CN111534543A - 一种真核生物CRISPR/Cas9敲除系统、基础载体、载体及细胞系 - Google Patents

一种真核生物CRISPR/Cas9敲除系统、基础载体、载体及细胞系 Download PDF

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CN111534543A
CN111534543A CN202010378948.6A CN202010378948A CN111534543A CN 111534543 A CN111534543 A CN 111534543A CN 202010378948 A CN202010378948 A CN 202010378948A CN 111534543 A CN111534543 A CN 111534543A
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马三垣
常珈菘
夏庆友
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Abstract

本发明涉及一种真核生物CRISPR/Cas9敲除系统、基础载体、载体及细胞系，该敲除系统包含两个关键子系统：一个是piggyBac转座子系统作为递送系统；另一个是CRISPR/Cas9基因敲除系统。基因piggyBac转座子系统有强大的承载能力，本发明建立了all‑in‑one载体系统pB‑CRISPR用于表达CRISPR/Cas9系统，能够高效、经济地实现真核生物的基因敲除。

Description

一种真核生物CRISPR/Cas9敲除系统、基础载体、载体及细 胞系
技术领域
本发明属于真核生物基因敲除技术领域，涉及一种真核生物CRISPR/Cas9敲除系统、基础载体、载体及细胞系。
背景技术
自从人类基因组计划完成以来，包括小鼠、果蝇、家蚕、拟南芥、水稻等越来越多的模式生物和非模式生物的完成全基因组测序，面对海量的基因组信息，解读功能基因组成为了后基因组时代的重要课题，各种遗传操作技术(转基因、RNAi等)为功能基因组研究提供了基础平台，在众多的遗传操作技术中，基因敲除技术和转基因技术是研究功能基因的两个关键遗传操作技术。
基因编辑技术是近年来发展起来的重要的遗传操作技术，目前已经发展了四代，包括大范围核酸酶、锌指核酸酶类、转录激活因子样效应物核酸酶、CRISPR等。不同与前面三代基因编辑技术(依赖蛋白质-核苷酸相互识别)，CRISPR技术是基于RNA与DNA碱基互补配对的全新的基于编辑技术。CRISPR系统发明以来，已经在包括人，小鼠，果蝇，斑马鱼，家蚕，拟南芥，烟草，水稻等许多生物中成功实现了基因敲除。高效的基因敲除离不开高效的递送系统，目前在动物中应用最广泛的递送系统是慢病毒介导的递送系统，已经成功地在人、小鼠等哺乳动物中递送了CRISPR系统。
但是慢病毒系统有两个显著的缺点，一个是慢病毒系统的作用物种有限，在哺乳动物以外的物种中效率很低；二是慢病毒系统的承载能力有限，仅仅数千个核苷酸，而且随着外源基因的增大，病毒滴度明显降低，递送能力显著下降。为了在更多种类的真核生物中实现高效的基因敲除，急需开发一种高效递送CRISPR的系统。
piggyBac转座子系统是来源于粉纹夜蛾的一种Ⅱ型转座子，全长2476bp，包括两个末端反向重复序列(inverted terminal repeat,ITR)和一个编码转座酶的表达框。piggyBac转座子系统采用了“剪切-黏贴”的模式来实现转座，转座效率高。目前，piggyBac转座子系统已经被证明可以在从昆虫到哺乳动物的许多物种中高效转座。piggyBac转座子系统具有强大的承载能力，已有报道显示最大可承载超过207kb的核苷酸，承载能力远远超过慢病毒系统。由于piggyBac转座子是以“剪切-黏贴”的方式来实现来实现转座，因此可以通过控制转座子浓度等方式来控制piggyBac转座子系统携带的外源基因整合到宿主细胞的拷贝数。尤其是在构建全基因组敲除细胞库时，可以方便的控制每个细胞只整合一个外源sgRNA。因此，开发piggyBac转座子系统介导的真核生物CRISPR敲除系统具有很广阔的应用前景。
发明内容
有鉴于此，本发明的目的在于提供一种真核生物CRISPR/Cas9敲除系统、基础载体、载体及细胞系。
为达到上述目的，本发明提供如下技术方案：
1、piggyBac转座子系统介导的真核生物CRISPR/Cas9敲除系统，是由基因元件递送系统和基因敲除系统两部分构成，两者分别为piggyBac转座子系统和CRISPR/Cas9系统。
作为优选的技术方案之一，所述piggyBac转座子系统包括：
两个转座臂(包含piggyBac转座子的末端反向重复序列，inverted terminalrepeat,ITR)，其核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示；
筛选标记Zeocin的抗性基因表达框，其核苷酸序列如SEQ ID NO.3所示。
作为优选的技术方案之一，所述CRISPR/Cas9系统包括两部分元件：
Hr3 CQ Enhancer-Hsp70启动子启动的spCas9蛋白表达框，其核苷酸序列如SEQID NO.4所示；
家蚕U6启动子启动的sgRNA表达框，其核苷酸序列如SEQ ID NO.5所示。
2、包含上述系统的一种真核生物基因敲除基础载体，其核苷酸序列如SEQ IDNO.6所示，命名为pB-CRISPR。
3、上述一种真核生物基因敲除基础载体的构建方法，具体方法如下：
(1)合成包含Zeocin抗性基因表达框的载体PUC57-IE2-Zeocin-Ser1PA；
(2)将IE2-Zeocin-Ser1PA表达框连接到piggyBac转座子基础载体piggyBacModify上，构建成中间载体pB-Modified{IE2-Zeocin-Ser1PA}；
(3)将hr3-hsp70-Cas9-sv40表达框从载体pUC57-hr3-hsp70-Cas9-sv40上扩增出来；然后用无缝克隆的方法连接到pB-Modified{IE2-Zeocin-Ser1PA}的AscI位点，构建成中间载体pB-Modified{IE2-Zeocin-Ser1PA}{hr3-hsp70-Cas9-SV40}；
(4)将U6-gRNA从载体pUC57-U6-gRNA扩增出来，用酶切连接的方法连接到载体pB-Modified{IE2-Zeocin-Ser1PA}{hr3-hsp70-Cas9-SV40}的AscI/NheI位点，构建成真核生物基因敲除基础载体pB-Modified{IE2-Zeocin-Ser1PA}{U6-gRNA}{hr3-hsp70-Cas9-SV40},命名为pB-CRISPR；
其中，载体PUC57-IE2-Zeocin-Ser1PA，核苷酸序列如SEQ ID NO.7所示；
piggyBac转座子基础载体piggyBacModify，核苷酸序列如SEQ ID NO.8所示；
中间载体pB-Modified{IE2-Zeocin-Ser1PA}，核苷酸序列如SEQ ID NO.9所示；
载体pUC57-hr3-hsp70-Cas9-sv40，核苷酸序列如SEQ ID NO.10所示；
中间载体pB-Modified{IE2-Zeocin-Ser1PA}{hr3-hsp70-Cas9-SV40}，核苷酸序列如SEQ ID NO.11所示；
载体pUC57-U6-gRNA，核苷酸序列如SEQ ID NO.12所示。
4、利用上述基础载体构建的一种真核生物基因敲除载体，是将上述基础载体pB-CRISPR用核酸内切酶AarI消化作为骨架，利用引物组合成并且梯度退火成包含粘性末端的双链DNA，然后连接到所述骨架上即得，命名为pB-CRISPR-X。
作为优选的技术方案之一，所述引物组包括：
正向引物X-F，5’-AAGT-NNNNNNNNNNNNNNNNNNNN-3’，如SEQ ID NO.13所示；
反向引物X-R，5-AAAC-NNNNNNNNNNNNNNNNNNNN-3’，如SEQ ID NO.14所示；
其中两条引物中的“N”为反向互补序列，“AAGT”和“AAAC”为粘性末端序列；
以及用于合成敲除真核生物蛋白编码基因的打靶位点的引物对：
EGFP-1F，5’-AAGTGGGCGAGGAGCTGTTCACCG-3’，如SEQ ID NO.15所示；
EGFP-1R，5’-AAACCGGTGAACAGCTCCTCGCCC-3’，如SEQ ID NO.16所示；
EGFP-2F，5’-AAGTGAGCTGGACGGCGACGTAAA-3’，如SEQ ID NO.17所示，
EGFP-2R，5’-AAACTTTACGTCGCCGTCCAGCTC-3’，如SEQ ID NO.18所示；
EGFP-3F，5’-AAGTGGCCACAAGTTCAGCGTGTC-3’，如SEQ ID NO.19所示；
EGFP-3R，5’-AAACGACACGCTGAACTTGTGGCC-3’，如SEQ ID NO.20所示；
NS-1F，5’-AAGTCGCTTCAAGGTGCACATGGA-3’，如SEQ ID NO.21所示；
NS-1R，5’-AAACTCCATGTGCACCTTGAAGCG-3’，如SEQ ID NO.22所示；
NS-2F，5’-AAGTGCGGAAGAACGCCTGCGGCT-3’，如SEQ ID NO.23所示
NS-2R，5’-AAACAGCCGCAGGCGTTCTTCCGC-3’，如SEQ ID NO.24所示。
作为进一步优选的技术方案之一，所述打靶位点的核苷酸具有如下规律：
5’-NNNNNNNNNNNNNNNNNNN-NGG-3’，具体的，所述打靶位点包括：3个敲除绿色荧光蛋白编码基因的打靶位点EGFP-1、EGFP-2、EGFP-3，和2个阴性对照打靶位点NS-1、NS-2，它们的核苷酸序列如下：
EGFP-1，5’-GGGCGAGGAGCTGTTCACCG-3’，如SEQ ID NO.25所示；
EGFP-2，5’-GAGCTGGACGGCGACGTAAA-3’，如SEQ ID NO.26所示；
EGFP-3，5’-GGCCACAAGTTCAGCGTGTC-3’，如SEQ ID NO.27所示；
NS-1，5’-CGCTTCAAGGTGCACATGGAGGG-3’，如SEQ ID NO.28所示；
NS-2，5’-GCGGAAGAACGCCTGCGGCTCGG-3’，如SEQ ID NO.29所示。
作为优选的技术方案之一，所述基础载体pB-CRISPR用核酸内切酶AarI消化所得产物用琼脂糖凝胶电泳后切胶回收。
作为优选的技术方案之一，所述骨架与双链DNA的摩尔比为1：50。
作为优选的技术方案之一，所述骨架与双链DNA利用DNA连接酶连接。
作为优选的技术方案之一，所述骨架与双链DNA连接所得连接产物转化大肠杆菌后挑选单克隆。
作为进一步优选的技术方案之一，设计引物对对挑选的单克隆进行检测，若PCR扩增产物的大小约1100bp时，判断为阳性克隆并挑选出来；所述引物对包括：
正向引物K-SerP1-F1，5’-CGAGTCCTTTCCGATGTGT-3’，如SEQ ID NO.31所示；
反向引物K-hr3-R1，5’-CGTTTTGTATTTGTCATTGCC-3’，如SEQ ID NO.32所示。
作为更进一步优选的技术方案之一，上述挑选的阳性克隆用sanger法测序分析，寻找能够敲除真核生物编码蛋白基因X的基因敲除载体pB-CRISPR-X。
5、利用上述载体构建的一种真核生物基因敲除细胞系，是将上述载体pB-CRISPR-X和核苷酸序列如SEQ ID NO.30所示的piggyBac transposon表达载体A3-helper按照摩尔比1：1转染真核细胞，将转染后的细胞用Zeocin筛选2个月得到。
作为优选的技术方案之一，在真核生物基因X的打靶位点两侧设计引物对，以上述细胞系基因组为模板扩增约1000bp的DNA片段，T-A克隆后用sanger法可以检测基因敲除情况。
正向引物EGFP-F，5’-ATGGTGAGCAAGGGCG-3’，如SEQ ID NO.33所示；
反向引物EGFP-R，5’-TTACTTGTACAGCTCGTCCATG-3’，如SEQ ID NO.34所示。
作为优选的技术方案之一，所述真核生物包括但不限于家蚕、果蝇等。
本发明的有益效果在于：
本发明包含两个关键子系统：一个是piggyBac转座子系统作为递送系统；另一个是CRISPR/Cas9系统。基因piggyBac转座子系统有强大的承载能力，本发明建立了all-in-one载体系统pB-CRISPR用于表达CRISPR/Cas9系统，能够高效、经济地实现真核生物的基因敲除。现有的真核生物CRISPR/Cas9敲除系统主要是由慢病毒系统递送的，由于慢病毒主要在哺乳动物中有较高的转基因效率在其他物种中转基因效率极低，而且慢病毒系统承载容量一般只有几kb，CRISPR/Cas9系统的两个组成部分：Cas9蛋白表达框和sgRNA表达框通常需要分两次整合到宿主细胞的基因组上。这些缺点极大的限制了慢病毒递送的CRISPR/Cas9系统的应用。由于piggyBac转座子是以“剪切-黏贴”的方式来实现来实现转座，因此可以通过控制转座子浓度等方式来控制piggyBac转座子系统携带的外源基因整合到宿主细胞的拷贝数。尤其是在构建全基因组敲除细胞库时，可以方便的控制每个细胞只整合一个外源sgRNA。与现有的慢病毒递送CRISPR/Cas9系统相比，本发明具有极大的优势。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为载体pB-CRISPR图谱，包括：piggyBacL/piggyBacR，piggyBac转座臂；IE2，IE2启动子；Zeocin，Zeocin抗性基因；Ser1PA，家蚕丝胶1(Ser1)基因polyA；U6，U6启动子；gRNA，sgRNA scaffold；Hr3-hsp70，Hr3增强子和hsp70启动子；spCas9，spCas9蛋白；SV40PA，SV40 polyA；
图2为流式细胞仪检测pB-CRISPR系统敲除家蚕BmE细胞基因效率，其中，上面的图片表示流式细胞仪散点图，下面的图表示统计数据柱状图。。
图3为sanger测序分析pB-CRISPR系统敲除家蚕BmE细胞基因效率，结果显示全部单克隆都实现了基因敲除，突变形式主要是小片段缺失，少数是小片段插入。
具体实施方式
下面将结合附图，对本发明的优选实施例进行详细的描述。
以下凡是未注明的具体实验方法，都按照公认的实验方法与条件实施，例如，按照试剂耗材厂商提供的说明书操作，或者按照经典实验书籍《分子克隆实验指南》(第三版，J.萨姆布鲁克等著)来完成实验。
实施例：
本实例中所用到的家蚕胚胎细胞系(The Bombyx mori embryonic cell line,BmE)为生物实验中常用细胞系(PMID:17570024)。
构建piggyBac转座子系统介导的家蚕CRISPR/Cas9敲除系统，打靶位点为EGFP。
1、构建一个以piggyBac转座子系统为递送系统和以CRISPR/Cas9系统为基因敲除系统的家蚕基因敲除基础载体，命名为pB-CRISPR。其构建方法如下：
(1)合成包含Zeocin抗性基因表达框的载体PUC57-IE2-Zeocin-Ser1PA，核苷酸序列如SEQ ID NO.7所示；
(2)将载体PUC57-IE2-Zeocin-Ser1PA上的Zeocin抗性基因表达框IE2-Zeocin-Ser1PA表达框连接到piggyBac转座子基础载体piggyBacModify(核苷酸序列如SEQ IDNO.8所示)上，构建成中间载体pB-Modified{IE2-Zeocin-Ser1PA}，核苷酸序列如SEQ IDNO.9所示；
(3)将hr3-hsp70-Cas9-sv40表达框从载体pUC57-hr3-hsp70-Cas9-sv40上扩增出来。然后用无缝克隆的方法连接到pB-Modified{IE2-Zeocin-Ser1PA}的AscI位点，构建成中间载体pB-Modified{IE2-Zeocin-Ser1PA}{hr3-hsp70-Cas9-SV40}，核苷酸序列如SEQID NO.11所示；
(4)将U6-gRNA从载体pUC57-U6-gRNA(核苷酸序列如SEQ ID NO.12所示)扩增出来，用酶切连接的方法连接到载体pB-Modified{IE2-Zeocin-Ser1PA}{hr3-hsp70-Cas9-SV40}的AscI/NheI位点，构建成真核生物基因敲除基础载体pB-Modified{IE2-Zeocin-Ser1PA}{U6-gRNA}{hr3-hsp70-Cas9-SV40},命名为pB-CRISPR。
载体图谱如图1所示。
2、根据CRISPR/Cas9(spCas9)作用规律，设计3个敲除绿色荧光蛋白编码基因的打靶位点EGFP-1、EGFP-2、EGFP-3，和2个阴性对照打靶位点NS-1、NS-2，具体如下：
EGFP-1，5’-GGGCGAGGAGCTGTTCACCG-3’，如SEQ ID NO.25所示；
EGFP-2，5’-GAGCTGGACGGCGACGTAAA-3’，如SEQ ID NO.26所示；
EGFP-3，5’-GGCCACAAGTTCAGCGTGTC-3’，如SEQ ID NO.27所示；
NS-1，5’-CGCTTCAAGGTGCACATGGAGGG-3’，如SEQ ID NO.28所示；
NS-2，5’-GCGGAAGAACGCCTGCGGCTCGG-3’，如SEQ ID NO.29所示。
3、根据步骤2，设计用于构建家蚕基因敲除的载体的成对的引物，具体如下：
正向引物X-F，5’-AAGT-NNNNNNNNNNNNNNNNNNNN-3’，如SEQ ID NO.13所示；
反向引物X-R，5-AAAC-NNNNNNNNNNNNNNNNNNNN-3’，如SEQ ID NO.14所示；
其中两条引物中的“N”为反向互补序列，“AAGT”和“AAAC”为粘性末端序列；
以及用于合成敲除真核生物蛋白编码基因的打靶位点的引物对：
EGFP-1F，5’-AAGTGGGCGAGGAGCTGTTCACCG-3’，如SEQ ID NO.15所示；
EGFP-1R，5’-AAACCGGTGAACAGCTCCTCGCCC-3’，如SEQ ID NO.16所示；
EGFP-2F，5’-AAGTGAGCTGGACGGCGACGTAAA-3’，如SEQ ID NO.17所示，
EGFP-2R，5’-AAACTTTACGTCGCCGTCCAGCTC-3’，如SEQ ID NO.18所示；
EGFP-3F，5’-AAGTGGCCACAAGTTCAGCGTGTC-3’，如SEQ ID NO.19所示；
EGFP-3R，5’-AAACGACACGCTGAACTTGTGGCC-3’，如SEQ ID NO.20所示；
NS-1F，5’-AAGTCGCTTCAAGGTGCACATGGA-3’，如SEQ ID NO.21所示；
NS-1R，5’-AAACTCCATGTGCACCTTGAAGCG-3’，如SEQ ID NO.22所示；
NS-2F，5’-AAGTGCGGAAGAACGCCTGCGGCT-3’，如SEQ ID NO.23所示
NS-2R，5’-AAACAGCCGCAGGCGTTCTTCCGC-3’，如SEQ ID NO.24所示。
4、将步骤3设计的成对的引物合成，然后稀释到10μM，每对引物各自混合(两条引物各自10μL)并梯度退火成包含粘性末端的双链DNA。梯度退火条件如下：95℃、10min然后室温冷却。
5、将步骤1构建的真核生物基因敲除基础载体pB-CRISPR用核酸内切酶AarI消化。酶切条件为20μL体系，包含1μg的载体pB-CRISPR，AarI酶1μL，用双蒸水补齐20μL，酶切条件为37℃酶切16小时。
6、将步骤5核酸内切酶AarI消化产物用琼脂糖凝胶电泳后切胶回收，然后和步骤4退火形成的包含粘性末端的DNA双链连接，DNA连接酶为T4 DNA连接酶，连接总体系为50μL，其中骨架和片段按照摩尔比1：10添加，总质量为2μg，T4 DNA连接酶缓冲液5μL，T4 DNA连接酶1μL，用双蒸水补齐50μL，16℃连接4小时，T4 DNA连接酶采购自NEB公司。将连接产物转化大肠杆菌后挑选单克隆。转化所用的感受态为Trans1-T1感受态细胞，购买自全式金公司，按照公司说明书来执行转化实验。
7、正确单克隆的挑选。首先是每个打靶载体各自挑选约24个单克隆，用含有氨苄青霉素的LB液体培养基在37℃、220rmp摇10小时，然后用菌液PCR的方式挑选阳性克隆，氨苄青霉素工作浓度为50μg/ml。最后将阳性单克隆执行sanger测序，最终挑选到正确的单克隆。
1)根据步骤1构建的真核生物基因敲除基础载体pB-CRISPR，设计用于检测载体的引物对：
正向引物K-SerP1-F1，5’-CGAGTCCTTTCCGATGTGT-3’，如SEQ ID NO.31所示；
反向引物K-hr3-R1，5’-CGTTTTGTATTTGTCATTGCC-3’，如SEQ ID NO.32所示。
2)PCR酶为普通rTaq酶，PCR反应条件为：94℃预变性4min；94℃变性30s，55℃退火30s，72℃延伸90s；35个循环；72℃延伸10min；12℃保存；若PCR扩增产物的大小约1100bp时，基本可以判断为阳性克隆。
3)sanger测序方法为双向测序，测序引物：
正向引物K-SerP1-F1，5’-CGAGTCCTTTCCGATGTGT-3’，如SEQ ID NO.31所示；
反向引物K-hr3-R1，5’-CGTTTTGTATTTGTCATTGCC-3’，如SEQ ID NO.32所示。
8、将步骤7挑选的正确克隆分别命名为：pB-CRISPR-EGFP-1，pB-CRISPR-EGFP-2，pB-CRISPR-EGFP-3，pB-CRISPR-NS-1，pB-CRISPR-NS-2。用超纯质粒抽提试剂盒分别抽提以上质粒和piggyBac transposase表达载体A3-helper(核苷酸序列如SEQ ID NO.30所示)。
9、将过表达绿色荧光蛋白的家蚕胚胎细胞系(The Bombyx mori embryonic cellline BmE)BmE-Mi-puro-EGFP接种到一个6孔板中。
10、将步骤8描述的5个敲除载体piggyBac transposon表达载体A3-helper(核苷酸序列如SEQ ID NO.30所示)按照摩尔比1：1转染家蚕BmE-Mi-puro-EGFP，作为实验组，另一个孔的细胞用完全培养基在正常条件下培养，作为实验组。将转染后的细胞用Zeocin筛选2个月(对照组一直用不含Zeocin的完全培养基在正常条件下培养)，即可得到家蚕基因敲除细胞系。
1)细胞转染方法包括脂质体转染法、电穿孔转染法等。
2)细胞完全培养基为包含体积浓度10％胎牛血清(fetal bovine serum,FBS)和青霉素-链霉素(Penicillin-Streptomycin，20万单位/升，赛默飞世尔公司)的Grace昆虫培养基(Grace's Insect Medium)。培养条件为27℃。
3)Zeocin工作浓度为200μg/ml。
11、pB-CRISPR系统敲除家蚕BmE细胞基因效率的检测
1)流式细胞仪检测法
①将以上步骤描述的6组细胞分别收集105个，用磷酸缓冲盐溶液(phosphatebuffer saline，PBS)清洗后用1ml PBS重悬。
②用流式细胞仪分别检测6组细胞发绿色荧光蛋白的效率，激发光波长为488nm，接受光波长为510-550nm。
③流式细胞仪检测结果显示，pB-CRISPR系统敲除家蚕BmE细胞基因效率极高，如图2所示。
2)sanger测序检测法
①根据EGFP核苷酸序列，设计引物对扩增打靶位点区域。引物序列如下：
正向引物EGFP-F，5’-ATGGTGAGCAAGGGCG-3’，如SEQ ID NO.33所示；
反向引物EGFP-R，5’-TTACTTGTACAGCTCGTCCATG-3’，如SEQ ID NO.34所示。
②将三组转染EGFP敲除载体的细胞收集，抽提基因组DNA，用引物对EGFP-F/EGFP-R分别做PCR扩增，PCR扩增产物连接T载体后挑选单克隆执行sanger测序。
③sanger测序结果显示，pB-CRISPR系统敲除家蚕BmE细胞基因效率极高，全部单克隆都实现了基因敲除，突变形式主要是小片段缺失，少数是小片段插入，如图3所示。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
序列表
<110> 西南大学
<120> 一种真核生物CRISPR/Cas9敲除系统、基础载体、载体及细胞系
<130> 2020
<160> 34
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial
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ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc 60
atgtgtttta tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt 120
tattatattt acacttacat actaataata aattcaacaa acaatttatt tatgtttatt 180
tatttattaa aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aacttttaaa 240
