著降低。
基于以上技术方案，本发明的第一个目的是提供在CRISPR/SaCas9特异性敲除人CCR5基因中用于特异性靶向CCR5基因的sgRNA,所述sgRNA在CCR5基因上的靶序列符合5’-G(21N)NNGRRT-3’或者5’-ARRCNN(21N)C-3’的序列排列规则，下划线代表PAM，21N代表靶位点的21个碱基序列，其对应的DNA序列如SEQIDNO.1-4任意一条序列所示，其中SEQIDNO.1-3在人与恒河猴的基因位点上保守。
本发明的目的还在于提供含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的慢病毒载体，其具有以下特征：
①所述靶向敲除载体是将Addgene公司PX601载体上的SaCas9基因通过PCR扩增后转移到lentiCRISPR-V2载体上，取代原来的SpCas9；将PX601载体上的SaCas9对应的crRNA通过重叠PCR扩增后转移到lentiCRISPR-V2载体上，取代原来的SpCas9对应的crRNA。
②此载体含有一个人的U6启动子，该启动子控制着sgRNA的表达，用BsmBI核酸内切酶对此载体进行消化，然后再插入含有粘性末端和长为21bp的靶sgRNA片段，靶序列如SEQIDNO.1-4任意一条所示；
③在酶切位点BsmBI后有长为81bp的crRNA核酸序列为sgRNA的骨架序列；
④crRNA核酸序列后的EFS的启动子控制人类编码最优的SaCas9的表达；
⑤含有自切割多肽P2A和带有筛选标记的基因Puromycin的编码序列。
以上所述的含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的慢病毒载体的序列如SEQIDNO.15-18任意一项所示。
本发明还提供靶向敲除人CCR5基因的慢病毒载体的慢病毒。
本发明提供的含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的腺相关病毒载体，其特征在于：
①选择的腺相关病毒载体为Addgene公司PX601；
②此载体同样含有一个U6启动子，控制着sgRNA的表达，用BsaI核酸内切酶切割载体后，插入含有粘性末端和长为21bp的靶sgRNA片段，靶序列如SEQIDNO.1-4任意一条所示；
③含有一个TBG启动子来调控SaCas9的表达。
本发明提供含有权利要求4所述靶向敲除人CCR5基因的腺相关病毒载体的腺相关病毒。
本发明的目的还在于提供在CRISPR/SaCas9特异性敲除人CCR5基因中用于特异性靶向CCR5基因的sgRNA在非诊疗目的的敲除人或恒河猴CCR5基因中的应用。
本发明的目的是提供含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的慢病毒载体或腺相关病毒载体在非诊疗目的的敲除人或恒河猴CCR5基因中的应用。
本发明的目的还在于含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的慢病毒载体或腺相关病毒载体在制备抗艾滋病药物中的应用。
以上的研究结果表明：本发明CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体可以用于艾滋病基因治疗。本发明的有益之处有：
(1)以上实验筛选出来的具有高效切除效率的CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体可以用于基于造血干细胞的艾滋病的基因治疗。在某一方面可以运用CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体转导入艾滋病病人的造血干细胞，然后分选出转导成功的改造后的细胞把它回输到病人的体内，从而进行艾滋病的基因治疗。
(2)本发明所提供的CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体能够表达针对于人和恒河猴同一基因的CCR5靶点的SaCas9-RNA，使得应用范围更广，效率更高。
(3)本发明所提供多个靶点的CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体能靶向艾滋病毒侵入相关的宿主膜蛋白受体CCR5，可以更有效地抑制病毒的侵入，阻止疾病的发生发展。
附图说明
图1CRISPR/SaCas9的慢病毒载体和腺相关病毒载体系统的构建流程图。
通过BsmBI酶切位点将用化学方法合成的编码不同靶位点的寡核苷酸片段经过退火合成含有与载体酶切后末端互补的粘性末端的sgRNA寡聚核苷酸双链后连入lentiCRISPR载体，同样的方法通过BsaI酶切位点把经过退火合成sgRNA寡聚核苷酸双链连入AAVCRISPR载体，分别得到不同靶CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体。
图2T7EN1酶切鉴定不同靶点的CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体对CCR5基因的基因敲除效果图
图2A通过T7EndonucleaseI酶切实验，con为阴性对照组。结果表明：在T7E1实验中，CRISPR/SaCas9重组慢病毒载体的2、6、8、11号靶点有作用，但6、11号作用最强；
图2BCRISPR/SaCas9重组腺相关病毒载体的2、3、5、8、11、12号靶点有作用。
图3T克隆测序验证不同靶点的CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体对CCR5基因的基因敲除效果图
图3A通过T克隆测序显示CRISPR/SaCas9重组慢病毒载体的2、6、8、11号都有碱基的插入或缺失以及突变，6号效果最为明显；
图3BCRISPR/SaCas9重组腺相关病毒载体的8、11号有插入、缺失或突变(图3B)；
图4流式细胞仪技术检测CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体建立的基于TZM-bl细胞的稳定细胞系的CCR5膜蛋白表达水平图
图4A未经改造的con细胞其APC-CCR5阳性细胞率为100％，而经lentiCRISPR-2、6、8、11改造的细胞其APC-CCR5阳性细胞率分别为46.6％、39.5％、43.7％和36.6％。相比于对照分别下调了53.4％、60.5％、56.3％和63.4％；
图4B未经改造的con细胞其APC-CCR5阳性细胞率为100％，而经AAV-CRISPR-8、11改造的细胞其APC-CCR5阳性细胞率分别为84.8％、和86.4％。相比于对照分别下调了15.2％和13.6％。
图5单荧光素酶报告基因检测HIV在经Lenti-CRISPR慢病毒和AAV-CRISPR腺相关病毒改造的TZM-bl细胞中的复制情况
图5A是HIV-1感染经Lenti-CRISPR慢病毒改造的TZM-bl细胞后5天后收样品检测细胞内HIV的复制情况，从上图我们可以发现与阴性对照Con相比，lenti-CRISPR-2、6、8、11号细胞中HIV-1的复制水平明显下降；
图5B是HIV-1感染细胞后5天后收样品检测经AAV-CRISPR腺相关病毒改造的TZM-bl细胞内HIV的复制情况，从上图我们可以发现与阴性对照Con相比，AAV-CRISPR-8、11号细胞中HIV-1的复制水平明显下降。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容。
【实施例1】CRISPR/SaCas9特异性敲除人CCR5基因中用于特异性靶向CCR5的sgRNA的设计及靶位点序列保守性的分析
在本发明选择了HIV-1病毒的辅助受体蛋白即宿主蛋白CCR5(NCBI基因号：NM_000579)作为靶标，依据SaCas9编辑规则(F.AnnRanetal.2015)筛选出了多个在恒河猴和人的基因都保守的靶位点，13个靶位点见附表1，为了证明申请人所选的CCR5靶位点序列在人和恒河猴中具有保守性，申请人把针对于CCR5的13个靶位点在NationalCenterForBiotechnologyInformationDatabase中的BLAST软件进行了序列保守性的分析，结果显示：其中10个靶位点在人与恒河猴的基因位点上保守，有一个碱基不一样的有3个靶位点，即在附表1中分别是7、9和11号以粗体显示的靶点，序列中括号内是恒河猴基因组上对应的碱基)。
所述sgRNA在CCR5基因上的靶序列符合5’-G(21N)NNGRRT-3’(下划线代表PAM)或者5’-ARRCNN(21N)C-3’(下划线代表PAM)的序列排列规则；由此获得了在CRISPR/SaCas9特异性敲除人CCR5基因中用于特异性靶向CCR5基因的sgRNA,如附表1所示。
附表1、人和恒河猴CCR5基因的保守靶位点分析
靶编号 靶序列5’-3’
1 TGGTGTTCATCTTTGGTTTTG 116-136
2 GACAGGGCTCTATTTTATAGG 312-322
3 CTATTTTATAGGCTTCTTCTC 321-331
4 CTGACAATCGATAGGTACCTG 354-364
5 GGTGGTGACAAGTGTGATCAC 435-445
6 GGCTGTGTTTGCGTCTCTCCC 455-465
7 GAAGAATTTC(T)CAGACATTAAA 550-560
8 TACAGTCAGTATCAATTCTGG 570-580
9 GAATCCTA(G)AAAACTCTGCTTC 624-634
10 TGTCATGGTCATCTGCTACTC 637-647
11 CTTCGGTGTCGAAAT(C)GAGAAG 644-654
12 GTCCTTCTCCTGAACACCTTCC 661-671
13 TATGCAGGTGACAGAGACTCT 834-844
【实施例2】重组lenti-CRISPR和AAV-CRISPR构建以及基因编辑效果鉴定
本发明中选择的慢病毒载体为lentiCRISPR/SaCas9，该载体由本实验室构建，具体是将Addgene公司AAV-CRISPR-SaCas9(也叫PX601)载体上的SaCas9基因通过PCR扩增后转移到lentiCRISPR-V2载体上，取代原来的SpCas9。将PX601载体上的SaCas9对应的crRNA通过重叠PCR扩增后转移到lentiCRISPR-V2载体上，取代原来的SpCas9对应的crRNA。
该载体包含一个人的U6启动子主要调控sgRNA的表达，还包含一个EFs启动子来调控SaCas9的表达。而改造的腺相关病毒载体为pAAV-CRISPR，该载体也包含着一个控制sgRNA表达的U6启动子，但含有一个TBG启动子来调控SaCas9的表达。
a、化学方法合成寡核苷酸链，格式如下：
有义链：5’-G(21N)NNGRRT-3’或者5’-ARRCNN(21N)C-3’(引物不含PAM)
PAMPAM
反义链：与有义链互补(引物不含PAM)，在有义链引物的5’端加上CACC,在反义链引物的5’端加上AAAC以形成BsmBI酶切位点和BsaI酶切位点的粘性末端；(注：21N代表靶位点的21个碱基序列)
b、把有义链引物和反义链引物经过退火，形成带有BsmBI酶切位点和BsaI酶切位点的粘性末端和平末端的小片段；
退火体系如下：
退火程序：95℃5min，90℃5min，85℃5min，80℃5min，72℃10min，65℃5min，60℃5min，55℃5min，50℃5min，45℃5min，40℃5min，35℃5min，30℃5min，4℃保存。
c、将退火形成的小片段连入由BsmBI酶和BsaI酶进行酶切的载体lentiCRISPR和AAVCRISPR(参见图1)；
d、将连接产物转化进大肠杆菌Stbl3感受态细胞中(Stbl3菌株购自Invitrogen公司)；
e、挑取单克隆，经初步鉴定后把阳性结果送公司进行测序验证。
f、申请人将所构建的CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体转染TZM-bl细胞(购买自ATCC)，72小时后收集细胞提取其基因组，PCR扩增CCR5基因，把回收的产物经T7EndonucleaseI(购买自BioLabs)酶切鉴定(参见图2)，结果表明：申请人所设计的针对CCR5基因的各个靶位点有不同程度的突变。申请人进一步将PCR产物连接至T-easy载体(购买自Promega)并测序鉴定(参见图3)，结果证实CCR5靶位点具有不同程度的基因插入或缺失和突变，甚至移码突变。CCR5基因的5’引物为：TTATCAAGTGTCAAGTC(SEQIDNO.6)所示，3’引物为：TCTGTGGGCTTG(SEQIDNO.7)
PCR体系如下：
PCR程序：95℃5min，95℃30s→60℃30s→68℃1min共进行30个循环，最后68℃10min，4℃保存。所有循环结束后，暂停反应，在反应体系中加0.5μltaq酶，72℃40min给PCR片段加A尾。
将上述PCR片段用胶回收试剂盒回收(购买自OMEGA)，回收的13个PCR片段经T7EndonucleaseI(购买自BioLabs)酶切鉴定。
酶切体系：
37℃反应60min。
酶切产物进行1.5％琼脂糖凝胶电泳鉴定。
T克隆连接体系：
将上述连接产物转化大肠杆菌Stbl3感受态细胞，筛选阳性克隆并测序鉴定；
慢病毒重组载体的4个靶位点和打靶效率：
CXCR5基因靶点 基因突变率
靶点2GACAGGGCTCTATTTTATAGG 50％
靶点6GGCTGTGTTTGCGTCTCTCCC 60％
靶点8TACAGTCAGTATCAATTCTGG 10％
靶点11CTTCGGTGTCGAAATGAGAAG 50％
腺相关病毒重组载体的6个靶位点和打靶效率：
【实施例3】CRISPR/SaCas9慢病毒系统和腺相关病毒系统的包装
为了使CRISPR/SaCas9重组慢病毒和腺相关病毒载体的切除效率达到更好的效果，申请人把CRISPR/SaCas9重组慢病毒和腺相关病毒载体在HEK-293T细胞中包装成慢病毒和腺相关病毒。
慢病毒系统的包装体系如下：
psPAX2慢病毒辅助包装质粒(购自Addgene)
pMD2.G慢病毒辅助包膜质粒(购自Addgene)
不同靶点的lentiCRISPR载体
具体过程如下：
I、转染前24小时铺HEK-293T细胞(购自CCTCC)于一个直径10cm的细胞培养皿中；
II、待细胞密度细胞达到80～90％为时，以标准的Lipofectamine2000转染试剂将上述质粒共转染细胞，转染后6～8小时换液。
转染体系：
在500μlOpti-MEM中加入三种不同量的质粒DNA，同样用500μlOpti-MEM稀释Lipidreagent，将稀释后的DNA按1:1的比例加入到转染试剂中，混匀静止放置20min后，加入到培养皿里，培养7小时后换液。
转染后72小时收集病毒上清(即细胞培养基)过滤，然后对病毒上清进行浓缩，在4℃，24000rpm，高速离心2小时，弃去上清后加入一定量的培养基重悬，分装后-80℃保存。
III、病毒滴度检测：将病毒上清以10倍梯度稀释，利用病毒计数仪进行病毒滴度的初步测定。将病毒分别感染293T细胞，48小时之后药物检测阳性细胞比率，并以比率在0.1-10％之间的稀释度进一步精确计算出病毒滴度。
腺相关病毒系统的包装体系如下：
pDF腺相关病毒辅助包装质粒(购自Addgene)
AAV6腺相关病毒辅助包膜质粒(购自Addgene)
不同靶点的AAVCRISPR载体
具体过程如下：
I、转染前24小时铺HEK-293T细胞(购自CCTCC)于一个直径10cm的细胞培养皿中；
II、待细胞密度细胞达到80～90％为时，以标准的Lipofectamine2000转染将上述质粒共转染细胞，转染后6～8小时换液。
转染体系：
在500μlOpti-MEM中加入三种不同量的质粒DNA，同样用500μlOpti-MEM稀释Lipidreagent，将稀释后的DNA按1:1的比例加入到转染试剂中，混匀静止放置20min后，加入到培养皿里，培养7小时后换液。
转染后72小时收集病毒上清(即细胞培养基)过滤，然后对病毒上清进行浓缩，在4℃，24000rpm，高速离心2小时，弃去上清后加入一定量的培养基重悬，分装后-80℃保存。
III、病毒滴度检测：将病毒上清以10倍梯度稀释，利用病毒计数仪进行病毒滴度的初步测定。将病毒分别感染293T细胞，48小时之后药物检测阳性细胞比率，并以比率在0.1-10％之间的稀释度进一步精确计算出病毒滴度。
【实施例4】利用CRISPR/Cas9重组慢病毒和腺相关病毒建立稳定敲除CCR5基因的细胞系。
用上述中包装的慢病毒和腺相关病毒以m.o.i.为100(1ml病毒上清加到含4mlDMEM细胞中)感染TZM-bl细胞，72小时后流式检测CCR5阳性细胞比率，流式检测APC-CCR5阳性细胞比率，以检测改造后的效率(参见图4)。
【实施例5】检测CRISPR/SaCas9重组慢病毒载体和腺相关病毒载体改造后的细胞系TZM-bl对HIV-1感染的抑制水平检测。
申请人利用pYU2质粒在HEK-293T细胞中包装成HIV-1型病毒。
I、转染前24小时铺HEK-293T细胞(购自CCTCC)于一个直径10cm的细胞培养皿中；
II、待细胞密度达到80～90％时，以PEI转染试剂操作将上述质粒转染细胞6～8小时后换液。
转染体系：
在500μlOpti-MEM中加入质粒DNA，同样用500μlOpti-MEM稀释PEIreagent，将稀释后的DNA按1:1的比例加入到转染试剂中，混匀静止放置20min后，加入到培养皿里，培养7小时后换液。
转染后72小时收集病毒上清(即细胞培养基)过滤分装，-80℃保存备用。
①病毒滴度检测：将病毒上清以10倍梯度稀释，利用病毒计数仪进行病毒滴度的初步测定。用滴度测定的病毒感染GhostCD4/CXCR4细胞，48小时之后流式检测GFP阳性细胞比率，并以比率在0.1-10％之间的稀释度进一步精确计算出病毒滴度。
②用上述包装的HIV-1病毒以m.o.i.为0.1感染TZM-bl细胞和改造成功的TZMBI细胞系，分别利用单荧光素酶报告基因检测HIV病毒的入侵、转录以及基因组复制水平(参见图5)，和野生型TZM-bl细胞(con)相比，慢病毒载体的2、6、8、11号和腺相关病毒载体的8、11号改造的TZM-bl细胞系中HIV的复制水平显著降低。这说明该发明方法能够成功地改造细胞，可用于改造包括造血干细胞、胚胎干细胞等以阻止HIV入侵，对艾滋病进行临床基因治疗。
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gaccgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccg6660
tacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccgg6720
accgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcg6780
acatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccgg6840
agagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcg6900
gttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaagg6960
agcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgg7020
gcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcc7080
tggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccg7140
ccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcct7200
gaacgcgttaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggta7260
ttcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatc7320
atgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgt7380
ctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttg7440
ctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactt7500
tcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgct7560
ggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgt7620
cctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgct7680
acgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgc7740
ggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcct7800
ccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccacttttt7860
aaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttt7920
tgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaact7980
agggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgc8040
ccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaa8100
aatctctagcagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgcc8160
agccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactccca8220
ctgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattcta8280
ttctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggc8340
atgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctcta8400
gggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgc8460
gcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccctt8520
cctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttag8580
ggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggtt8640
cacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgt8700
tctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctatt8760
cttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgattt8820
aacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtc8880
cccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccag8940
gtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaatta9000
gtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttc9060
cgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgc9120
ctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttg9180
caaaaagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaat9240
taatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatg9300
gccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgag9360
ttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtg9420
gtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaac9480
accctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtc9540
gtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccg9600
tgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgag9660
gagcaggactgacacgtgctacgagatttcgattccaccgccgccttctatgaaaggttg9720
ggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatg9780
ctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagc9840
aatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttg9900
tccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttg9960
gcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacac10020
aacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactc10080
acattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctg10140
cattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgct10200
tcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcac10260
tcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtga10320
gcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccat10380
aggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaac10440
ccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcct10500
gttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcg10560
ctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctg10620
ggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgt10680
cttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacagg10740
attagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactac10800
ggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcgga10860
aaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggttttttt10920
gtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttt10980
tctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgaga11040
ttatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatc11100
taaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacct11160
atctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagata11220
actacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagaccca11280
cgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcaga11340
agtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctaga11400
gtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtg11460
gtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcga11520
gttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgtt11580
gtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattct11640
cttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtca11700
ttctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataat11760
accgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcga11820
aaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcaccc11880
aactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaagg11940
caaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttc12000
ctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatattt12060
gaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgcca12120
cctgac12126
<210>15
<211>12122
<212>DNA
<213>人工序列
<400>15
gtcgacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctg60
atgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagt120
gcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatc180
tgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgac240
attgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccat300
atatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacg360
acccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactt420
tccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaag480
tgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggc540
attatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattag600
tcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggt660
ttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggc720
accaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgg780
gcggtaggcgtgtacggtgggaggtctatataagcagcgcgttttgcctgtactgggtct840
ctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgctt900
aagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgac960
tctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggc1020
gcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactc1080
ggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaa1140
ttttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcggg1200
ggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatata1260
aattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcc1320
tgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcaga1380
caggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatc1440
aaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaaca1500
aaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatg1560
agggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattagga1620
gtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaata1680
ggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatg1740
acgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttg1800
ctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcag1860
ctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatt1920
tggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagt1980
aataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaatt2040
aacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaag2100
aatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacata2160
acaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttta2220
agaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccatta2280
tcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaa2340
gaaggtggagagagagacagagacagatccattcgattagtgaacggatcggcactgcgt2400
gcgccaattctgcagacaaatggcagtattcatccacaattttaaaagaaaaggggggat2460
tggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaa2520
agaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcag2580
agatccagtttggttaattaaggtaccgagggcctatttcccatgattccttcatatttg2640
catatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaag2700
atattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttta2760
aaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttc2820