cattctctct tttacaaaaa taaacttatt ttgtacttta aaaacagtca tgttgtatta 300
taaaataagt aattagctta acttatacat aatagaaaca aattatactt attagtcagt 360
cagaaacaac tttggcacat atcaatatta tgctctcgac aaataacttt tttgcatttt 420
ttgcacgatg catttgcctt tcgccttatt ttagaggggc agtaagtaca gtaagtacgt 480
tttttcatta ctggctcttc agtactgtca tctgatgtac caggcacttc atttggcaaa 540
atattagaga tattatcgcg caaatatctc ttcaaagtag gagcttctaa acgcttacgc 600
ataaacgatg acgtcaggct catgtaaagg tttctcataa attttttgcg actttgaacc 660
ttttctccct tgctactgac attatggctg tatataataa aagaatttat gcaggcaatg 720
tttatcattc cgtacaataa tgccataggc cacctattcg tcctcctact gcaggtcatc 780
acagaacaca tttggtctag cgtgtccact ccgcctttag tttgattata atacataacc 840
atttgcggtt taccggtact ttcgttgata gaagcatcct catcacaaga tgataataag 900
tataccatct tagctggctt cggtttatat gagacgagag taaggggtcc gtcaaaacaa 960
aacatcgatg ttcccactgg cctggagcga ctgtttttca gtacttccgg tatctcgcgt 1020
ttgtt 1025
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gatctgacaa tgttcagtgc agagactcgg ctacgcctcg tggactttga agttgaccaa 60
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aatgaagaac ctggttgttc aataacattt tgttcgtcta atatttcact accgcttgac 180
gttggctgca cttcatgtac ctcatctata aacgcttctt ctgtatcgct ctggacgtca 240
tcttcactta cgtgatctga tatttcactg tcagaatcct caccaacaag ctcgtcatcg 300
ctttgcagaa gagcagagag gatatgctca tcgtctaaag aactacccat tttattatat 360
attagtcacg atatctataa caagaaaata tatatataat aagttatcac gtaagtagaa 420
catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat 480
catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt 540
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ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg 660
cagactatct ttctaggg 678
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catgatgata aacaatgtat ggtgctaatg ttgcttcaac aacaattctg ttgaactgtg 60
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gcactgcaaa aaaacacgct tttgcacgcg ggcccataca tagtacaaac tctacgtttc 180
gtagactatt ttacataaat agtctacacc gttgtatacg ctccaaatac actaccacac 240
attgaacctt tttgcagtgc aaaaaagtac gtgtcggcag tcacgtaggc cggccttatc 300
gggtcgcgtc ctgtcacgta cgaatcacat tatcggaccg gacgagtgtt gtcttatcgt 360
gacaggacgc cagcttcctg tgttgctaac cgcagccgga cgcaactcct tatcggaaca 420
ggacgcgcct ccatatcagc cgcgcgttat ctcatgcgcg tgaccggaca cgaggcgccc 480
gtcccgctta tcgcgcctat aaatacagcc cgcaacgatc tggtaaacac agttgaacag 540
catctgttcg aaatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc 600
gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac 660
gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag 720
gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc 780
gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag 840
atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg 900
cacttcgtgg ccgaggagca ggactaaagc tttacaacta aacacgactt ggagtattcc 960
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accaaagacc cctttaagat gggcctaatg ggcttaagtc gagtcctttc cgatgtgtta 1200
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cagcgtcgtg aaaagaggca atgacaaata caaaacgacg tatgagcaga cccgtcgcca 60
agacgggtct acctctaaga tgatgtcatt tgttttttaa aactaactcg ctttacgagt 120
agaattctac gtgtaaaaca taatcaagag atgatgtcat ttgtttttca aaaccaaact 180
cgctttacga gtagaattct acgtgtaaaa cacaatcaaa agatgatgtc attcgttttt 240
caaaaccgaa tttaagaaat gatgtcattt gtttttcaaa accaaactcg ctttacgagc 300
agaattctac gtgtaaaaca caatcaagag atgatgtcat ttgtttttca aaactgaatg 360
atgtcatttg tttttcaaaa ctaaacttgc tttgcgagta gaattctacg tgtaaaacac 420
agtcaagaga tgatgtcatt tgtttttcaa aactgaaccg gctttacgag tagaattcta 480
cttgtaaaac ataatcaaga gatgatgtca tttgtttttc aaaactgaac tggctttacg 540
agtagaattc tacgtgtaaa acataatcaa gagatgatgt catcattaaa ctgatgtcat 600
tttatacacg attgttaaca tgtttaataa tgactaattt gtttttccaa attaaactcg 660
ctttacgagt agaattctac ttgtaacgca cgattaagta tgaatcataa gctgatgtca 720
tttgttttcg acataaaatg tttatacaat ggaatcttct tgtaaattat ccaaataata 780
taatttatcc gattctacgt tacatttaaa ttcgttgtta tcgtacaatt cttcaggaca 840
cgccatgtat tggtcatttt tagcgtgcaa ccaacgattg tatttgacgc cgtcgttgga 900
ttgcgtgttc aggttggcgt acacgtgact gggcacggct tctttttcca tgggacgtcg 960
accgagaaat ttctctggcc gttattcgtt attctctctt ttctttttgg gtctctccct 1020
ctctgcacta atgctctctc actctgtcac acagtaaacg gcatactgct ctcgttggtt 1080
cgagagagcg cgcctcgaat gttcgcgaaa agagcgccgg agtataaata gaggcgcttc 1140
gtctacggag cgacaattca attcaaacaa gcaaagtgaa cacgtcgcta agcgaaagct 1200
aagcaaataa acaagcgcag ctgaacaagc taaacaatct gcagtaaagt gcaagttaaa 1260
gtgaatcaat taaaagtaac cagcaaccaa gtaaatcaac tgcaactact gaaatctgcc 1320
aagaagtaat tattgaatac aagaagagaa ctctggggga tctctagtcc agtgtggtgg 1380
aattcgccat ggccccaaag aaaaagagaa aggttgatta caaagaccac gacggagact 1440
acaaagacca cgacattgat tataaagatg atgatgataa aggaacgatg gacaaaaagt 1500
atagcatcgg tctggatatt ggaactaact ccgtcggctg ggctgtaatc accgacgaat 1560
acaaggtccc gtcaaaaaag ttcaaggtat tgggtaacac agatcgtcac tctatcaaaa 1620
agaatctcat tggagctctg ttgttcgaca gcggcgaaac agctgaggcc actagactga 1680
agcgcaccgc cagacgccgt tacacgagga gaaagaacag aatctgctac ttgcaagaaa 1740
tattctcaaa cgagatggcc aaagtggacg attcgttctt tcataggtta gaagagagtt 1800
tccttgttga agaggataaa aagcacgaaa gacatccgat atttggaaac atcgtggacg 1860
aagttgctta tcacgagaag taccccacga tctatcatct gcgtaaaaag ttggtggact 1920
cgacagataa ggccgacctc aggttaatat accttgcact ggcgcacatg atcaaattca 1980
gaggccattt tctgattgaa ggtgacctga accctgacaa tagtgatgtg gacaaactct 2040
tcattcaatt agttcagacc tacaatcaac tgtttgaaga gaaccctatc aacgcttcag 2100
gagttgacgc taaggccatc cttagtgcga gactgagcaa atcccgccgt ctcgaaaact 2160
taatcgcaca gttgcctgga gagaaaaaga acggtttgtt cggaaatctc attgcgttgt 2220
cactcggact cacgccaaac ttcaagtcta acttcgattt ggcagaagac gcgaaactgc 2280
aactgagcaa agacacatat gacgatgacc tcgataacct cttagctcag atcggcgatc 2340
aatacgccga cttgttcctc gctgccaaaa atctgtcgga cgctatactt ctgagtgata 2400
tcttgcgcgt caacacagaa attactaagg ctcctctgtc ggccagtatg ataaaacgct 2460
atgacgaaca ccatcaggat ttgacattgc tcaaagccct cgtgcgtcaa cagctcccag 2520
aaaagtacaa ggagattttc tttgatcagt ccaagaatgg ctacgcaggt tatatagacg 2580
gtggagcgtc gcaagaagag ttctacaagt tcatcaagcc aatattagaa aagatggacg 2640
gcacggaaga gttacttgtt aagctgaatc gtgaggacct gttgcgtaaa cagaggacat 2700
tcgataacgg atcaattccg caccaaatac atcttggcga actgcacgct atcctcagga 2760
gacaagagga cttctacccc tttttaaagg ataaccgtga aaagatcgag aaaatcctga 2820
ctttcaggat tccttactat gtcggcccac tggctcgtgg taatagcagg tttgcctgga 2880
tgaccaggaa gtccgaagag acaattactc cgtggaactt cgaagaggtg gttgataaag 2940
gagcatcagc gcagtctttc atagaacgca tgacaaattt tgacaagaac ttaccgaatg 3000
agaaggtcct tcccaaacac tcactcctct acgaatactt cacagtatac aacgagctca 3060
ctaaagtcaa gtacgtaacc gagggtatgc gcaaacccgc tttcctgtct ggagagcaga 3120
aaaaggccat cgtggacctt ctgttcaaga caaaccgtaa ggtcactgta aagcaactca 3180
aggaagacta cttcaaaaag atagagtgtt tcgattcagt ggaaatctct ggcgttgagg 3240
acagatttaa cgcttccttg ggtacttacc acgatttgct caagatcatt aaagataagg 3300
acttcctcga caacgaagag aacgaagata tcttagagga catagttctc acccttacgc 3360
tgtttgaaga tagagagatg attgaagagc gcctgaagac ttatgctcat ttgttcgatg 3420
acaaagtcat gaagcaactg aaacgccgta ggtacaccgg ctggggtaga ttatcgcgca 3480
aacttattaa tggtataagg gacaagcagt cgggaaaaac gatattggac tttctcaaga 3540
gtgatggttt cgccaacaga aattttatgc aactcataca cgatgacagc ttaacattca 3600
aggaagatat ccaaaaagca caggtgtcgg gacagggcga cagtttgcac gaacatattg 3660
ctaacctcgc cggctccccg gcgataaaaa agggtatcct tcagactgtg aaagtcgtag 3720
atgaactggt gaaggttatg ggtcgtcata aacccgagaa catagttatc gaaatggcta 3780
gggagaatca aacaactcag aagggacaga aaaactcaag agaacgcatg aagcgcattg 3840
aagagggtat caaagagctt ggcagtcaaa tcctgaagga acaccctgtc gagaacacgc 3900
aacttcagaa cgaaaaattg tacctctact atctgcagaa tggtagagat atgtacgtag 3960
accaagaatt ggatattaac cgcctctcag attacgacgt ggatcatata gttccgcagt 4020
cattcttgaa ggatgactct atcgacaaca aagtcctcac aagatcagac aagaaccgcg 4080
gaaaatcaga taatgtaccc tctgaagagg tggttaaaaa gatgaaaaac tactggagac 4140
agttacttaa cgctaagttg atcacgcaaa gaaagttcga taacctcaca aaggctgaac 4200
gcggcggttt aagcgagctt gacaaggccg gtttcataaa acgtcagtta gtcgaaacca 4260
ggcaaattac gaaacacgta gcccaaatat tggattcccg catgaacact aaatacgatg 4320
aaaatgacaa gctcatccgt gaggtcaaag taattaccct gaaaagcaag ttggtgtccg 4380
acttcagaaa ggatttccag ttctacaaag ttcgcgaaat caacaactac caccatgcac 4440
atgacgctta cctgaacgca gtcgtaggca ctgcgttaat taaaaagtac cctaaactgg 4500
aatctgagtt cgtgtacggt gactataaag tgtacgatgt tagaaagatg atcgctaaaa 4560
gcgaacagga gattggaaag gctaccgcca agtatttctt ttactccaac atcatgaatt 4620
tctttaagac cgaaatcacg ttagcaaatg gcgagatacg taaaaggcca cttatcgaaa 4680
caaacggaga aactggcgag atagtgtggg acaagggtag agattttgcc actgtccgca 4740
aagtactgtc gatgccgcaa gtgaatatcg ttaaaaagac cgaagttcaa acgggaggct 4800
tcagcaaaga gtccatcctg cccaagcgta acagtgataa attgatagct aggaaaaagg 4860
actgggatcc taaaaagtat ggtggattcg acagcccaac tgtcgcatac tccgtattgg 4920
tggttgcgaa agtcgaaaaa ggaaagagca aaaagctcaa gtccgtaaaa gagctgttgg 4980
gcattaccat aatggaaaga tcatctttcg agaagaatcc tatcgatttt ctggaagcca 5040
agggatataa agaggtcaaa aaggacctca taatcaagtt accaaaatac agtctgttcg 5100
aattggagaa cggcagaaaa cgcatgcttg catcagcggg tgaactgcaa aagggaaatg 5160
agttagcact tccttctaaa tacgtcaact tcctgtattt ggcgtcacac tacgaaaaac 5220
tgaagggctc tccagaagat aacgagcaaa agcagttatt tgtggaacag cacaaacatt 5280
accttgacga aattatagag caaatctcgg agttcagtaa gagagtgatt ttggctgacg 5340
ccaatcttga taaagttctg tctgcttaca acaagcaccg tgataaaccg attagggaac 5400
aggccgagaa catcatacat ctcttcacac tcactaacct tggtgcaccc gcagcgttca 5460
aatattttga caccacgata gatcgtaaga ggtacaccag cacgaaagaa gttttggacg 5520
cgacactcat ccatcaatca atcacgggcc tgtacgagac cagaatcgac ctgtcccagc 5580
tcggtggcga ctagcggccg cgactctaga tcataatcag ccatgcggcc gcgactctag 5640
accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg 5700
aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac 5760
aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 5820
tgtggtttgt ccaaactcat caatgtatct taaagcttat cgatacgcgt a 5871
<210> 5
<211> 1206
<212> DNA
<213> Artificial
<400> 5
agctgtccaa ggaatgcgta gcagctttct ccagcaatac atttcaaacg cctcaatctt 60
tttgcgttcc tttttcctga gacaccaagt ctcctaaagt catgatgatt gacctaaaag 120
aatcaataca gtttaataaa tttataagta ttaggttatg tagtacacat tgttgtaaat 180
cactgaattg ttttagatga ttttaacaat tagtacttat taatattaaa taagtacata 240
ccttgagaat ttaaaaatcg tcaactataa gccatacgaa tttaagcttg gtacttggct 300
tatagataag gacagaataa gaattgttaa cgtgtaagac aaggtcagat agtcatagtg 360
attttgtcaa agtaataaca gatggcgctg tacaaaccat aactgttttc atttgttttt 420
atggatttta ttacaaattc taaaggtttt attgttatta tttaatttcg ttttaattat 480
attatatatc tttaatagaa tatgttaaga gtttttgctc tttttgaata atctttgtaa 540
agtcgagtgt tgttgtaaat cacgctttca atagtttagt ttttttaggt atatatacaa 600
aatatcgtgc tctacaagtg atggcaggtg gcattggtaa ctgtcagacc aagtttactc 660
atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 720
cctttttgat aatctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 780
aggggttccg cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca 840
gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaataatt cagaagaact 900