ttggctttatatatcttgtggaaaggacgaaacaccgcctataaaatagagccctgtcgt2880
tttagtactctggaaacagaatctactaaaacaaggcaaaatgccgtgtttatctcgtca2940
acttgttggcgagatttttgaattcgctagctaggtcttgaaaggagtgggaattggctc3000
cggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagg3060
ggtcggcaattgatccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgt3120
cgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagt3180
cgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggaccggttctagag3240
cgctgccaccatggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagc3300
caagcggaactacatcctgggcctggacatcggcatcaccagcgtgggctacggcatcat3360
cgactacgagacacgggacgtgatcgatgccggcgtgcggctgttcaaagaggccaacgt3420
ggaaaacaacgagggcaggcggagcaagagaggcgccagaaggctgaagcggcggaggcg3480
gcatagaatccagagagtgaagaagctgctgttcgactacaacctgctgaccgaccacag3540
cgagctgagcggcatcaacccctacgaggccagagtgaagggcctgagccagaagctgag3600
cgaggaagagttctctgccgccctgctgcacctggccaagagaagaggcgtgcacaacgt3660
gaacgaggtggaagaggacaccggcaacgagctgtccaccaaagagcagatcagccggaa3720
cagcaaggccctggaagagaaatacgtggccgaactgcagctggaacggctgaagaaaga3780
cggcgaagtgcggggcagcatcaacagattcaagaccagcgactacgtgaaagaagccaa3840
acagctgctgaaggtgcagaaggcctaccaccagctggaccagagcttcatcgacaccta3900
catcgacctgctggaaacccggcggacctactatgagggacctggcgagggcagcccctt3960
cggctggaaggacatcaaagaatggtacgagatgctgatgggccactgcacctacttccc4020
cgaggaactgcggagcgtgaagtacgcctacaacgccgacctgtacaacgccctgaacga4080
cctgaacaatctcgtgatcaccagggacgagaacgagaagctggaatattacgagaagtt4140
ccagatcatcgagaacgtgttcaagcagaagaagaagcccaccctgaagcagatcgccaa4200
agaaatcctcgtgaacgaagaggatattaagggctacagagtgaccagcaccggcaagcc4260
cgagttcaccaacctgaaggtgtaccacgacatcaaggacattaccgcccggaaagagat4320
tattgagaacgccgagctgctggatcagattgccaagatcctgaccatctaccagagcag4380
cgaggacatccaggaagaactgaccaatctgaactccgagctgacccaggaagagatcga4440
gcagatctctaatctgaagggctataccggcacccacaacctgagcctgaaggccatcaa4500
cctgatcctggacgagctgtggcacaccaacgacaaccagatcgctatcttcaaccggct4560
gaagctggtgcccaagaaggtggacctgtcccagcagaaagagatccccaccaccctggt4620
ggacgacttcatcctgagccccgtcgtgaagagaagcttcatccagagcatcaaagtgat4680
caacgccatcatcaagaagtacggcctgcccaacgacatcattatcgagctggcccgcga4740
gaagaactccaaggacgcccagaaaatgatcaacgagatgcagaagcggaaccggcagac4800
caacgagcggatcgaggaaatcatccggaccaccggcaaagagaacgccaagtacctgat4860
cgagaagatcaagctgcacgacatgcaggaaggcaagtgcctgtacagcctggaagccat4920
ccctctggaagatctgctgaacaaccccttcaactatgaggtggaccacatcatccccag4980
aagcgtgtccttcgacaacagcttcaacaacaaggtgctcgtgaagcaggaagaaaacag5040
caagaagggcaaccggaccccattccagtacctgagcagcagcgacagcaagatcagcta5100
cgaaaccttcaagaagcacatcctgaatctggccaagggcaagggcagaatcagcaagac5160
caagaaagagtatctgctggaagaacgggacatcaacaggttctccgtgcagaaagactt5220
catcaaccggaacctggtggataccagatacgccaccagaggcctgatgaacctgctgcg5280
gagctacttcagagtgaacaacctggacgtgaaagtgaagtccatcaatggcggcttcac5340
cagctttctgcggcggaagtggaagtttaagaaagagcggaacaaggggtacaagcacca5400
cgccgaggacgccctgatcattgccaacgccgatttcatcttcaaagagtggaagaaact5460
ggacaaggccaaaaaagtgatggaaaaccagatgttcgaggaaaagcaggccgagagcat5520
gcccgagatcgaaaccgagcaggagtacaaagagatcttcatcaccccccaccagatcaa5580
gcacattaaggacttcaaggactacaagtacagccaccgggtggacaagaagcctaatag5640
agagctgattaacgacaccctgtactccacccggaaggacgacaagggcaacaccctgat5700
cgtgaacaatctgaacggcctgtacgacaaggacaatgacaagctgaaaaagctgatcaa5760
caagagccccgaaaagctgctgatgtaccaccacgacccccagacctaccagaaactgaa5820
gctgattatggaacagtacggcgacgagaagaatcccctgtacaagtactacgaggaaac5880
cgggaactacctgaccaagtactccaaaaaggacaacggccccgtgatcaagaagattaa5940
gtattacggcaacaaactgaacgcccatctggacatcaccgacgactaccccaacagcag6000
aaacaaggtcgtgaagctgtccctgaagccctacagattcgacgtgtacctggacaatgg6060
cgtgtacaagttcgtgaccgtgaagaatctggatgtgatcaaaaaagaaaactactacga6120
agtgaatagcaagtgctatgaggaagctaagaagctgaagaagatcagcaaccaggccga6180
gtttatcgcctccttctacaacaacgatctgatcaagatcaacggcgagctgtatagagt6240
gatcggcgtgaacaacgacctgctgaaccggatcgaagtgaacatgatcgacatcaccta6300
ccgcgagtacctggaaaacatgaacgacaagaggccccccaggatcattaagacaatcgc6360
ctccaagacccagagcattaagaagtacagcacagacattctgggcaacctgtatgaagt6420
gaaatctaagaagcaccctcagatcatcaaaaagggcaaaaggccggcggccacgaaaaa6480
ggccggccaggcaaaaaagaaaaaggattacaaagacgatgacgataagctcgagggatc6540
cggcgcaacaaacttctctctgctgaaacaagccggagatgtcgaagagaatcctggacc6600
gaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacg6660
caccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccg6720
ccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacat6780
cggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagag6840
cgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttc6900
ccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcc6960
cgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcag7020
cgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctgga7080
gacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccga7140
cgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaac7200
gcgttaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattct7260
taactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgc7320
tattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctct7380
ttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctga7440
cgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgc7500
tttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggac7560
aggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctt7620
tccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgt7680
cccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcc7740
tcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctcccc7800
gcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaa7860
gaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgct7920
tgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactaggg7980
aacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgt8040
ctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatc8100
tctagcagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagcc8160
atctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgt8220
cctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattct8280
ggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgc8340
tggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggg8400
gtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcag8460
cgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctt8520
tctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggtt8580
ccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacg8640
tagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctt8700
taatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttt8760
tgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaaca8820
aaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtcccca8880
ggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgt8940
ggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtca9000
gcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcc9060
cattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctct9120
gcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaa9180
aagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaat9240
catcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggcca9300
agttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttct9360
ggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtcc9420
gggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccc9480
tggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgt9540
ccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtggg9600
ggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagc9660
aggactgacacgtgctacgagatttcgattccaccgccgccttctatgaaaggttgggct9720
tcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctgg9780
agttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaata9840
gcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtcca9900
aactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgt9960
aatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaaca10020
tacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacat10080
taattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcatt10140
aatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcct10200
cgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaa10260
aggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaa10320
aaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggc10380
tccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccga10440
caggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttc10500
cgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgcttt10560
ctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggct10620
gtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttg10680
agtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatta10740
gcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggct10800
acactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaa10860
gagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgttt10920
gcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttcta10980
cggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattat11040
caaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaa11100
gtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatct11160
cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataacta11220
cgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgct11280
caccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtg11340
gtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaa11400
gtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgt11460
cacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagtta11520
catgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtca11580
gaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctctta11640
ctgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattct11700
gagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccg11760
cgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaac11820
tctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaact11880
gatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaa11940
atgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttccttt12000
ttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaat12060
gtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctg12120
ac12122
<210>16
<211>12121
<212>DNA
<213>人工序列
<400>16
gtcgacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctg60
atgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagt120
gcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatc180
tgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgac240
attgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccat300
atatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacg360
acccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactt420
tccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaag480
tgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggc540
attatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattag600
tcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggt660
ttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggc720
accaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgg780
gcggtaggcgtgtacggtgggaggtctatataagcagcgcgttttgcctgtactgggtct840
ctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgctt900
aagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgac960
tctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggc1020
gcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactc1080
ggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaa1140
ttttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcggg1200
ggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatata1260
aattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcc1320
tgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcaga1380
caggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatc1440
aaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaaca1500
aaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatg1560
agggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattagga1620
gtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaata1680
ggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatg1740
acgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttg1800
ctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcag1860
ctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatt1920
tggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagt1980
aataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaatt2040
aacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaag2100
aatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacata2160
acaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttta2220
agaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccatta2280
tcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaa2340
gaaggtggagagagagacagagacagatccattcgattagtgaacggatcggcactgcgt2400
gcgccaattctgcagacaaatggcagtattcatccacaattttaaaagaaaaggggggat2460
tggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaa2520
agaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcag2580
agatccagtttggttaattaaggtaccgagggcctatttcccatgattccttcatatttg2640
catatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaag2700
atattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttta2760
aaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttc2820
ttggctttatatatcttgtggaaaggacgaaacaccggctgtgtttgcgtctctcccgtt2880
ttagtactctggaaacagaatctactaaaacaaggcaaaatgccgtgtttatctcgtcaa2940
cttgttggcgagatttttgaattcgctagctaggtcttgaaaggagtgggaattggctcc3000
ggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggg3060
gtcggcaattgatccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtc3120
gtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtc3180
gccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggaccggttctagagc3240
gctgccaccatggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagcc3300
aagcggaactacatcctgggcctggacatcggcatcaccagcgtgggctacggcatcatc3360
gactacgagacacgggacgtgatcgatgccggcgtgcggctgttcaaagaggccaacgtg3420
gaaaacaacgagggcaggcggagcaagagaggcgccagaaggctgaagcggcggaggcgg3480
catagaatccagagagtgaagaagctgctgttcgactacaacctgctgaccgaccacagc3540
gagctgagcggcatcaacccctacgaggccagagtgaagggcctgagccagaagctgagc3600
gaggaagagttctctgccgccctgctgcacctggccaagagaagaggcgtgcacaacgtg3660
aacgaggtggaagaggacaccggcaacgagctgtccaccaaagagcagatcagccggaac3720
agcaaggccctggaagagaaatacgtggccgaactgcagctggaacggctgaagaaagac3780
ggcgaagtgcggggcagcatcaacagattcaagaccagcgactacgtgaaagaagccaaa3840
cagctgctgaaggtgcagaaggcctaccaccagctggaccagagcttcatcgacacctac3900
atcgacctgctggaaacccggcggacctactatgagggacctggcgagggcagccccttc3960
ggctggaaggacatcaaagaatggtacgagatgctgatgggccactgcacctacttcccc4020
gaggaactgcggagcgtgaagtacgcctacaacgccgacctgtacaacgccctgaacgac4080
ctgaacaatctcgtgatcaccagggacgagaacgagaagctggaatattacgagaagttc4140
cagatcatcgagaacgtgttcaagcagaagaagaagcccaccctgaagcagatcgccaaa4200
gaaatcctcgtgaacgaagaggatattaagggctacagagtgaccagcaccggcaagccc4260
gagttcaccaacctgaaggtgtaccacgacatcaaggacattaccgcccggaaagagatt4320
attgagaacgccgagctgctggatcagattgccaagatcctgaccatctaccagagcagc4380
gaggacatccaggaagaactgaccaatctgaactccgagctgacccaggaagagatcgag4440
cagatctctaatctgaagggctataccggcacccacaacctgagcctgaaggccatcaac4500
ctgatcctggacgagctgtggcacaccaacgacaaccagatcgctatcttcaaccggctg4560
aagctggtgcccaagaaggtggacctgtcccagcagaaagagatccccaccaccctggtg4620
gacgacttcatcctgagccccgtcgtgaagagaagcttcatccagagcatcaaagtgatc4680
aacgccatcatcaagaagtacggcctgcccaacgacatcattatcgagctggcccgcgag4740
aagaactccaaggacgcccagaaaatgatcaacgagatgcagaagcggaaccggcagacc4800
aacgagcggatcgaggaaatcatccggaccaccggcaaagagaacgccaagtacctgatc4860
gagaagatcaagctgcacgacatgcaggaaggcaagtgcctgtacagcctggaagccatc4920
cctctggaagatctgctgaacaaccccttcaactatgaggtggaccacatcatccccaga4980
agcgtgtccttcgacaacagcttcaacaacaaggtgctcgtgaagcaggaagaaaacagc5040
aagaagggcaaccggaccccattccagtacctgagcagcagcgacagcaagatcagctac5100
gaaaccttcaagaagcacatcctgaatctggccaagggcaagggcagaatcagcaagacc5160
aagaaagagtatctgctggaagaacgggacatcaacaggttctccgtgcagaaagacttc5220
atcaaccggaacctggtggataccagatacgccaccagaggcctgatgaacctgctgcgg5280
agctacttcagagtgaacaacctggacgtgaaagtgaagtccatcaatggcggcttcacc5340
agctttctgcggcggaagtggaagtttaagaaagagcggaacaaggggtacaagcaccac5400
gccgaggacgccctgatcattgccaacgccgatttcatcttcaaagagtggaagaaactg5460
gacaaggccaaaaaagtgatggaaaaccagatgttcgaggaaaagcaggccgagagcatg5520
cccgagatcgaaaccgagcaggagtacaaagagatcttcatcaccccccaccagatcaag5580
cacattaaggacttcaaggactacaagtacagccaccgggtggacaagaagcctaataga5640
gagctgattaacgacaccctgtactccacccggaaggacgacaagggcaacaccctgatc5700
gtgaacaatctgaacggcctgtacgacaaggacaatgacaagctgaaaaagctgatcaac5760
aagagccccgaaaagctgctgatgtaccaccacgacccccagacctaccagaaactgaag5820
ctgattatggaacagtacggcgacgagaagaatcccctgtacaagtactacgaggaaacc5880
gggaactacctgaccaagtactccaaaaaggacaacggccccgtgatcaagaagattaag5940
tattacggcaacaaactgaacgcccatctggacatcaccgacgactaccccaacagcaga6000
aacaaggtcgtgaagctgtccctgaagccctacagattcgacgtgtacctggacaatggc6060
gtgtacaagttcgtgaccgtgaagaatctggatgtgatcaaaaaagaaaactactacgaa6120
gtgaatagcaagtgctatgaggaagctaagaagctgaagaagatcagcaaccaggccgag6180
tttatcgcctccttctacaacaacgatctgatcaagatcaacggcgagctgtatagagtg6240
atcggcgtgaacaacgacctgctgaaccggatcgaagtgaacatgatcgacatcacctac6300
cgcgagtacctggaaaacatgaacgacaagaggccccccaggatcattaagacaatcgcc6360