cgtcaagaca cctgccagtg ttttagagct agaaatagca agttaaaata aggctagtcc 960
gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagaaca attttataac 1020
atacatcgga ttttttaatt agtttaaaaa tatatttgat tcgttatcaa atgttaacat 1080
aaatattaat actagataaa cagtttatgt ataaaaaatt gtttattttt ttaaataaaa 1140
aaacaaatat tatcctattt ttggtcaagc ttttgctttt ggctaaatcg ataaagatct 1200
ttcatt 1206
<210> 6
<211> 13408
<212> DNA
<213> Artificial
<400> 6
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420
tgcgtcgaga taagagggtt aaaaaatata ttttacgcac catatacgca tcgggttgat 480
atcgttaata tggatcaatt tgaacagttg attaacgtgt ctctgctcaa gtctttgatc 540
aaaacgcaaa tcgacgaaaa tgtgtcggac aatatcaagt cgatgagcga aaaactaaaa 600
aggctagaat acgacaatct cacagacagc gttgagatat acggtattca cgacagcagg 660
ctgaataata aaaaaattag aaactattat ttaaccctag aaagataatc atattgtgac 720
gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga 780
ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt acatactaat 840
aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc 900
aaaatttctt ctataaagta acaaaacttt taaacattct ctcttttaca aaaataaact 960
tattttgtac tttaaaaaca gtcatgttgt attataaaat aagtaattag cttaacttat 1020
acataataga aacaaattat acttattagt cagtcagaaa caactttggc acatatcaat 1080
attatgctct cgacaaataa cttttttgca ttttttgcac gatgcatttg cctttcgcct 1140
tattttagag gggcagtaag tacagtaagt acgttttttc attactggct cttcagtact 1200
gtcatctgat gtaccaggca cttcatttgg caaaatatta gagatattat cgcgcaaata 1260
tctcttcaaa gtaggagctt ctaaacgctt acgcataaac gatgacgtca ggctcatgta 1320
aaggtttctc ataaattttt tgcgactttg aaccttttct cccttgctac tgacattatg 1380
gctgtatata ataaaagaat ttatgcaggc aatgtttatc attccgtaca ataatgccat 1440
aggccaccta ttcgtcctcc tactgcaggt catcacagaa cacatttggt ctagcgtgtc 1500
cactccgcct ttagtttgat tataatacat aaccatttgc ggtttaccgg tactttcgtt 1560
gatagaagca tcctcatcac aagatgataa taagtatacc atcttagctg gcttcggttt 1620
atatgagacg agagtaaggg gtccgtcaaa acaaaacatc gatgttccca ctggcctgga 1680
gcgactgttt ttcagtactt ccggtatctc gcgtttgttc ctgcaggatc atgatgataa 1740
acaatgtatg gtgctaatgt tgcttcaaca acaattctgt tgaactgtgt tttcatgttt 1800
gccaacaagc acctttatac tcggtggcct ccccaccacc aacttttttg cactgcaaaa 1860
aaacacgctt ttgcacgcgg gcccatacat agtacaaact ctacgtttcg tagactattt 1920
tacataaata gtctacaccg ttgtatacgc tccaaataca ctaccacaca ttgaaccttt 1980
ttgcagtgca aaaaagtacg tgtcggcagt cacgtaggcc ggccttatcg ggtcgcgtcc 2040
tgtcacgtac gaatcacatt atcggaccgg acgagtgttg tcttatcgtg acaggacgcc 2100
agcttcctgt gttgctaacc gcagccggac gcaactcctt atcggaacag gacgcgcctc 2160
catatcagcc gcgcgttatc tcatgcgcgt gaccggacac gaggcgcccg tcccgcttat 2220
cgcgcctata aatacagccc gcaacgatct ggtaaacaca gttgaacagc atctgttcga 2280
aatggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt 2340
cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg 2400
tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga 2460
caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga 2520
ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca 2580
gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc 2640
cgaggagcag gactaaagct ttacaactaa acacgacttg gagtattcct tgtagtgttt 2700
aagattttaa atcttactta atgacttcga acgattttaa cgataacttt ctctttgttt 2760
aactttaatc agcatacata aaaagccccg gttttgtatc gggaagaaaa aaaatgtaat 2820
tgtgttgcct agataataaa cgtattatca aagtgtgtgg ttttccttta ccaaagaccc 2880
ctttaagatg ggcctaatgg gcttaagtcg agtcctttcc gatgtgttaa atacacattt 2940
attacactga tgcgtcgaat gtacactttt aataggatag ctccactaaa aattatttta 3000
tttatttaat ttgttgcacc aaaactgata cattgacgaa acgcgtatgc tagcaatgaa 3060
agatctttat cgatttagcc aaaagcaaaa gcttgaccaa aaataggata atatttgttt 3120
ttttatttaa aaaaataaac aattttttat acataaactg tttatctagt attaatattt 3180
atgttaacat ttgataacga atcaaatata tttttaaact aattaaaaaa tccgatgtat 3240
gttataaaat tgttctagaa aaaaagcacc gactcggtgc cactttttca agttgataac 3300
ggactagcct tattttaact tgctatttct agctctaaaa cactggcagg tgtcttgacg 3360
agttcttctg aattattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc 3420
tgtgcggtat ttcacaccgc atcaggtggc acttttcggg gaaatgtgcg cggaacccct 3480
atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagatt atcaaaaagg 3540
atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 3600
gagtaaactt ggtctgacag ttaccaatgc cacctgccat cacttgtaga gcacgatatt 3660
ttgtatatat acctaaaaaa actaaactat tgaaagcgtg atttacaaca acactcgact 3720
ttacaaagat tattcaaaaa gagcaaaaac tcttaacata ttctattaaa gatatataat 3780
ataattaaaa cgaaattaaa taataacaat aaaaccttta gaatttgtaa taaaatccat 3840
aaaaacaaat gaaaacagtt atggtttgta cagcgccatc tgttattact ttgacaaaat 3900
cactatgact atctgacctt gtcttacacg ttaacaattc ttattctgtc cttatctata 3960
agccaagtac caagcttaaa ttcgtatggc ttatagttga cgatttttaa attctcaagg 4020
tatgtactta tttaatatta ataagtacta attgttaaaa tcatctaaaa caattcagtg 4080
atttacaaca atgtgtacta cataacctaa tacttataaa tttattaaac tgtattgatt 4140
cttttaggtc aatcatcatg actttaggag acttggtgtc tcaggaaaaa ggaacgcaaa 4200
aagattgagg cgtttgaaat gtattgctgg agaaagctgc tacgcattcc ttggacagct 4260
tggcgcgccc agcgtcgtga aaagaggcaa tgacaaatac aaaacgacgt atgagcagac 4320
ccgtcgccaa gacgggtcta cctctaagat gatgtcattt gttttttaaa actaactcgc 4380
tttacgagta gaattctacg tgtaaaacat aatcaagaga tgatgtcatt tgtttttcaa 4440
aaccaaactc gctttacgag tagaattcta cgtgtaaaac acaatcaaaa gatgatgtca 4500
ttcgtttttc aaaaccgaat ttaagaaatg atgtcatttg tttttcaaaa ccaaactcgc 4560
tttacgagca gaattctacg tgtaaaacac aatcaagaga tgatgtcatt tgtttttcaa 4620
aactgaatga tgtcatttgt ttttcaaaac taaacttgct ttgcgagtag aattctacgt 4680
gtaaaacaca gtcaagagat gatgtcattt gtttttcaaa actgaaccgg ctttacgagt 4740
agaattctac ttgtaaaaca taatcaagag atgatgtcat ttgtttttca aaactgaact 4800
ggctttacga gtagaattct acgtgtaaaa cataatcaag agatgatgtc atcattaaac 4860
tgatgtcatt ttatacacga ttgttaacat gtttaataat gactaatttg tttttccaaa 4920
ttaaactcgc tttacgagta gaattctact tgtaacgcac gattaagtat gaatcataag 4980
ctgatgtcat ttgttttcga cataaaatgt ttatacaatg gaatcttctt gtaaattatc 5040
caaataatat aatttatccg attctacgtt acatttaaat tcgttgttat cgtacaattc 5100
ttcaggacac gccatgtatt ggtcattttt agcgtgcaac caacgattgt atttgacgcc 5160
gtcgttggat tgcgtgttca ggttggcgta cacgtgactg ggcacggctt ctttttccat 5220
gggacgtcga ccgagaaatt tctctggccg ttattcgtta ttctctcttt tctttttggg 5280
tctctccctc tctgcactaa tgctctctca ctctgtcaca cagtaaacgg catactgctc 5340
tcgttggttc gagagagcgc gcctcgaatg ttcgcgaaaa gagcgccgga gtataaatag 5400
aggcgcttcg tctacggagc gacaattcaa ttcaaacaag caaagtgaac acgtcgctaa 5460
gcgaaagcta agcaaataaa caagcgcagc tgaacaagct aaacaatctg cagtaaagtg 5520
caagttaaag tgaatcaatt aaaagtaacc agcaaccaag taaatcaact gcaactactg 5580
aaatctgcca agaagtaatt attgaataca agaagagaac tctgggggat ctctagtcca 5640
gtgtggtgga attcgccatg gccccaaaga aaaagagaaa ggttgattac aaagaccacg 5700
acggagacta caaagaccac gacattgatt ataaagatga tgatgataaa ggaacgatgg 5760
acaaaaagta tagcatcggt ctggatattg gaactaactc cgtcggctgg gctgtaatca 5820
ccgacgaata caaggtcccg tcaaaaaagt tcaaggtatt gggtaacaca gatcgtcact 5880
ctatcaaaaa gaatctcatt ggagctctgt tgttcgacag cggcgaaaca gctgaggcca 5940
ctagactgaa gcgcaccgcc agacgccgtt acacgaggag aaagaacaga atctgctact 6000
tgcaagaaat attctcaaac gagatggcca aagtggacga ttcgttcttt cataggttag 6060
aagagagttt ccttgttgaa gaggataaaa agcacgaaag acatccgata tttggaaaca 6120
tcgtggacga agttgcttat cacgagaagt accccacgat ctatcatctg cgtaaaaagt 6180
tggtggactc gacagataag gccgacctca ggttaatata ccttgcactg gcgcacatga 6240
tcaaattcag aggccatttt ctgattgaag gtgacctgaa ccctgacaat agtgatgtgg 6300
acaaactctt cattcaatta gttcagacct acaatcaact gtttgaagag aaccctatca 6360
acgcttcagg agttgacgct aaggccatcc ttagtgcgag actgagcaaa tcccgccgtc 6420
tcgaaaactt aatcgcacag ttgcctggag agaaaaagaa cggtttgttc ggaaatctca 6480
ttgcgttgtc actcggactc acgccaaact tcaagtctaa cttcgatttg gcagaagacg 6540
cgaaactgca actgagcaaa gacacatatg acgatgacct cgataacctc ttagctcaga 6600
tcggcgatca atacgccgac ttgttcctcg ctgccaaaaa tctgtcggac gctatacttc 6660
tgagtgatat cttgcgcgtc aacacagaaa ttactaaggc tcctctgtcg gccagtatga 6720
taaaacgcta tgacgaacac catcaggatt tgacattgct caaagccctc gtgcgtcaac 6780
agctcccaga aaagtacaag gagattttct ttgatcagtc caagaatggc tacgcaggtt 6840
atatagacgg tggagcgtcg caagaagagt tctacaagtt catcaagcca atattagaaa 6900
agatggacgg cacggaagag ttacttgtta agctgaatcg tgaggacctg ttgcgtaaac 6960
agaggacatt cgataacgga tcaattccgc accaaataca tcttggcgaa ctgcacgcta 7020
tcctcaggag acaagaggac ttctacccct ttttaaagga taaccgtgaa aagatcgaga 7080
aaatcctgac tttcaggatt ccttactatg tcggcccact ggctcgtggt aatagcaggt 7140
ttgcctggat gaccaggaag tccgaagaga caattactcc gtggaacttc gaagaggtgg 7200
ttgataaagg agcatcagcg cagtctttca tagaacgcat gacaaatttt gacaagaact 7260
taccgaatga gaaggtcctt cccaaacact cactcctcta cgaatacttc acagtataca 7320
acgagctcac taaagtcaag tacgtaaccg agggtatgcg caaacccgct ttcctgtctg 7380
gagagcagaa aaaggccatc gtggaccttc tgttcaagac aaaccgtaag gtcactgtaa 7440
agcaactcaa ggaagactac ttcaaaaaga tagagtgttt cgattcagtg gaaatctctg 7500
gcgttgagga cagatttaac gcttccttgg gtacttacca cgatttgctc aagatcatta 7560
aagataagga cttcctcgac aacgaagaga acgaagatat cttagaggac atagttctca 7620
cccttacgct gtttgaagat agagagatga ttgaagagcg cctgaagact tatgctcatt 7680
tgttcgatga caaagtcatg aagcaactga aacgccgtag gtacaccggc tggggtagat 7740
tatcgcgcaa acttattaat ggtataaggg acaagcagtc gggaaaaacg atattggact 7800
ttctcaagag tgatggtttc gccaacagaa attttatgca actcatacac gatgacagct 7860
taacattcaa ggaagatatc caaaaagcac aggtgtcggg acagggcgac agtttgcacg 7920
aacatattgc taacctcgcc ggctccccgg cgataaaaaa gggtatcctt cagactgtga 7980
aagtcgtaga tgaactggtg aaggttatgg gtcgtcataa acccgagaac atagttatcg 8040
aaatggctag ggagaatcaa acaactcaga agggacagaa aaactcaaga gaacgcatga 8100
agcgcattga agagggtatc aaagagcttg gcagtcaaat cctgaaggaa caccctgtcg 8160
agaacacgca acttcagaac gaaaaattgt acctctacta tctgcagaat ggtagagata 8220
tgtacgtaga ccaagaattg gatattaacc gcctctcaga ttacgacgtg gatcatatag 8280
ttccgcagtc attcttgaag gatgactcta tcgacaacaa agtcctcaca agatcagaca 8340
agaaccgcgg aaaatcagat aatgtaccct ctgaagaggt ggttaaaaag atgaaaaact 8400
actggagaca gttacttaac gctaagttga tcacgcaaag aaagttcgat aacctcacaa 8460
aggctgaacg cggcggttta agcgagcttg acaaggccgg tttcataaaa cgtcagttag 8520
tcgaaaccag gcaaattacg aaacacgtag cccaaatatt ggattcccgc atgaacacta 8580
aatacgatga aaatgacaag ctcatccgtg aggtcaaagt aattaccctg aaaagcaagt 8640
tggtgtccga cttcagaaag gatttccagt tctacaaagt tcgcgaaatc aacaactacc 8700
accatgcaca tgacgcttac ctgaacgcag tcgtaggcac tgcgttaatt aaaaagtacc 8760
ctaaactgga atctgagttc gtgtacggtg actataaagt gtacgatgtt agaaagatga 8820
tcgctaaaag cgaacaggag attggaaagg ctaccgccaa gtatttcttt tactccaaca 8880
tcatgaattt ctttaagacc gaaatcacgt tagcaaatgg cgagatacgt aaaaggccac 8940
ttatcgaaac aaacggagaa actggcgaga tagtgtggga caagggtaga gattttgcca 9000
ctgtccgcaa agtactgtcg atgccgcaag tgaatatcgt taaaaagacc gaagttcaaa 9060
cgggaggctt cagcaaagag tccatcctgc ccaagcgtaa cagtgataaa ttgatagcta 9120
ggaaaaagga ctgggatcct aaaaagtatg gtggattcga cagcccaact gtcgcatact 9180
ccgtattggt ggttgcgaaa gtcgaaaaag gaaagagcaa aaagctcaag tccgtaaaag 9240
agctgttggg cattaccata atggaaagat catctttcga gaagaatcct atcgattttc 9300
tggaagccaa gggatataaa gaggtcaaaa aggacctcat aatcaagtta ccaaaataca 9360
gtctgttcga attggagaac ggcagaaaac gcatgcttgc atcagcgggt gaactgcaaa 9420