tccaagacccagagcattaagaagtacagcacagacattctgggcaacctgtatgaagtg6420
aaatctaagaagcaccctcagatcatcaaaaagggcaaaaggccggcggccacgaaaaag6480
gccggccaggcaaaaaagaaaaaggattacaaagacgatgacgataagctcgagggatcc6540
ggcgcaacaaacttctctctgctgaaacaagccggagatgtcgaagagaatcctggaccg6600
accgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgc6660
accctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgc6720
cacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatc6780
ggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagc6840
gtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcc6900
cggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagccc6960
gcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcagc7020
gccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggag7080
acctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgac7140
gtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaacg7200
cgttaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattctt7260
aactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgct7320
attgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctt7380
tatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgac7440
gcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgct7500
ttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggaca7560
ggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtccttt7620
ccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtc7680
ccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcct7740
cttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccg7800
cgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaag7860
aaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgctt7920
gtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactaggga7980
acccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtc8040
tgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatct8100
ctagcagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagcca8160
tctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtc8220
ctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctg8280
gggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgct8340
ggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctaggggg8400
tatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagc8460
gtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttccttt8520
ctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc8580
cgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgt8640
agtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttcttt8700
aatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattctttt8760
gatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaa8820
aaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccag8880
gctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtg8940
gaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcag9000
caaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgccc9060
attctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctg9120
cctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaa9180
agctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaatc9240
atcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaa9300
gttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctg9360
gaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccg9420
ggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccct9480
ggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtc9540
cacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggg9600
gcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagca9660
ggactgacacgtgctacgagatttcgattccaccgccgccttctatgaaaggttgggctt9720
cggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctgga9780
gttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatag9840
catcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaa9900
actcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgta9960
atcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacat10020
acgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacatt10080
aattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcatta10140
atgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctc10200
gctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaa10260
ggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaa10320
aggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggct10380
ccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgac10440
aggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttcc10500
gaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttc10560
tcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctg10620
tgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttga10680
gtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattag10740
cagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggcta10800
cactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaag10860
agttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttg10920
caagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctac10980
ggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatc11040
aaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaag11100
tatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctc11160
agcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactac11220
gatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctc11280
accggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtgg11340
tcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaag11400
tagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtc11460
acgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttac11520
atgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcag11580
aagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttac11640
tgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctg11700
agaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgc11760
gccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaact11820
ctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactg11880
atcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaa11940
tgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttt12000
tcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatg12060
tatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctga12120
c12121
<210>17
<211>12122
<212>DNA
<213>人工序列
<400>17
gtcgacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctg60
atgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagt120
gcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatc180
tgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgac240
attgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccat300
atatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacg360
acccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactt420
tccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaag480
tgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggc540
attatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattag600
tcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggt660
ttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggc720
accaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgg780
gcggtaggcgtgtacggtgggaggtctatataagcagcgcgttttgcctgtactgggtct840
ctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgctt900
aagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgac960
tctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggc1020
gcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactc1080
ggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaa1140
ttttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcggg1200
ggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatata1260
aattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcc1320
tgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcaga1380
caggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatc1440
aaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaaca1500
aaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatg1560
agggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattagga1620
gtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaata1680
ggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatg1740
acgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttg1800
ctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcag1860
ctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatt1920
tggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagt1980
aataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaatt2040
aacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaag2100
aatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacata2160
acaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttta2220
agaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccatta2280
tcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaa2340
gaaggtggagagagagacagagacagatccattcgattagtgaacggatcggcactgcgt2400
gcgccaattctgcagacaaatggcagtattcatccacaattttaaaagaaaaggggggat2460
tggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaa2520
agaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcag2580
agatccagtttggttaattaaggtaccgagggcctatttcccatgattccttcatatttg2640
catatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaag2700
atattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttta2760
aaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttc2820
ttggctttatatatcttgtggaaaggacgaaacaccgtacagtcagtatcaattctgggt2880
tttagtactctggaaacagaatctactaaaacaaggcaaaatgccgtgtttatctcgtca2940
acttgttggcgagatttttgaattcgctagctaggtcttgaaaggagtgggaattggctc3000
cggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagg3060
ggtcggcaattgatccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgt3120
cgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagt3180
cgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggaccggttctagag3240
cgctgccaccatggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagc3300
caagcggaactacatcctgggcctggacatcggcatcaccagcgtgggctacggcatcat3360
cgactacgagacacgggacgtgatcgatgccggcgtgcggctgttcaaagaggccaacgt3420
ggaaaacaacgagggcaggcggagcaagagaggcgccagaaggctgaagcggcggaggcg3480
gcatagaatccagagagtgaagaagctgctgttcgactacaacctgctgaccgaccacag3540
cgagctgagcggcatcaacccctacgaggccagagtgaagggcctgagccagaagctgag3600
cgaggaagagttctctgccgccctgctgcacctggccaagagaagaggcgtgcacaacgt3660
gaacgaggtggaagaggacaccggcaacgagctgtccaccaaagagcagatcagccggaa3720
cagcaaggccctggaagagaaatacgtggccgaactgcagctggaacggctgaagaaaga3780
cggcgaagtgcggggcagcatcaacagattcaagaccagcgactacgtgaaagaagccaa3840
acagctgctgaaggtgcagaaggcctaccaccagctggaccagagcttcatcgacaccta3900
catcgacctgctggaaacccggcggacctactatgagggacctggcgagggcagcccctt3960
cggctggaaggacatcaaagaatggtacgagatgctgatgggccactgcacctacttccc4020
cgaggaactgcggagcgtgaagtacgcctacaacgccgacctgtacaacgccctgaacga4080
cctgaacaatctcgtgatcaccagggacgagaacgagaagctggaatattacgagaagtt4140
ccagatcatcgagaacgtgttcaagcagaagaagaagcccaccctgaagcagatcgccaa4200
agaaatcctcgtgaacgaagaggatattaagggctacagagtgaccagcaccggcaagcc4260
cgagttcaccaacctgaaggtgtaccacgacatcaaggacattaccgcccggaaagagat4320
tattgagaacgccgagctgctggatcagattgccaagatcctgaccatctaccagagcag4380
cgaggacatccaggaagaactgaccaatctgaactccgagctgacccaggaagagatcga4440
gcagatctctaatctgaagggctataccggcacccacaacctgagcctgaaggccatcaa4500
cctgatcctggacgagctgtggcacaccaacgacaaccagatcgctatcttcaaccggct4560
gaagctggtgcccaagaaggtggacctgtcccagcagaaagagatccccaccaccctggt4620
ggacgacttcatcctgagccccgtcgtgaagagaagcttcatccagagcatcaaagtgat4680
caacgccatcatcaagaagtacggcctgcccaacgacatcattatcgagctggcccgcga4740
gaagaactccaaggacgcccagaaaatgatcaacgagatgcagaagcggaaccggcagac4800
caacgagcggatcgaggaaatcatccggaccaccggcaaagagaacgccaagtacctgat4860
cgagaagatcaagctgcacgacatgcaggaaggcaagtgcctgtacagcctggaagccat4920
ccctctggaagatctgctgaacaaccccttcaactatgaggtggaccacatcatccccag4980
aagcgtgtccttcgacaacagcttcaacaacaaggtgctcgtgaagcaggaagaaaacag5040
caagaagggcaaccggaccccattccagtacctgagcagcagcgacagcaagatcagcta5100
cgaaaccttcaagaagcacatcctgaatctggccaagggcaagggcagaatcagcaagac5160
caagaaagagtatctgctggaagaacgggacatcaacaggttctccgtgcagaaagactt5220
catcaaccggaacctggtggataccagatacgccaccagaggcctgatgaacctgctgcg5280
gagctacttcagagtgaacaacctggacgtgaaagtgaagtccatcaatggcggcttcac5340
cagctttctgcggcggaagtggaagtttaagaaagagcggaacaaggggtacaagcacca5400
cgccgaggacgccctgatcattgccaacgccgatttcatcttcaaagagtggaagaaact5460
ggacaaggccaaaaaagtgatggaaaaccagatgttcgaggaaaagcaggccgagagcat5520
gcccgagatcgaaaccgagcaggagtacaaagagatcttcatcaccccccaccagatcaa5580
gcacattaaggacttcaaggactacaagtacagccaccgggtggacaagaagcctaatag5640
agagctgattaacgacaccctgtactccacccggaaggacgacaagggcaacaccctgat5700
cgtgaacaatctgaacggcctgtacgacaaggacaatgacaagctgaaaaagctgatcaa5760
caagagccccgaaaagctgctgatgtaccaccacgacccccagacctaccagaaactgaa5820
gctgattatggaacagtacggcgacgagaagaatcccctgtacaagtactacgaggaaac5880
cgggaactacctgaccaagtactccaaaaaggacaacggccccgtgatcaagaagattaa5940
gtattacggcaacaaactgaacgcccatctggacatcaccgacgactaccccaacagcag6000
aaacaaggtcgtgaagctgtccctgaagccctacagattcgacgtgtacctggacaatgg6060
cgtgtacaagttcgtgaccgtgaagaatctggatgtgatcaaaaaagaaaactactacga6120
agtgaatagcaagtgctatgaggaagctaagaagctgaagaagatcagcaaccaggccga6180
gtttatcgcctccttctacaacaacgatctgatcaagatcaacggcgagctgtatagagt6240
gatcggcgtgaacaacgacctgctgaaccggatcgaagtgaacatgatcgacatcaccta6300
ccgcgagtacctggaaaacatgaacgacaagaggccccccaggatcattaagacaatcgc6360
ctccaagacccagagcattaagaagtacagcacagacattctgggcaacctgtatgaagt6420
gaaatctaagaagcaccctcagatcatcaaaaagggcaaaaggccggcggccacgaaaaa6480
ggccggccaggcaaaaaagaaaaaggattacaaagacgatgacgataagctcgagggatc6540
cggcgcaacaaacttctctctgctgaaacaagccggagatgtcgaagagaatcctggacc6600
gaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacg6660
caccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccg6720
ccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacat6780
cggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagag6840
cgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttc6900
ccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcc6960
cgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcag7020
cgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctgga7080
gacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccga7140
cgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaac7200
gcgttaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattct7260
taactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgc7320
tattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctct7380
ttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctga7440
cgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgc7500
tttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggac7560
aggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctt7620
tccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgt7680
cccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcc7740
tcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctcccc7800
gcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaa7860
gaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgct7920
tgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactaggg7980
aacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgt8040
ctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatc8100
tctagcagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagcc8160
atctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgt8220
cctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattct8280
ggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgc8340
tggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggg8400
gtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcag8460
cgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctt8520
tctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggtt8580
ccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacg8640
tagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctt8700
taatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttt8760
tgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaaca8820
aaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtcccca8880
ggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgt8940
ggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtca9000
gcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcc9060
cattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctct9120
gcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaa9180
aagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaat9240
catcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggcca9300
agttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttct9360
ggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtcc9420
gggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccc9480
tggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgt9540
ccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtggg9600
ggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagc9660
aggactgacacgtgctacgagatttcgattccaccgccgccttctatgaaaggttgggct9720
tcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctgg9780
agttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaata9840
gcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtcca9900
aactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgt9960
aatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaaca10020
tacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacat10080
taattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcatt10140
aatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcct10200
cgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaa10260
aggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaa10320
aaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggc10380
tccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccga10440
caggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttc10500
cgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgcttt10560
ctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggct10620
gtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttg10680
agtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatta10740
gcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggct10800
acactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaa10860
gagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgttt10920
gcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttcta10980
cggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattat11040
caaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaa11100
gtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatct11160
cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataacta11220
cgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgct11280
caccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtg11340
gtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaa11400
gtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgt11460
cacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagtta11520
catgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtca11580
gaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctctta11640
ctgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattct11700
gagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccg11760
cgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaac11820
tctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaact11880
gatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaa11940
atgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttccttt12000
ttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaat12060
gtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctg12120
ac12122
<210>18
<211>12122
<212>DNA
<213>人工序列
<400>18
gtcgacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctg60
atgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagt120
gcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatc180
tgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgac240
attgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccat300
atatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacg360
acccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactt420
tccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaag480
tgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggc540
attatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattag600
tcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggt660
ttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggc720
accaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgg780
gcggtaggcgtgtacggtgggaggtctatataagcagcgcgttttgcctgtactgggtct840
ctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgctt900
aagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgac960
tctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggc1020
gcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactc1080
ggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaa1140
ttttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcggg1200
ggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatata1260
aattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcc1320
tgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcaga1380
caggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatc1440
aaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaaca1500
aaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatg1560
agggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattagga1620
gtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaata1680
ggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatg1740
acgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttg1800
ctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcag1860
ctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatt1920
tggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagt1980
aataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaatt2040
aacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaag2100
aatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacata2160
acaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttta2220
agaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccatta2280
tcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaa2340
gaaggtggagagagagacagagacagatccattcgattagtgaacggatcggcactgcgt2400
gcgccaattctgcagacaaatggcagtattcatccacaattttaaaagaaaaggggggat2460
tggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaa2520
agaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcag2580
agatccagtttggttaattaaggtaccgagggcctatttcccatgattccttcatatttg2640
catatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaag2700
atattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttta2760
aaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttc2820
ttggctttatatatcttgtggaaaggacgaaacaccgcttctcatttcgacaccgaaggt2880
tttagtactctggaaacagaatctactaaaacaaggcaaaatgccgtgtttatctcgtca2940
acttgttggcgagatttttgaattcgctagctaggtcttgaaaggagtgggaattggctc3000
cggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagg3060
ggtcggcaattgatccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgt3120
cgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagt3180
cgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggaccggttctagag3240
cgctgccaccatggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagc3300
caagcggaactacatcctgggcctggacatcggcatcaccagcgtgggctacggcatcat3360
cgactacgagacacgggacgtgatcgatgccggcgtgcggctgttcaaagaggccaacgt3420
ggaaaacaacgagggcaggcggagcaagagaggcgccagaaggctgaagcggcggaggcg3480
gcatagaatccagagagtgaagaagctgctgttcgactacaacctgctgaccgaccacag3540
cgagctgagcggcatcaacccctacgaggccagagtgaagggcctgagccagaagctgag3600
cgaggaagagttctctgccgccctgctgcacctggccaagagaagaggcgtgcacaacgt3660
gaacgaggtggaagaggacaccggcaacgagctgtccaccaaagagcagatcagccggaa3720
cagcaaggccctggaagagaaatacgtggccgaactgcagctggaacggctgaagaaaga3780
cggcgaagtgcggggcagcatcaacagattcaagaccagcgactacgtgaaagaagccaa3840
acagctgctgaaggtgcagaaggcctaccaccagctggaccagagcttcatcgacaccta3900
catcgacctgctggaaacccggcggacctactatgagggacctggcgagggcagcccctt3960
cggctggaaggacatcaaagaatggtacgagatgctgatgggccactgcacctacttccc4020
cgaggaactgcggagcgtgaagtacgcctacaacgccgacctgtacaacgccctgaacga4080
cctgaacaatctcgtgatcaccagggacgagaacgagaagctggaatattacgagaagtt4140
ccagatcatcgagaacgtgttcaagcagaagaagaagcccaccctgaagcagatcgccaa4200
agaaatcctcgtgaacgaagaggatattaagggctacagagtgaccagcaccggcaagcc4260
cgagttcaccaacctgaaggtgtaccacgacatcaaggacattaccgcccggaaagagat4320
tattgagaacgccgagctgctggatcagattgccaagatcctgaccatctaccagagcag4380
cgaggacatccaggaagaactgaccaatctgaactccgagctgacccaggaagagatcga4440
gcagatctctaatctgaagggctataccggcacccacaacctgagcctgaaggccatcaa4500
cctgatcctggacgagctgtggcacaccaacgacaaccagatcgctatcttcaaccggct4560
gaagctggtgcccaagaaggtggacctgtcccagcagaaagagatccccaccaccctggt4620
ggacgacttcatcctgagccccgtcgtgaagagaagcttcatccagagcatcaaagtgat4680
caacgccatcatcaagaagtacggcctgcccaacgacatcattatcgagctggcccgcga4740
gaagaactccaaggacgcccagaaaatgatcaacgagatgcagaagcggaaccggcagac4800
caacgagcggatcgaggaaatcatccggaccaccggcaaagagaacgccaagtacctgat4860
cgagaagatcaagctgcacgacatgcaggaaggcaagtgcctgtacagcctggaagccat4920
ccctctggaagatctgctgaacaaccccttcaactatgaggtggaccacatcatccccag4980
aagcgtgtccttcgacaacagcttcaacaacaaggtgctcgtgaagcaggaagaaaacag5040
caagaagggcaaccggaccccattccagtacctgagcagcagcgacagcaagatcagcta5100
cgaaaccttcaagaagcacatcctgaatctggccaagggcaagggcagaatcagcaagac5160
caagaaagagtatctgctggaagaacgggacatcaacaggttctccgtgcagaaagactt5220
catcaaccggaacctggtggataccagatacgccaccagaggcctgatgaacctgctgcg5280
gagctacttcagagtgaacaacctggacgtgaaagtgaagtccatcaatggcggcttcac5340
cagctttctgcggcggaagtggaagtttaagaaagagcggaacaaggggtacaagcacca5400
cgccgaggacgccctgatcattgccaacgccgatttcatcttcaaagagtggaagaaact5460
ggacaaggccaaaaaagtgatggaaaaccagatgttcgaggaaaagcaggccgagagcat5520
gcccgagatcgaaaccgagcaggagtacaaagagatcttcatcaccccccaccagatcaa5580
gcacattaaggacttcaaggactacaagtacagccaccgggtggacaagaagcctaatag5640
agagctgattaacgacaccctgtactccacccggaaggacgacaagggcaacaccctgat5700
cgtgaacaatctgaacggcctgtacgacaaggacaatgacaagctgaaaaagctgatcaa5760