agggaaatga gttagcactt ccttctaaat acgtcaactt cctgtatttg gcgtcacact 9480
acgaaaaact gaagggctct ccagaagata acgagcaaaa gcagttattt gtggaacagc 9540
acaaacatta ccttgacgaa attatagagc aaatctcgga gttcagtaag agagtgattt 9600
tggctgacgc caatcttgat aaagttctgt ctgcttacaa caagcaccgt gataaaccga 9660
ttagggaaca ggccgagaac atcatacatc tcttcacact cactaacctt ggtgcacccg 9720
cagcgttcaa atattttgac accacgatag atcgtaagag gtacaccagc acgaaagaag 9780
ttttggacgc gacactcatc catcaatcaa tcacgggcct gtacgagacc agaatcgacc 9840
tgtcccagct cggtggcgac tagcggccgc gactctagat cataatcagc catgcggccg 9900
cgactctaga ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 9960
ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 10020
aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 10080
cattctagtt gtggtttgtc caaactcatc aatgtatctt aaagcttatc gatacgcgta 10140
cctaggccgg ccgatctcgg atctgacaat gttcagtgca gagactcggc tacgcctcgt 10200
ggactttgaa gttgaccaac aatgtttatt cttacctcta atagtcctct gtggcaaggt 10260
caagattctg ttagaagcca atgaagaacc tggttgttca ataacatttt gttcgtctaa 10320
tatttcacta ccgcttgacg ttggctgcac ttcatgtacc tcatctataa acgcttcttc 10380
tgtatcgctc tggacgtcat cttcacttac gtgatctgat atttcactgt cagaatcctc 10440
accaacaagc tcgtcatcgc tttgcagaag agcagagagg atatgctcat cgtctaaaga 10500
actacccatt ttattatata ttagtcacga tatctataac aagaaaatat atatataata 10560
agttatcacg taagtagaac atgaaataac aatataatta tcgtatgagt taaatcttaa 10620
aagtcacgta aaagataatc atgcgtcatt ttgactcacg cggtcgttat agttcaaaat 10680
cagtgacact taccgcattg acaagcacgc ctcacgggag ctccaagcgg cgactgagat 10740
gtcctaaatg cacagcgacg gattcgcgct atttagaaag agagagcaat atttcaagaa 10800
tgcatgcgtc aattttacgc agactatctt tctagggtta aaaaagattt gcgctttact 10860
cgacctaaac tttaaacacg tcatagaatc ttcgtttgac aaaaaccaca ttgtggccaa 10920
gctgtgtgac gcgacgcgcg ctaaagaatg gcaaaccaag tcgcgcgagc gtcgactcta 10980
gaggatcccc gggtaccgag ctcgaattcg taatcatggt catagctgtt tcctgtgtga 11040
aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 11100
tggggtgcct aatgagtgag ctaactcaca tcggatgccg ggaccgacga gtgcagaggc 11160
gtgcaagcga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 11220
ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 11280
tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 11340
ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 11400
gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 11460
gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 11520
ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 11580
ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 11640
cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 11700
ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 11760
tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 11820
gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 11880
tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 11940
gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 12000
tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 12060
ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 12120
agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 12180
gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 12240
attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 12300
agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 12360
atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 12420
cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 12480
ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 12540
agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 12600
tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 12660
gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 12720
caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 12780
ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 12840
gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 12900
tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 12960
tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 13020
cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 13080
cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 13140
gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 13200
atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 13260
agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 13320
ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 13380
aataggcgta tcacgaggcc ctttcgtc 13408
<210> 7
<211> 1383
<212> DNA
<213> Artificial
<400> 7
atcgatgttc ccactggcct ggagcgactg tttttcagta cttccggtat ctcgcgtttg 60
ttcctgcagg atcatgatga taaacaatgt atggtgctaa tgttgcttca acaacaattc 120
tgttgaactg tgttttcatg tttgccaaca agcaccttta tactcggtgg cctccccacc 180
accaactttt ttgcactgca aaaaaacacg cttttgcacg cgggcccata catagtacaa 240
actctacgtt tcgtagacta ttttacataa atagtctaca ccgttgtata cgctccaaat 300
acactaccac acattgaacc tttttgcagt gcaaaaaagt acgtgtcggc agtcacgtag 360
gccggcctta tcgggtcgcg tcctgtcacg tacgaatcac attatcggac cggacgagtg 420
ttgtcttatc gtgacaggac gccagcttcc tgtgttgcta accgcagccg gacgcaactc 480
cttatcggaa caggacgcgc ctccatatca gccgcgcgtt atctcatgcg cgtgaccgga 540
cacgaggcgc ccgtcccgct tatcgcgcct ataaatacag cccgcaacga tctggtaaac 600
acagttgaac agcatctgtt cgaaatggcc aagttgacca gtgccgttcc ggtgctcacc 660
gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac 720
ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg 780
gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac 840
gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg 900
gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc 960
ggcaactgcg tgcacttcgt ggccgaggag caggactaaa gctttacaac taaacacgac 1020
ttggagtatt ccttgtagtg tttaagattt taaatcttac ttaatgactt cgaacgattt 1080
taacgataac tttctctttg tttaacttta atcagcatac ataaaaagcc ccggttttgt 1140
atcgggaaga aaaaaaatgt aattgtgttg cctagataat aaacgtatta tcaaagtgtg 1200
tggttttcct ttaccaaaga cccctttaag atgggcctaa tgggcttaag tcgagtcctt 1260
tccgatgtgt taaatacaca tttattacac tgatgcgtcg aatgtacact tttaatagga 1320
tagctccact aaaaattatt ttatttattt aatttgttgc accaaaactg atacattgac 1380
gaa 1383
<210> 8
<211> 6291
<212> DNA
<213> Artificial
<400> 8
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420
tgcgtcgaga taagagggtt aaaaaatata ttttacgcac catatacgca tcgggttgat 480
atcgttaata tggatcaatt tgaacagttg attaacgtgt ctctgctcaa gtctttgatc 540
aaaacgcaaa tcgacgaaaa tgtgtcggac aatatcaagt cgatgagcga aaaactaaaa 600
aggctagaat acgacaatct cacagacagc gttgagatat acggtattca cgacagcagg 660
ctgaataata aaaaaattag aaactattat ttaaccctag aaagataatc atattgtgac 720
gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga 780
ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt acatactaat 840
aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc 900
aaaatttctt ctataaagta acaaaacttt taaacattct ctcttttaca aaaataaact 960
tattttgtac tttaaaaaca gtcatgttgt attataaaat aagtaattag cttaacttat 1020
acataataga aacaaattat acttattagt cagtcagaaa caactttggc acatatcaat 1080
attatgctct cgacaaataa cttttttgca ttttttgcac gatgcatttg cctttcgcct 1140
tattttagag gggcagtaag tacagtaagt acgttttttc attactggct cttcagtact 1200
gtcatctgat gtaccaggca cttcatttgg caaaatatta gagatattat cgcgcaaata 1260
tctcttcaaa gtaggagctt ctaaacgctt acgcataaac gatgacgtca ggctcatgta 1320
aaggtttctc ataaattttt tgcgactttg aaccttttct cccttgctac tgacattatg 1380
gctgtatata ataaaagaat ttatgcaggc aatgtttatc attccgtaca ataatgccat 1440
aggccaccta ttcgtcctcc tactgcaggt catcacagaa cacatttggt ctagcgtgtc 1500
cactccgcct ttagtttgat tataatacat aaccatttgc ggtttaccgg tactttcgtt 1560
gatagaagca tcctcatcac aagatgataa taagtatacc atcttagctg gcttcggttt 1620
atatgagacg agagtaaggg gtccgtcaaa acaaaacatc gatgttccca ctggcctgga 1680
gcgactgttt ttcagtactt ccggtatctc gcgtttgttt gatcgcacgg ttcccacaat 1740
ggttaattcg agctcgcccg gggatctaat tcaattagag actaattcaa ttagagctaa 1800
ttcaattagg atccaagctt atcgatttcg aaccctcgac cgccggagta taaatagagg 1860
cgcttcgtct acggagcgac aattcaattc aaacaagcaa agtgaacacg tcgctaagcg 1920
aaagctaagc aaataaacaa gcgcagctga acaagctaaa caatcggggt accgctagag 1980
tcgacggtac cgcgggcccg ggatccaccg gtcgccacca tggtgagcaa gggcgaggag 2040
ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 2100
ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 2160
atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 2220
ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 2280
gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 2340
aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 2400
ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 2460
agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 2520
atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 2580
cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 2640
ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 2700
gccgggatca ctctcggcat ggacgagctg tacaagtaac ggccgcgact ctagatcata 2760
atcagccatg cggccgcgac tctagaccac atttgtagag gttttacttg ctttaaaaaa 2820
cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt 2880
gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 2940
agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaaag 3000
cttatcgata cgcgtacggc gcgcctaggc cggccgatct cggatctgac aatgttcagt 3060
gcagagactc ggctacgcct cgtggacttt gaagttgacc aacaatgttt attcttacct 3120
ctaatagtcc tctgtggcaa ggtcaagatt ctgttagaag ccaatgaaga acctggttgt 3180
tcaataacat tttgttcgtc taatatttca ctaccgcttg acgttggctg cacttcatgt 3240
acctcatcta taaacgcttc ttctgtatcg ctctggacgt catcttcact tacgtgatct 3300
gatatttcac tgtcagaatc ctcaccaaca agctcgtcat cgctttgcag aagagcagag 3360
aggatatgct catcgtctaa agaactaccc attttattat atattagtca cgatatctat 3420
aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa 3480
ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc 3540
acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg 3600
gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga 3660
aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg 3720
ttaaaaaaga tttgcgcttt actcgaccta aactttaaac acgtcataga atcttcgttt 3780
gacaaaaacc acattgtggc caagctgtgt gacgcgacgc gcgctaaaga atggcaaacc 3840
aagtcgcgcg agcgtcgact ctagaggatc cccgggtacc gagctcgaat tcgtaatcat 3900
ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 3960
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acatcggatg 4020
ccgggaccga cgagtgcaga ggcgtgcaag cgagcttggc gtaatcatgg tcatagctgt 4080
ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 4140
agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 4200
tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 4260
cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 4320
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4380
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4440
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 4500
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 4560
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 4620
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 4680
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 4740
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 4800
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 4860
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 4920
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 4980
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5040
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5100
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 5160
agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 5220
ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 5280
gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 5340
catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 5400
cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 5460
cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 5520
gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 5580
tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 5640
gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 5700
tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 5760
gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 5820
gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 5880
taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 5940
tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6000
ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6060
taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 6120
tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 6180
aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 6240
ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c 6291
<210> 9
<211> 6334
<212> DNA
<213> Artificial
<400> 9
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420
tgcgtcgaga taagagggtt aaaaaatata ttttacgcac catatacgca tcgggttgat 480
atcgttaata tggatcaatt tgaacagttg attaacgtgt ctctgctcaa gtctttgatc 540
aaaacgcaaa tcgacgaaaa tgtgtcggac aatatcaagt cgatgagcga aaaactaaaa 600
aggctagaat acgacaatct cacagacagc gttgagatat acggtattca cgacagcagg 660
ctgaataata aaaaaattag aaactattat ttaaccctag aaagataatc atattgtgac 720
gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga 780
ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt acatactaat 840
aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc 900
aaaatttctt ctataaagta acaaaacttt taaacattct ctcttttaca aaaataaact 960
tattttgtac tttaaaaaca gtcatgttgt attataaaat aagtaattag cttaacttat 1020
acataataga aacaaattat acttattagt cagtcagaaa caactttggc acatatcaat 1080
attatgctct cgacaaataa cttttttgca ttttttgcac gatgcatttg cctttcgcct 1140
tattttagag gggcagtaag tacagtaagt acgttttttc attactggct cttcagtact 1200
gtcatctgat gtaccaggca cttcatttgg caaaatatta gagatattat cgcgcaaata 1260
tctcttcaaa gtaggagctt ctaaacgctt acgcataaac gatgacgtca ggctcatgta 1320
aaggtttctc ataaattttt tgcgactttg aaccttttct cccttgctac tgacattatg 1380
gctgtatata ataaaagaat ttatgcaggc aatgtttatc attccgtaca ataatgccat 1440
aggccaccta ttcgtcctcc tactgcaggt catcacagaa cacatttggt ctagcgtgtc 1500
cactccgcct ttagtttgat tataatacat aaccatttgc ggtttaccgg tactttcgtt 1560
gatagaagca tcctcatcac aagatgataa taagtatacc atcttagctg gcttcggttt 1620
atatgagacg agagtaaggg gtccgtcaaa acaaaacatc gatgttccca ctggcctgga 1680
gcgactgttt ttcagtactt ccggtatctc gcgtttgttc ctgcaggatc atgatgataa 1740
acaatgtatg gtgctaatgt tgcttcaaca acaattctgt tgaactgtgt tttcatgttt 1800
gccaacaagc acctttatac tcggtggcct ccccaccacc aacttttttg cactgcaaaa 1860
aaacacgctt ttgcacgcgg gcccatacat agtacaaact ctacgtttcg tagactattt 1920
tacataaata gtctacaccg ttgtatacgc tccaaataca ctaccacaca ttgaaccttt 1980
ttgcagtgca aaaaagtacg tgtcggcagt cacgtaggcc ggccttatcg ggtcgcgtcc 2040
tgtcacgtac gaatcacatt atcggaccgg acgagtgttg tcttatcgtg acaggacgcc 2100
agcttcctgt gttgctaacc gcagccggac gcaactcctt atcggaacag gacgcgcctc 2160
catatcagcc gcgcgttatc tcatgcgcgt gaccggacac gaggcgcccg tcccgcttat 2220
cgcgcctata aatacagccc gcaacgatct ggtaaacaca gttgaacagc atctgttcga 2280
aatggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt 2340
cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg 2400
tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga 2460
caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga 2520
ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca 2580
gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc 2640
cgaggagcag gactaaagct ttacaactaa acacgacttg gagtattcct tgtagtgttt 2700
aagattttaa atcttactta atgacttcga acgattttaa cgataacttt ctctttgttt 2760
aactttaatc agcatacata aaaagccccg gttttgtatc gggaagaaaa aaaatgtaat 2820
tgtgttgcct agataataaa cgtattatca aagtgtgtgg ttttccttta ccaaagaccc 2880
ctttaagatg ggcctaatgg gcttaagtcg agtcctttcc gatgtgttaa atacacattt 2940
attacactga tgcgtcgaat gtacactttt aataggatag ctccactaaa aattatttta 3000
tttatttaat ttgttgcacc aaaactgata cattgacgaa acgcgtatgc tagcaatgaa 3060
ggcgcgccta ggccggccga tctcggatct gacaatgttc agtgcagaga ctcggctacg 3120
cctcgtggac tttgaagttg accaacaatg tttattctta cctctaatag tcctctgtgg 3180
caaggtcaag attctgttag aagccaatga agaacctggt tgttcaataa cattttgttc 3240
gtctaatatt tcactaccgc ttgacgttgg ctgcacttca tgtacctcat ctataaacgc 3300
ttcttctgta tcgctctgga cgtcatcttc acttacgtga tctgatattt cactgtcaga 3360
atcctcacca acaagctcgt catcgctttg cagaagagca gagaggatat gctcatcgtc 3420
taaagaacta cccattttat tatatattag tcacgatatc tataacaaga aaatatatat 3480
ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt atgagttaaa 3540
tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt cgttatagtt 3600
caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc aagcggcgac 3660
tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag agcaatattt 3720
caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaaaaa agatttgcgc 3780
tttactcgac ctaaacttta aacacgtcat agaatcttcg tttgacaaaa accacattgt 3840
ggccaagctg tgtgacgcga cgcgcgctaa agaatggcaa accaagtcgc gcgagcgtcg 3900
actctagagg atccccgggt accgagctcg aattcgtaat catggtcata gctgtttcct 3960
gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 4020
aaagcctggg gtgcctaatg agtgagctaa ctcacatcgg atgccgggac cgacgagtgc 4080
agaggcgtgc aagcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 4140
tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 4200
gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 4260
ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 4320
cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 4380
cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 4440
aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 4500
gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 4560
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 4620
agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 4680
ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 4740
taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 4800
gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 4860
gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 4920
ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 4980
ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 5040
gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 5100
caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 5160
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 5220
aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 5280
tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 5340
tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 5400
gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 5460
gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 5520
aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 5580
gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 5640
ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 5700
tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 5760
atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 5820
ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 5880
ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 5940
ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 6000
atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 6060
gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 6120
tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 6180
ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 6240
acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc 6300
tataaaaata ggcgtatcac gaggcccttt cgtc 6334
<210> 10
<211> 5898
<212> DNA
<213> Artificial
<400> 10
ggcgcgccta tactcgagca gcgtcgtgaa aagaggcaat gacaaataca aaacgacgta 60
tgagcagacc cgtcgccaag acgggtctac ctctaagatg atgtcatttg ttttttaaaa 120
ctaactcgct ttacgagtag aattctacgt gtaaaacata atcaagagat gatgtcattt 180
gtttttcaaa accaaactcg ctttacgagt agaattctac gtgtaaaaca caatcaaaag 240
atgatgtcat tcgtttttca aaaccgaatt taagaaatga tgtcatttgt ttttcaaaac 300
caaactcgct ttacgagcag aattctacgt gtaaaacaca atcaagagat gatgtcattt 360
gtttttcaaa actgaatgat gtcatttgtt tttcaaaact aaacttgctt tgcgagtaga 420
attctacgtg taaaacacag tcaagagatg atgtcatttg tttttcaaaa ctgaaccggc 480
tttacgagta gaattctact tgtaaaacat aatcaagaga tgatgtcatt tgtttttcaa 540
aactgaactg gctttacgag tagaattcta cgtgtaaaac ataatcaaga gatgatgtca 600
tcattaaact gatgtcattt tatacacgat tgttaacatg tttaataatg actaatttgt 660
ttttccaaat taaactcgct ttacgagtag aattctactt gtaacgcacg attaagtatg 720
aatcataagc tgatgtcatt tgttttcgac ataaaatgtt tatacaatgg aatcttcttg 780
taaattatcc aaataatata atttatccga ttctacgtta catttaaatt cgttgttatc 840
gtacaattct tcaggacacg ccatgtattg gtcattttta gcgtgcaacc aacgattgta 900
tttgacgccg tcgttggatt gcgtgttcag gttggcgtac acgtgactgg gcacggcttc 960
tttttccatg ggacgtcgac cgagaaattt ctctggccgt tattcgttat tctctctttt 1020
ctttttgggt ctctccctct ctgcactaat gctctctcac tctgtcacac agtaaacggc 1080
atactgctct cgttggttcg agagagcgcg cctcgaatgt tcgcgaaaag agcgccggag 1140
tataaataga ggcgcttcgt ctacggagcg acaattcaat tcaaacaagc aaagtgaaca 1200
cgtcgctaag cgaaagctaa gcaaataaac aagcgcagct gaacaagcta aacaatctgc 1260
agtaaagtgc aagttaaagt gaatcaatta aaagtaacca gcaaccaagt aaatcaactg 1320
caactactga aatctgccaa gaagtaatta ttgaatacaa gaagagaact ctgggggatc 1380
tctagtccag tgtggtggaa ttcgccatgg ccccaaagaa aaagagaaag gttgattaca 1440
aagaccacga cggagactac aaagaccacg acattgatta taaagatgat gatgataaag 1500
gaacgatgga caaaaagtat agcatcggtc tggatattgg aactaactcc gtcggctggg 1560
ctgtaatcac cgacgaatac aaggtcccgt caaaaaagtt caaggtattg ggtaacacag 1620
atcgtcactc tatcaaaaag aatctcattg gagctctgtt gttcgacagc ggcgaaacag 1680
ctgaggccac tagactgaag cgcaccgcca gacgccgtta cacgaggaga aagaacagaa 1740
tctgctactt gcaagaaata ttctcaaacg agatggccaa agtggacgat tcgttctttc 1800
ataggttaga agagagtttc cttgttgaag aggataaaaa gcacgaaaga catccgatat 1860
ttggaaacat cgtggacgaa gttgcttatc acgagaagta ccccacgatc tatcatctgc 1920
gtaaaaagtt ggtggactcg acagataagg ccgacctcag gttaatatac cttgcactgg 1980
cgcacatgat caaattcaga ggccattttc tgattgaagg tgacctgaac cctgacaata 2040
gtgatgtgga caaactcttc attcaattag ttcagaccta caatcaactg tttgaagaga 2100
accctatcaa cgcttcagga gttgacgcta aggccatcct tagtgcgaga ctgagcaaat 2160
cccgccgtct cgaaaactta atcgcacagt tgcctggaga gaaaaagaac ggtttgttcg 2220
gaaatctcat tgcgttgtca ctcggactca cgccaaactt caagtctaac ttcgatttgg 2280
cagaagacgc gaaactgcaa ctgagcaaag acacatatga cgatgacctc gataacctct 2340
tagctcagat cggcgatcaa tacgccgact tgttcctcgc tgccaaaaat ctgtcggacg 2400
ctatacttct gagtgatatc ttgcgcgtca acacagaaat tactaaggct cctctgtcgg 2460
ccagtatgat aaaacgctat gacgaacacc atcaggattt gacattgctc aaagccctcg 2520