caagagccccgaaaagctgctgatgtaccaccacgacccccagacctaccagaaactgaa5820
gctgattatggaacagtacggcgacgagaagaatcccctgtacaagtactacgaggaaac5880
cgggaactacctgaccaagtactccaaaaaggacaacggccccgtgatcaagaagattaa5940
gtattacggcaacaaactgaacgcccatctggacatcaccgacgactaccccaacagcag6000
aaacaaggtcgtgaagctgtccctgaagccctacagattcgacgtgtacctggacaatgg6060
cgtgtacaagttcgtgaccgtgaagaatctggatgtgatcaaaaaagaaaactactacga6120
agtgaatagcaagtgctatgaggaagctaagaagctgaagaagatcagcaaccaggccga6180
gtttatcgcctccttctacaacaacgatctgatcaagatcaacggcgagctgtatagagt6240
gatcggcgtgaacaacgacctgctgaaccggatcgaagtgaacatgatcgacatcaccta6300
ccgcgagtacctggaaaacatgaacgacaagaggccccccaggatcattaagacaatcgc6360
ctccaagacccagagcattaagaagtacagcacagacattctgggcaacctgtatgaagt6420
gaaatctaagaagcaccctcagatcatcaaaaagggcaaaaggccggcggccacgaaaaa6480
ggccggccaggcaaaaaagaaaaaggattacaaagacgatgacgataagctcgagggatc6540
cggcgcaacaaacttctctctgctgaaacaagccggagatgtcgaagagaatcctggacc6600
gaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacg6660
caccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccg6720
ccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacat6780
cggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagag6840
cgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttc6900
ccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcc6960
cgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcag7020
cgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctgga7080
gacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccga7140
cgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaac7200
gcgttaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattct7260
taactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgc7320
tattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctct7380
ttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctga7440
cgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgc7500
tttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggac7560
aggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctt7620
tccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgt7680
cccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcc7740
tcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctcccc7800
gcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaa7860
gaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgct7920
tgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactaggg7980
aacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgt8040
ctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatc8100
tctagcagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagcc8160
atctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgt8220
cctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattct8280
ggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgc8340
tggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggg8400
gtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcag8460
cgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctt8520
tctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggtt8580
ccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacg8640
tagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctt8700
taatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttt8760
tgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaaca8820
aaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtcccca8880
ggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgt8940
ggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtca9000
gcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcc9060
cattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctct9120
gcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaa9180
aagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaat9240
catcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggcca9300
agttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttct9360
ggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtcc9420
gggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccc9480
tggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgt9540
ccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtggg9600
ggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagc9660
aggactgacacgtgctacgagatttcgattccaccgccgccttctatgaaaggttgggct9720
tcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctgg9780
agttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaata9840
gcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtcca9900
aactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgt9960
aatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaaca10020
tacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacat10080
taattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcatt10140
aatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcct10200
cgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaa10260
aggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaa10320
aaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggc10380
tccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccga10440
caggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttc10500
cgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgcttt10560
ctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggct10620
gtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttg10680
agtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatta10740
gcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggct10800
acactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaa10860
gagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgttt10920
gcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttcta10980
cggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattat11040
caaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaa11100
gtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatct11160
cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataacta11220
cgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgct11280
caccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtg11340
gtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaa11400
gtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgt11460
cacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagtta11520
catgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtca11580
gaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctctta11640
ctgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattct11700
gagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccg11760
cgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaac11820
tctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaact11880
gatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaa11940
atgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttccttt12000
ttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaat12060
gtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctg12120
ac12122

Claims (11)

1.在CRISPR/SaCas9特异性敲除人CCR5基因中用于特异性靶向CCR5基因的sgRNA,其特征在于：所述sgRNA在CCR5基因上的靶序列符合5’-G(21N)NNGRRT-3’或者5’-ARRCNN(21N)C-3’的序列排列规则，下划线代表PAM，21N代表靶位点的21个碱基序列，其对应的DNA序列如SEQIDNO.1-4任意一条序列所示，其中SEQIDNO.1-3在人与恒河猴的基因位点上保守。
2.含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的慢病毒载体，其特征在于：
①所述靶向敲除载体是将Addgene公司PX601载体上的SaCas9基因通过PCR扩增后转移到lentiCRISPR-V2载体上，取代原来的SpCas9；将PX601载体上的SaCas9对应的crRNA通过重叠PCR扩增后转移到lentiCRISPR-V2载体上，取代原来的SpCas9对应的crRNA；
②此载体含有一个人的U6启动子，该启动子控制着sgRNA的表达，用BsmBI核酸内切酶对此载体进行消化，然后再插入含有突出粘性末端和长为21bp的靶序列sgRNA片段；
③在酶切位点BsmBI后有长为81bp的crRNA核酸序列为sgRNA的骨架序列；
④crRNA核酸序列后的EFS的启动子控制人类编码最优的SaCas9的表达；
⑤含有自切割多肽P2A和带有筛选标记的基因Puromycin的编码序列。
3.根据权利要求2所述的含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的慢病毒载体，其特征在于：所述的慢病毒载体的序列如SEQIDNO.15-18任意一项所示。
4.含有权利要求3所述靶向敲除人CCR5基因的慢病毒载体的慢病毒。
5.含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的腺相关病毒载体，其特征在于：
①选择的腺相关病毒载体为Addgene公司PX601；
②此载体含有一个U6启动子，控制着sgRNA的表达，用BsaI核酸内切酶切割载体后，插入含有突出粘性末端和长为21bp的靶序列sgRNA片段，靶序列如SEQIDNO.1-4任意一项所示；
③含有一个TBG启动子来调控SaCas9的表达。
6.含有权利要求5所述靶向敲除人CCR5基因的腺相关病毒载体的腺相关病毒。
7.权利要求1所述的在CRISPR/SaCas9特异性敲除人CCR5基因中用于特异性靶向CCR5基因的sgRNA在非诊疗目的的敲除人或恒河猴CCR5基因中的应用。
8.权利要求2或3所述的含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的慢病毒载体在非诊疗目的的敲除人或恒河猴CCR5基因中的应用。
9.权利要求5所述的含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的腺相关病毒载体在非诊疗目的的敲除人或恒河猴CCR5基因中的应用。
10.权利要求2或3所述的含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的慢病毒载体在制备抗艾滋病药物中的应用。
11.权利要求5所述的含有CRISPR/SaCas9系统的靶向敲除人CCR5基因的腺相关病毒载体在制备抗艾滋病药物中的应用。
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Cited By (31)

* Cited by examiner, † Cited by third party
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