tgcgtcaaca gctcccagaa aagtacaagg agattttctt tgatcagtcc aagaatggct 2580
acgcaggtta tatagacggt ggagcgtcgc aagaagagtt ctacaagttc atcaagccaa 2640
tattagaaaa gatggacggc acggaagagt tacttgttaa gctgaatcgt gaggacctgt 2700
tgcgtaaaca gaggacattc gataacggat caattccgca ccaaatacat cttggcgaac 2760
tgcacgctat cctcaggaga caagaggact tctacccctt tttaaaggat aaccgtgaaa 2820
agatcgagaa aatcctgact ttcaggattc cttactatgt cggcccactg gctcgtggta 2880
atagcaggtt tgcctggatg accaggaagt ccgaagagac aattactccg tggaacttcg 2940
aagaggtggt tgataaagga gcatcagcgc agtctttcat agaacgcatg acaaattttg 3000
acaagaactt accgaatgag aaggtccttc ccaaacactc actcctctac gaatacttca 3060
cagtatacaa cgagctcact aaagtcaagt acgtaaccga gggtatgcgc aaacccgctt 3120
tcctgtctgg agagcagaaa aaggccatcg tggaccttct gttcaagaca aaccgtaagg 3180
tcactgtaaa gcaactcaag gaagactact tcaaaaagat agagtgtttc gattcagtgg 3240
aaatctctgg cgttgaggac agatttaacg cttccttggg tacttaccac gatttgctca 3300
agatcattaa agataaggac ttcctcgaca acgaagagaa cgaagatatc ttagaggaca 3360
tagttctcac ccttacgctg tttgaagata gagagatgat tgaagagcgc ctgaagactt 3420
atgctcattt gttcgatgac aaagtcatga agcaactgaa acgccgtagg tacaccggct 3480
ggggtagatt atcgcgcaaa cttattaatg gtataaggga caagcagtcg ggaaaaacga 3540
tattggactt tctcaagagt gatggtttcg ccaacagaaa ttttatgcaa ctcatacacg 3600
atgacagctt aacattcaag gaagatatcc aaaaagcaca ggtgtcggga cagggcgaca 3660
gtttgcacga acatattgct aacctcgccg gctccccggc gataaaaaag ggtatccttc 3720
agactgtgaa agtcgtagat gaactggtga aggttatggg tcgtcataaa cccgagaaca 3780
tagttatcga aatggctagg gagaatcaaa caactcagaa gggacagaaa aactcaagag 3840
aacgcatgaa gcgcattgaa gagggtatca aagagcttgg cagtcaaatc ctgaaggaac 3900
accctgtcga gaacacgcaa cttcagaacg aaaaattgta cctctactat ctgcagaatg 3960
gtagagatat gtacgtagac caagaattgg atattaaccg cctctcagat tacgacgtgg 4020
atcatatagt tccgcagtca ttcttgaagg atgactctat cgacaacaaa gtcctcacaa 4080
gatcagacaa gaaccgcgga aaatcagata atgtaccctc tgaagaggtg gttaaaaaga 4140
tgaaaaacta ctggagacag ttacttaacg ctaagttgat cacgcaaaga aagttcgata 4200
acctcacaaa ggctgaacgc ggcggtttaa gcgagcttga caaggccggt ttcataaaac 4260
gtcagttagt cgaaaccagg caaattacga aacacgtagc ccaaatattg gattcccgca 4320
tgaacactaa atacgatgaa aatgacaagc tcatccgtga ggtcaaagta attaccctga 4380
aaagcaagtt ggtgtccgac ttcagaaagg atttccagtt ctacaaagtt cgcgaaatca 4440
acaactacca ccatgcacat gacgcttacc tgaacgcagt cgtaggcact gcgttaatta 4500
aaaagtaccc taaactggaa tctgagttcg tgtacggtga ctataaagtg tacgatgtta 4560
gaaagatgat cgctaaaagc gaacaggaga ttggaaaggc taccgccaag tatttctttt 4620
actccaacat catgaatttc tttaagaccg aaatcacgtt agcaaatggc gagatacgta 4680
aaaggccact tatcgaaaca aacggagaaa ctggcgagat agtgtgggac aagggtagag 4740
attttgccac tgtccgcaaa gtactgtcga tgccgcaagt gaatatcgtt aaaaagaccg 4800
aagttcaaac gggaggcttc agcaaagagt ccatcctgcc caagcgtaac agtgataaat 4860
tgatagctag gaaaaaggac tgggatccta aaaagtatgg tggattcgac agcccaactg 4920
tcgcatactc cgtattggtg gttgcgaaag tcgaaaaagg aaagagcaaa aagctcaagt 4980
ccgtaaaaga gctgttgggc attaccataa tggaaagatc atctttcgag aagaatccta 5040
tcgattttct ggaagccaag ggatataaag aggtcaaaaa ggacctcata atcaagttac 5100
caaaatacag tctgttcgaa ttggagaacg gcagaaaacg catgcttgca tcagcgggtg 5160
aactgcaaaa gggaaatgag ttagcacttc cttctaaata cgtcaacttc ctgtatttgg 5220
cgtcacacta cgaaaaactg aagggctctc cagaagataa cgagcaaaag cagttatttg 5280
tggaacagca caaacattac cttgacgaaa ttatagagca aatctcggag ttcagtaaga 5340
gagtgatttt ggctgacgcc aatcttgata aagttctgtc tgcttacaac aagcaccgtg 5400
ataaaccgat tagggaacag gccgagaaca tcatacatct cttcacactc actaaccttg 5460
gtgcacccgc agcgttcaaa tattttgaca ccacgataga tcgtaagagg tacaccagca 5520
cgaaagaagt tttggacgcg acactcatcc atcaatcaat cacgggcctg tacgagacca 5580
gaatcgacct gtcccagctc ggtggcgact agcggccgcg actctagatc ataatcagcc 5640
atgcggccgc gactctagac cacatttgta gaggttttac ttgctttaaa aaacctccca 5700
cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt 5760
gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 5820
ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aagcttatcg 5880
atacgcgtac ggcgcgcc 5898
<210> 11
<211> 12207
<212> DNA
<213> Artificial
<400> 11
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420
tgcgtcgaga taagagggtt aaaaaatata ttttacgcac catatacgca tcgggttgat 480
atcgttaata tggatcaatt tgaacagttg attaacgtgt ctctgctcaa gtctttgatc 540
aaaacgcaaa tcgacgaaaa tgtgtcggac aatatcaagt cgatgagcga aaaactaaaa 600
aggctagaat acgacaatct cacagacagc gttgagatat acggtattca cgacagcagg 660
ctgaataata aaaaaattag aaactattat ttaaccctag aaagataatc atattgtgac 720
gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga 780
ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt acatactaat 840
aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc 900
aaaatttctt ctataaagta acaaaacttt taaacattct ctcttttaca aaaataaact 960
tattttgtac tttaaaaaca gtcatgttgt attataaaat aagtaattag cttaacttat 1020
acataataga aacaaattat acttattagt cagtcagaaa caactttggc acatatcaat 1080
attatgctct cgacaaataa cttttttgca ttttttgcac gatgcatttg cctttcgcct 1140
tattttagag gggcagtaag tacagtaagt acgttttttc attactggct cttcagtact 1200
gtcatctgat gtaccaggca cttcatttgg caaaatatta gagatattat cgcgcaaata 1260
tctcttcaaa gtaggagctt ctaaacgctt acgcataaac gatgacgtca ggctcatgta 1320
aaggtttctc ataaattttt tgcgactttg aaccttttct cccttgctac tgacattatg 1380
gctgtatata ataaaagaat ttatgcaggc aatgtttatc attccgtaca ataatgccat 1440
aggccaccta ttcgtcctcc tactgcaggt catcacagaa cacatttggt ctagcgtgtc 1500
cactccgcct ttagtttgat tataatacat aaccatttgc ggtttaccgg tactttcgtt 1560
gatagaagca tcctcatcac aagatgataa taagtatacc atcttagctg gcttcggttt 1620
atatgagacg agagtaaggg gtccgtcaaa acaaaacatc gatgttccca ctggcctgga 1680
gcgactgttt ttcagtactt ccggtatctc gcgtttgttc ctgcaggatc atgatgataa 1740
acaatgtatg gtgctaatgt tgcttcaaca acaattctgt tgaactgtgt tttcatgttt 1800
gccaacaagc acctttatac tcggtggcct ccccaccacc aacttttttg cactgcaaaa 1860
aaacacgctt ttgcacgcgg gcccatacat agtacaaact ctacgtttcg tagactattt 1920
tacataaata gtctacaccg ttgtatacgc tccaaataca ctaccacaca ttgaaccttt 1980
ttgcagtgca aaaaagtacg tgtcggcagt cacgtaggcc ggccttatcg ggtcgcgtcc 2040
tgtcacgtac gaatcacatt atcggaccgg acgagtgttg tcttatcgtg acaggacgcc 2100
agcttcctgt gttgctaacc gcagccggac gcaactcctt atcggaacag gacgcgcctc 2160
catatcagcc gcgcgttatc tcatgcgcgt gaccggacac gaggcgcccg tcccgcttat 2220
cgcgcctata aatacagccc gcaacgatct ggtaaacaca gttgaacagc atctgttcga 2280
aatggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt 2340
cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg 2400
tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga 2460
caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga 2520
ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca 2580
gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc 2640
cgaggagcag gactaaagct ttacaactaa acacgacttg gagtattcct tgtagtgttt 2700
aagattttaa atcttactta atgacttcga acgattttaa cgataacttt ctctttgttt 2760
aactttaatc agcatacata aaaagccccg gttttgtatc gggaagaaaa aaaatgtaat 2820
tgtgttgcct agataataaa cgtattatca aagtgtgtgg ttttccttta ccaaagaccc 2880
ctttaagatg ggcctaatgg gcttaagtcg agtcctttcc gatgtgttaa atacacattt 2940
attacactga tgcgtcgaat gtacactttt aataggatag ctccactaaa aattatttta 3000
tttatttaat ttgttgcacc aaaactgata cattgacgaa acgcgtatgc tagcaatgaa 3060
ggcgcgccca gcgtcgtgaa aagaggcaat gacaaataca aaacgacgta tgagcagacc 3120
cgtcgccaag acgggtctac ctctaagatg atgtcatttg ttttttaaaa ctaactcgct 3180
ttacgagtag aattctacgt gtaaaacata atcaagagat gatgtcattt gtttttcaaa 3240
accaaactcg ctttacgagt agaattctac gtgtaaaaca caatcaaaag atgatgtcat 3300
tcgtttttca aaaccgaatt taagaaatga tgtcatttgt ttttcaaaac caaactcgct 3360
ttacgagcag aattctacgt gtaaaacaca atcaagagat gatgtcattt gtttttcaaa 3420
actgaatgat gtcatttgtt tttcaaaact aaacttgctt tgcgagtaga attctacgtg 3480
taaaacacag tcaagagatg atgtcatttg tttttcaaaa ctgaaccggc tttacgagta 3540
gaattctact tgtaaaacat aatcaagaga tgatgtcatt tgtttttcaa aactgaactg 3600
gctttacgag tagaattcta cgtgtaaaac ataatcaaga gatgatgtca tcattaaact 3660
gatgtcattt tatacacgat tgttaacatg tttaataatg actaatttgt ttttccaaat 3720
taaactcgct ttacgagtag aattctactt gtaacgcacg attaagtatg aatcataagc 3780
tgatgtcatt tgttttcgac ataaaatgtt tatacaatgg aatcttcttg taaattatcc 3840
aaataatata atttatccga ttctacgtta catttaaatt cgttgttatc gtacaattct 3900
tcaggacacg ccatgtattg gtcattttta gcgtgcaacc aacgattgta tttgacgccg 3960
tcgttggatt gcgtgttcag gttggcgtac acgtgactgg gcacggcttc tttttccatg 4020
ggacgtcgac cgagaaattt ctctggccgt tattcgttat tctctctttt ctttttgggt 4080
ctctccctct ctgcactaat gctctctcac tctgtcacac agtaaacggc atactgctct 4140
cgttggttcg agagagcgcg cctcgaatgt tcgcgaaaag agcgccggag tataaataga 4200
ggcgcttcgt ctacggagcg acaattcaat tcaaacaagc aaagtgaaca cgtcgctaag 4260
cgaaagctaa gcaaataaac aagcgcagct gaacaagcta aacaatctgc agtaaagtgc 4320
aagttaaagt gaatcaatta aaagtaacca gcaaccaagt aaatcaactg caactactga 4380
aatctgccaa gaagtaatta ttgaatacaa gaagagaact ctgggggatc tctagtccag 4440
tgtggtggaa ttcgccatgg ccccaaagaa aaagagaaag gttgattaca aagaccacga 4500
cggagactac aaagaccacg acattgatta taaagatgat gatgataaag gaacgatgga 4560
caaaaagtat agcatcggtc tggatattgg aactaactcc gtcggctggg ctgtaatcac 4620
cgacgaatac aaggtcccgt caaaaaagtt caaggtattg ggtaacacag atcgtcactc 4680
tatcaaaaag aatctcattg gagctctgtt gttcgacagc ggcgaaacag ctgaggccac 4740
tagactgaag cgcaccgcca gacgccgtta cacgaggaga aagaacagaa tctgctactt 4800
gcaagaaata ttctcaaacg agatggccaa agtggacgat tcgttctttc ataggttaga 4860
agagagtttc cttgttgaag aggataaaaa gcacgaaaga catccgatat ttggaaacat 4920
cgtggacgaa gttgcttatc acgagaagta ccccacgatc tatcatctgc gtaaaaagtt 4980
ggtggactcg acagataagg ccgacctcag gttaatatac cttgcactgg cgcacatgat 5040
caaattcaga ggccattttc tgattgaagg tgacctgaac cctgacaata gtgatgtgga 5100
caaactcttc attcaattag ttcagaccta caatcaactg tttgaagaga accctatcaa 5160
cgcttcagga gttgacgcta aggccatcct tagtgcgaga ctgagcaaat cccgccgtct 5220
cgaaaactta atcgcacagt tgcctggaga gaaaaagaac ggtttgttcg gaaatctcat 5280
tgcgttgtca ctcggactca cgccaaactt caagtctaac ttcgatttgg cagaagacgc 5340
gaaactgcaa ctgagcaaag acacatatga cgatgacctc gataacctct tagctcagat 5400
cggcgatcaa tacgccgact tgttcctcgc tgccaaaaat ctgtcggacg ctatacttct 5460
gagtgatatc ttgcgcgtca acacagaaat tactaaggct cctctgtcgg ccagtatgat 5520
aaaacgctat gacgaacacc atcaggattt gacattgctc aaagccctcg tgcgtcaaca 5580
gctcccagaa aagtacaagg agattttctt tgatcagtcc aagaatggct acgcaggtta 5640
tatagacggt ggagcgtcgc aagaagagtt ctacaagttc atcaagccaa tattagaaaa 5700
gatggacggc acggaagagt tacttgttaa gctgaatcgt gaggacctgt tgcgtaaaca 5760
gaggacattc gataacggat caattccgca ccaaatacat cttggcgaac tgcacgctat 5820
cctcaggaga caagaggact tctacccctt tttaaaggat aaccgtgaaa agatcgagaa 5880
aatcctgact ttcaggattc cttactatgt cggcccactg gctcgtggta atagcaggtt 5940
tgcctggatg accaggaagt ccgaagagac aattactccg tggaacttcg aagaggtggt 6000
tgataaagga gcatcagcgc agtctttcat agaacgcatg acaaattttg acaagaactt 6060
accgaatgag aaggtccttc ccaaacactc actcctctac gaatacttca cagtatacaa 6120
cgagctcact aaagtcaagt acgtaaccga gggtatgcgc aaacccgctt tcctgtctgg 6180
agagcagaaa aaggccatcg tggaccttct gttcaagaca aaccgtaagg tcactgtaaa 6240
gcaactcaag gaagactact tcaaaaagat agagtgtttc gattcagtgg aaatctctgg 6300
cgttgaggac agatttaacg cttccttggg tacttaccac gatttgctca agatcattaa 6360
agataaggac ttcctcgaca acgaagagaa cgaagatatc ttagaggaca tagttctcac 6420
ccttacgctg tttgaagata gagagatgat tgaagagcgc ctgaagactt atgctcattt 6480
gttcgatgac aaagtcatga agcaactgaa acgccgtagg tacaccggct ggggtagatt 6540
atcgcgcaaa cttattaatg gtataaggga caagcagtcg ggaaaaacga tattggactt 6600
tctcaagagt gatggtttcg ccaacagaaa ttttatgcaa ctcatacacg atgacagctt 6660
aacattcaag gaagatatcc aaaaagcaca ggtgtcggga cagggcgaca gtttgcacga 6720
acatattgct aacctcgccg gctccccggc gataaaaaag ggtatccttc agactgtgaa 6780
agtcgtagat gaactggtga aggttatggg tcgtcataaa cccgagaaca tagttatcga 6840
aatggctagg gagaatcaaa caactcagaa gggacagaaa aactcaagag aacgcatgaa 6900
gcgcattgaa gagggtatca aagagcttgg cagtcaaatc ctgaaggaac accctgtcga 6960
gaacacgcaa cttcagaacg aaaaattgta cctctactat ctgcagaatg gtagagatat 7020
gtacgtagac caagaattgg atattaaccg cctctcagat tacgacgtgg atcatatagt 7080
tccgcagtca ttcttgaagg atgactctat cgacaacaaa gtcctcacaa gatcagacaa 7140
gaaccgcgga aaatcagata atgtaccctc tgaagaggtg gttaaaaaga tgaaaaacta 7200
ctggagacag ttacttaacg ctaagttgat cacgcaaaga aagttcgata acctcacaaa 7260
ggctgaacgc ggcggtttaa gcgagcttga caaggccggt ttcataaaac gtcagttagt 7320
cgaaaccagg caaattacga aacacgtagc ccaaatattg gattcccgca tgaacactaa 7380
atacgatgaa aatgacaagc tcatccgtga ggtcaaagta attaccctga aaagcaagtt 7440
ggtgtccgac ttcagaaagg atttccagtt ctacaaagtt cgcgaaatca acaactacca 7500
ccatgcacat gacgcttacc tgaacgcagt cgtaggcact gcgttaatta aaaagtaccc 7560
taaactggaa tctgagttcg tgtacggtga ctataaagtg tacgatgtta gaaagatgat 7620
cgctaaaagc gaacaggaga ttggaaaggc taccgccaag tatttctttt actccaacat 7680
catgaatttc tttaagaccg aaatcacgtt agcaaatggc gagatacgta aaaggccact 7740
tatcgaaaca aacggagaaa ctggcgagat agtgtgggac aagggtagag attttgccac 7800
tgtccgcaaa gtactgtcga tgccgcaagt gaatatcgtt aaaaagaccg aagttcaaac 7860
gggaggcttc agcaaagagt ccatcctgcc caagcgtaac agtgataaat tgatagctag 7920
gaaaaaggac tgggatccta aaaagtatgg tggattcgac agcccaactg tcgcatactc 7980
cgtattggtg gttgcgaaag tcgaaaaagg aaagagcaaa aagctcaagt ccgtaaaaga 8040
gctgttgggc attaccataa tggaaagatc atctttcgag aagaatccta tcgattttct 8100
ggaagccaag ggatataaag aggtcaaaaa ggacctcata atcaagttac caaaatacag 8160
tctgttcgaa ttggagaacg gcagaaaacg catgcttgca tcagcgggtg aactgcaaaa 8220
gggaaatgag ttagcacttc cttctaaata cgtcaacttc ctgtatttgg cgtcacacta 8280
cgaaaaactg aagggctctc cagaagataa cgagcaaaag cagttatttg tggaacagca 8340
caaacattac cttgacgaaa ttatagagca aatctcggag ttcagtaaga gagtgatttt 8400
ggctgacgcc aatcttgata aagttctgtc tgcttacaac aagcaccgtg ataaaccgat 8460
tagggaacag gccgagaaca tcatacatct cttcacactc actaaccttg gtgcacccgc 8520
agcgttcaaa tattttgaca ccacgataga tcgtaagagg tacaccagca cgaaagaagt 8580
tttggacgcg acactcatcc atcaatcaat cacgggcctg tacgagacca gaatcgacct 8640
gtcccagctc ggtggcgact agcggccgcg actctagatc ataatcagcc atgcggccgc 8700
gactctagac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 8760
tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 8820
atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 8880
attctagttg tggtttgtcc aaactcatca atgtatctta aagcttatcg atacgcgtac 8940
ctaggccggc cgatctcgga tctgacaatg ttcagtgcag agactcggct acgcctcgtg 9000
gactttgaag ttgaccaaca atgtttattc ttacctctaa tagtcctctg tggcaaggtc 9060
aagattctgt tagaagccaa tgaagaacct ggttgttcaa taacattttg ttcgtctaat 9120
atttcactac cgcttgacgt tggctgcact tcatgtacct catctataaa cgcttcttct 9180
gtatcgctct ggacgtcatc ttcacttacg tgatctgata tttcactgtc agaatcctca 9240
ccaacaagct cgtcatcgct ttgcagaaga gcagagagga tatgctcatc gtctaaagaa 9300
ctacccattt tattatatat tagtcacgat atctataaca agaaaatata tatataataa 9360
gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa 9420
agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc 9480
agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg 9540
tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat 9600
gcatgcgtca attttacgca gactatcttt ctagggttaa aaaagatttg cgctttactc 9660
gacctaaact ttaaacacgt catagaatct tcgtttgaca aaaaccacat tgtggccaag 9720
ctgtgtgacg cgacgcgcgc taaagaatgg caaaccaagt cgcgcgagcg tcgactctag 9780
aggatccccg ggtaccgagc tcgaattcgt aatcatggtc atagctgttt cctgtgtgaa 9840
attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 9900
ggggtgccta atgagtgagc taactcacat cggatgccgg gaccgacgag tgcagaggcg 9960
tgcaagcgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 10020
tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 10080
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 10140
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 10200
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 10260
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 10320
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 10380
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 10440
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 10500
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 10560
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 10620
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 10680
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 10740
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 10800
ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 10860
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 10920
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 10980
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 11040
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 11100
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 11160
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 11220
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 11280
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 11340
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 11400
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 11460
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 11520
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 11580
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 11640
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 11700
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 11760
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 11820
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 11880
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 11940
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 12000
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 12060
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 12120
cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 12180
ataggcgtat cacgaggccc tttcgtc 12207
<210> 12
<211> 3947
<212> DNA
<213> Artificial
<400> 12
gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60
cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120
tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180
aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240
ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300
ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360
tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420
tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480
actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540
gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600
acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660
gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720
acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780
gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840
ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900
gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960
cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020
agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080
catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140
tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200
cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260
gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320
taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560
cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620
agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680
gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800
gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860
gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920
ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980
cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040
cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100
acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160
cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220
accatgatta cgccaagctc gcttgcacgc ctctgcactc gtcggtcccg gcatccgatg 2280
acccaatctc gagttgctag caatgaaaga tctttatcga tttagccaaa agcaaaagct 2340
tgaccaaaaa taggataata tttgtttttt tatttaaaaa aataaacaat tttttataca 2400
taaactgttt atctagtatt aatatttatg ttaacatttg ataacgaatc aaatatattt 2460
ttaaactaat taaaaaatcc gatgtatgtt ataaaattgt tctagaaaaa aagcaccgac 2520
tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc tatttctagc 2580
tctaaaacac tggcaggtgt cttgacgagt tcttctgaat tattaacgct tacaatttcc 2640
tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcatc aggtggcact 2700
tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 2760
tatccgctca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 2820
agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgccac 2880
ctgccatcac ttgtagagca cgatattttg tatatatacc taaaaaaact aaactattga 2940
aagcgtgatt tacaacaaca ctcgacttta caaagattat tcaaaaagag caaaaactct 3000
taacatattc tattaaagat atataatata attaaaacga aattaaataa taacaataaa 3060
acctttagaa tttgtaataa aatccataaa aacaaatgaa aacagttatg gtttgtacag 3120
cgccatctgt tattactttg acaaaatcac tatgactatc tgaccttgtc ttacacgtta 3180
acaattctta ttctgtcctt atctataagc caagtaccaa gcttaaattc gtatggctta 3240
tagttgacga tttttaaatt ctcaaggtat gtacttattt aatattaata agtactaatt 3300
gttaaaatca tctaaaacaa ttcagtgatt tacaacaatg tgtactacat aacctaatac 3360
ttataaattt attaaactgt attgattctt ttaggtcaat catcatgact ttaggagact 3420
tggtgtctca ggaaaaagga acgcaaaaag attgaggcgt ttgaaatgta ttgctggaga 3480
aagctgctac gcattccttg gacagcttgg cgcgccatct cgacgcattc gcgaagtacc 3540
gatctccaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 3600
ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 3660
ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg 3720
gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac 3780
aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 3840
gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 3900
gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga 3947
<210> 13
<211> 24
<212> DNA
<213> Artificial
<220>
<221> misc_feature
<222> (5)..(24)
<223> n is a, c, g, or t
<400> 13
aagtnnnnnn nnnnnnnnnn nnnn 24
<210> 14
<211> 24
<212> DNA
<213> Artificial
<220>
<221> misc_feature
<222> (5)..(24)
<223> n is a, c, g, or t
<400> 14
aaacnnnnnn nnnnnnnnnn nnnn 24
<210> 15
<211> 24
<212> DNA
<213> Artificial
<400> 15
aagtgggcga ggagctgttc accg 24
<210> 16
<211> 24
<212> DNA
<213> Artificial
<400> 16
aaaccggtga acagctcctc gccc 24
<210> 17
<211> 24
<212> DNA
<213> Artificial
<400> 17
aagtgagctg gacggcgacg taaa 24
<210> 18
<211> 24
<212> DNA
<213> Artificial
<400> 18
aaactttacg tcgccgtcca gctc 24
<210> 19
<211> 24
<212> DNA
<213> Artificial
<400> 19
aagtggccac aagttcagcg tgtc 24
<210> 20
<211> 24
<212> DNA
<213> Artificial
<400> 20
aaacgacacg ctgaacttgt ggcc 24
<210> 21
<211> 24
<212> DNA
<213> Artificial
<400> 21
aagtcgcttc aaggtgcaca tgga 24
<210> 22
<211> 24
<212> DNA
<213> Artificial
<400> 22
aaactccatg tgcaccttga agcg 24
<210> 23
<211> 24
<212> DNA
<213> Artificial
<400> 23
aagtgcggaa gaacgcctgc ggct 24
<210> 24
<211> 24
<212> DNA
<213> Artificial
<400> 24
aaacagccgc aggcgttctt ccgc 24
<210> 25
<211> 20
<212> DNA
<213> Artificial
<400> 25
gggcgaggag ctgttcaccg 20
<210> 26
<211> 20
<212> DNA
<213> Artificial
<400> 26
gagctggacg gcgacgtaaa 20
<210> 27
<211> 20
<212> DNA
<213> Artificial
<400> 27
ggccacaagt tcagcgtgtc 20
<210> 28
<211> 23
<212> DNA
<213> Artificial
<400> 28
cgcttcaagg tgcacatgga ggg 23
<210> 29
<211> 23
<212> DNA
<213> Artificial
<400> 29
gcggaagaac gcctgcggct cgg 23
<210> 30
<211> 6161
<212> DNA
<213> Artificial
<400> 30
aaatcaactt gtgttatagt cacggatttg ccgtccaacg tgttcctcaa aaagttgaag 60
accaacaagt ttacggacac tattaattat ttgattttgc cccacttcat tttgtgggat 120
cacaattttg ttatattttt aaacaaagct tggcactggc cgtcgtttta caacgtcgtg 180
actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 240
gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 300
atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 360
gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 420
acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 480
gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 540
aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 600
taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 660
gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 720
tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 780
ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 840
taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 900
gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 960
aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 1020
gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 1080
ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 1140
ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 1200
acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 1260
taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 1320
tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 1380
cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 1440
ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 1500
gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 1560
gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 1620
aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 1680
aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 1740
actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 1800
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 1860
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 1920
atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 1980
ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 2040
gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 2100
cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 2160
tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 2220
cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 2280
ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 2340
gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 2400
tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 2460
ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 2520
gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg 2580
cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 2640
gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 2700
ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 2760
acagctatga catgattacg aattcgaatt cccatccccc tagaatccca aaacaaactg 2820
gttattgtgg taggtcattt gtttggcaga aagaaaactc gagaaatttc tctggccgtt 2880
attcgttatt ctctcttttc tttttgggtc tctccctctc tgcactaatg ctctctcact 2940
ctgtcacaca gtaaacggca tactgctctc gttggttcga gagagcgcgc ctcgaatgtt 3000
cgcgaaaaga gcgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt 3060
caaacaagca aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg 3120
aacaagctaa acaatctgca gtaaagtgca agttaaagtg aatcaattaa aagtaaccag 3180
caaccaagta aatcaactgc aactactgaa atctgccaag aagtaattat tgaatacaag 3240
aagagaactc tgggggatcc ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt 3300
ttgaactata acgaccgcgt gagtcaaaat gacgcatgat tatcttttac gtgactttta 3360
agatttaact catacgataa ttatattgtt atttcatgtt ctacttacgt gataacttat 3420
tatatatata ttttcttgtt atagatatcg tgactaatat ataataaaat gggtagttct 3480
ttagacgatg agcatatcct ctctgctctt ctgcaaagcg atgacgagct tgttggtgag 3540
gattctgaca gtgaaatatc agatcacgta agtgaagatg acgtccagag cgatacagaa 3600
gaagcgttta tagatgaggt acatgaagtg cagccaacgt caagcggtag tgaaatatta 3660
gacgaacaaa atgttattga acaaccaggt tcttcattgg cttctaacag aatcttgacc 3720
ttgccacaga ggactattag aggtaagaat aaacattgtt ggtcaacttc aaagtccacg 3780
aggcgtagcc gagtctctgc actgaacatt gtcagatctc aaagaggtcc gacgcgtatg 3840
tgccgcaata tatatgaccc acttttatgc ttcaaactat tttttactga tgagataatt 3900
tcggaaattg taaaatggac aaatgctgag atatcattga aacgtcggga atctatgaca 3960
ggtgctacat ttcgtgacac gaatgaagat gaaatctatg ctttctttgg tattctggta 4020
atgacagcag tgagaaaaga taaccacatg tccacagatg acctctttga tcgatctttg 4080
tcaatggtgt acgtctctgt aatgagtcgt gatcgttttg attttttgat acgatgtctt 4140
agaatggatg acaaaagtat acggcccaca cttcgagaaa acgatgtatt tactcctgtt 4200
agaaaaatat gggatctctt tatccatcag tgcatacaaa attacactcc aggggctcat 4260
ttgaccatag atgaacagtt acttggtttt agaggacggt gtccgtttag gatgtatatc 4320
ccaaacaagc caagtaagta tggaataaaa atcctcatga tgtgtgacag tggtacgaag 4380
tatatgataa atggaatgcc ttatttggga agaggaacac agaccaacgg agtaccactc 4440
ggtgaatact acgtgaagga gttatcaaag cctgtgcacg gtagttgtcg taatattacg 4500
tgtgacaatt ggttcacctc aatccctttg gcaaaaaact tactacaaga accgtataag 4560
ttaaccattg tgggaaccgt gcgatcaaac aaacgcgaga taccggaagt actgaaaaac 4620
agtcgctcca ggccagtggg aacatcgatg ttttgttttg acggacccct tactctcgtc 4680
tcatataaac cgaagccagc taagatggta tacttattat catcttgtga tgaggatgct 4740
tctatcaacg aaagtaccgg taaaccgcaa atggttatgt attataatca aactaaaggc 4800
ggagtggaca cgctagacca aatgtgttct gtgatgacct gcagtaggaa gacgaatagg 4860
tggcctatgg cattattgta cggaatgata aacattgcct gcataaattc ttttattata 4920
tacagccata atgtcagtag caagggagaa aaggttcaaa gtcgcaaaaa atttatgaga 4980
aacctttaca tgagcctgac gtcatcgttt atgcgtaagc gtttagaagc tcctactttg 5040
aagagatatt tgcgcgataa tatctctaat attttgccaa atgaagtgcc tggtacatca 5100
gatgacagta ctgaagagcc agtaatgaaa aaacgtactt actgtactta ctgcccctct 5160
aaaataaggc gaaaggcaaa tgcatcgtgc aaaaaatgca aaaaagttat ttgtcgagag 5220
cataatattg atatgtgcca aagttgtttc tgactgacta ataagtataa tttgtttcta 5280
ttatgtataa gttaagctaa ttacttattt tataatacaa catgactgtt tttaaagtac 5340
aaaataagtt tatttttgta aaagagagaa tgtttaaaag ttttgttact ttatagaaga 5400
aattttgagt ttttgttttt ttttaataaa taaataaaca taaataaatt gtttgttgaa 5460
tttattatta gtatgtaagt gtaaatataa taaaacttaa tatctattca aattaataaa 5520
taaacctcga tatacagacc gataaaacac atgcgtcaat tttacgcatg attatcttta 5580
acgtacgtca caatatgatt atctttctag ggttaaataa tagtttctaa tttttttatt 5640
attcagcctg ctgtcgtgaa taccgtatat ctcaacgctg tctgtgagat tgtcgtattc 5700
tagccttttt agtttttcgc tcatcgactt gatattgtcc gacacatttt cgtcgatttg 5760
cgttttgatc aaagacttga gcagagacac gttaatcaac tgttcaaatt gatccatatt 5820
aacgatatca acccgatgcg tatatggtgc gtaaaatata ttttttaacc ctcttatact 5880
ttgcactctg cgttaatacg cgttcgtgta cagacgtaat catgttttct tttttggata 5940
aaactcctac tgagtttgac ctcatattag accctcacaa gttgcaaaac gtggcatttt 6000
ttaccaatga agaatttaaa gttattttaa aaaatttcat cacagattta aagaagaacc 6060
aaaaattaaa ttatttcaac agtttaatcg accagttaat caacgtgtac acagacgcgt 6120
cggcaaaaaa cacgcagccc gacgtgttgg ctaaaattat t 6161
<210> 31
<211> 19
<212> DNA
<213> Artificial
<400> 31
cgagtccttt ccgatgtgt 19
<210> 32
<211> 21
<212> DNA
<213> Artificial
<400> 32
cgttttgtat ttgtcattgc c 21
<210> 33
<211> 16
<212> DNA
<213> Artificial
<400> 33
atggtgagca agggcg 16
<210> 34
<211> 22
<212> DNA
<213> Artificial
<400> 34
ttacttgtac agctcgtcca tg 22

Claims (9)

1.piggyBac转座子系统介导的真核生物CRISPR/Cas9敲除系统，其特征在于，是由基因元件递送系统和基因敲除系统两部分构成，两者分别为piggyBac转座子系统和CRISPR/Cas9系统。
2.根据权利要求1所述的敲除系统，其特征在于，所述piggyBac转座子系统包括：
两个转座臂，其核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示；
筛选标记Zeocin的抗性基因表达框，其核苷酸序列如SEQ ID NO.3所示。
3.根据权利要求1所述的敲除系统，其特征在于，所述CRISPR/Cas9系统包括两部分元件：
Hr3 CQ Enhancer-Hsp70启动子启动的spCas9蛋白表达框，其核苷酸序列如SEQ IDNO.4所示；
家蚕U6启动子启动的sgRNA表达框，其核苷酸序列如SEQ ID NO.5所示。
4.包含权利要求1～3中任一项所述系统的一种真核生物基因敲除基础载体，其特征在于，其核苷酸序列如SEQ ID NO.6所示，命名为pB-CRISPR。
5.权利要求4所述一种真核生物基因敲除基础载体的构建方法，其特征在于，具体方法如下：
(1)合成包含Zeocin抗性基因表达框的载体PUC57-IE2-Zeocin-Ser1PA；
(2)将IE2-Zeocin-Ser1PA表达框连接到piggyBac转座子基础载体piggyBacModify上，构建成中间载体pB-Modified{IE2-Zeocin-Ser1PA}；
(3)将hr3-hsp70-Cas9-sv40表达框从载体pUC57-hr3-hsp70-Cas9-sv40上扩增出来；然后用无缝克隆的方法连接到pB-Modified{IE2-Zeocin-Ser1PA}的AscI位点，构建成中间载体pB-Modified{IE2-Zeocin-Ser1PA}{hr3-hsp70-Cas9-SV40}；
(4)将U6-gRNA从载体pUC57-U6-gRNA扩增出来，用酶切连接的方法连接到载体pB-Modified{IE2-Zeocin-Ser1PA}{hr3-hsp70-Cas9-SV40}的AscI/NheI位点，构建成真核生物基因敲除基础载体pB-Modified{IE2-Zeocin-Ser1PA}{U6-gRNA}{hr3-hsp70-Cas9-SV40},命名为pB-CRISPR；
其中，载体PUC57-IE2-Zeocin-Ser1PA，核苷酸序列如SEQ ID NO.7所示；
piggyBac转座子基础载体piggyBacModify，核苷酸序列如SEQ ID NO.8所示；
中间载体pB-Modified{IE2-Zeocin-Ser1PA}，核苷酸序列如SEQ ID NO.9所示；
载体pUC57-hr3-hsp70-Cas9-sv40，核苷酸序列如SEQ ID NO.10所示；
中间载体pB-Modified{IE2-Zeocin-Ser1PA}{hr3-hsp70-Cas9-SV40}，核苷酸序列如SEQ IDNO.11所示；
载体pUC57-U6-gRNA，核苷酸序列如SEQ ID NO.12所示。
6.利用权利要求4所述基础载体构建的一种真核生物基因敲除载体，其特征在于，是将上述基础载体pB-CRISPR用核酸内切酶AarI消化作为骨架，利用引物组合成并且梯度退火成包含粘性末端的双链DNA，然后连接到所述骨架上即得，命名为pB-CRISPR-X。
7.根据权利要求6所述的载体，其特征在于，所述引物组包括：
正向引物X-F，5’-AAGT-NNNNNNNNNNNNNNNNNNNN-3’，如SEQ ID NO.13所示；
反向引物X-R，5-AAAC-NNNNNNNNNNNNNNNNNNNN-3’，如SEQ ID NO.14所示；
其中两条引物中的“N”为反向互补序列，“AAGT”和“AAAC”为粘性末端序列；
以及用于合成敲除真核生物蛋白编码基因的打靶位点的引物对：
EGFP-1F，5’-AAGTGGGCGAGGAGCTGTTCACCG-3’，如SEQ ID NO.15所示；
EGFP-1R，5’-AAACCGGTGAACAGCTCCTCGCCC-3’，如SEQ ID NO.16所示；
EGFP-2F，5’-AAGTGAGCTGGACGGCGACGTAAA-3’，如SEQ ID NO.17所示，
EGFP-2R，5’-AAACTTTACGTCGCCGTCCAGCTC-3’，如SEQ ID NO.18所示；
EGFP-3F，5’-AAGTGGCCACAAGTTCAGCGTGTC-3’，如SEQ ID NO.19所示；
EGFP-3R，5’-AAACGACACGCTGAACTTGTGGCC-3’，如SEQ ID NO.20所示；
NS-1F，5’-AAGTCGCTTCAAGGTGCACATGGA-3’，如SEQ ID NO.21所示；
NS-1R，5’-AAACTCCATGTGCACCTTGAAGCG-3’，如SEQ ID NO.22所示；
NS-2F，5’-AAGTGCGGAAGAACGCCTGCGGCT-3’，如SEQ ID NO.23所示
NS-2R，5’-AAACAGCCGCAGGCGTTCTTCCGC-3’，如SEQ ID NO.24所示。
8.根据权利要求7所述的载体，其特征在于，基于CRISPR/Cas9作用规律，所述打靶位点的核苷酸具有如下规律：5’-NNNNNNNNNNNNNNNNNNN-NGG-3’，具体的，所述打靶位点包括：3个敲除绿色荧光蛋白编码基因的打靶位点EGFP-1、EGFP-2、EGFP-3，和2个阴性对照打靶位点NS-1、NS-2，它们的核苷酸序列如下：
EGFP-1，5’-GGGCGAGGAGCTGTTCACCG-3’，如SEQ ID NO.25所示；
EGFP-2，5’-GAGCTGGACGGCGACGTAAA-3’，如SEQ ID NO.26所示；
EGFP-3，5’-GGCCACAAGTTCAGCGTGTC-3’，如SEQ ID NO.27所示；
NS-1，5’-CGCTTCAAGGTGCACATGGAGGG-3’，如SEQ ID NO.28所示；
NS-2，5’-GCGGAAGAACGCCTGCGGCTCGG-3’，如SEQ ID NO.29所示。
9.利用权利要求7所述载体构建的一种真核生物基因敲除细胞系，其特征在于，是将上述载体pB-CRISPR-X和核苷酸序列如SEQ ID NO.30所示的piggyBac transposon表达载体A3-helper按照摩尔比1：1转染真核细胞，将转染后的细胞用Zeocin筛选2个月得到。
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Application publication date: 20200814