CRIPSR/Cas9系统中重组sgRNA骨架载体的构建方法与流程

本发明涉及分子生物学和生物技术领域，特别涉及cripsr/cas9系统中重组sgrna骨架载体的构建方法。

背景技术：

cripsr/cas9系统，被称为第三代基因编辑技术，广泛应用于各类体内和体外体系的遗传学改造、转基因模式动物的构建，甚至基因治疗领域。在利用cripsr/cas9系统进行基因编辑操作时，需要选定一种sgrna的供给形式。以对哺乳动物细胞的基因编辑为例，可使用质粒或病毒为载体表达sgrna，也可以在体外将sgrna生产纯化出来后，直接转染进细胞实行功能。当然，以何种形式导入sgrna取决于实验需要以及细胞本身的特性，但在通常情况下直接将sgrna构建于表达载体之上更为经济和便捷。特别是可同时表达优化的cas9蛋白及sgrna的all-in-one载体的出现使得研究者们能够更加轻松的利用单个质粒实现基于cripsr/cas9系统的基因编辑[congl,ranfa,coxd,lins,barrettor,habibn,hsupd,wux,jiangw,marraffinila,zhangf.multiplexgenomeengineeringusingcrispr/cas-systems.science339,819-823(2013).]。

在sgrna骨架载体的构建过程中，最为重要的一步是质粒的线性化。几乎所有的sgrna骨架载体都会利用识别位点和切割位点不一致的iis型内切酶，使得sgrna寡核苷酸单链退火配对之后能够无缝插入。然而常用的iis型内切酶如bsmbi等酶切效果并不充分。为解决这一问题，一些载体，例如px330，其在使用过程中往往会在两个反向的iis型内切酶的酶切位点之间加入区分片段，从而可以通过胶回收的方法将线性化的载体和空质粒加以区分。尽管这一手段是有效且必要的，然而在实际操作中假阳性的产生依旧无法避免，尤其在胶回收过程中，对操作要求以及试剂配制要求较高，普通技术人员回收的线性化质粒中参杂了未充分酶切的载体，在与退火形成的具有粘性末端的sgrna短双链dna片段进行连接后，由于残留的少数空质粒转化效率要远远超过连接产物，因而转化大肠杆菌涂板后长出的大部分克隆都是含有空质粒的假阳性转化子，阳性率很低，往往在10%左右，需要挑较多的单克隆进行菌落pcr鉴定才能得到阳性克隆，费时费力。

技术实现要素：

针对上述问题，本发明提供了cripsr/cas9系统中重组sgrna骨架载体的构建方法，其能解决现有构建方法操作要求以及试剂配制要求较高受，普通技术人员获得的菌株阳性率低，阳性菌株挑选费时费力的技术问题。

其技术方案是这样的，cripsr/cas9系统中重组sgrna骨架载体的构建方法，其包括以下步骤：

（1）制备线性化的sgrna空质粒，采用的sgrna空质粒具有一对对称的iis型内切酶酶切位点，所述sgrna空质粒通过iis型内切酶进行酶切，胶回收酶切后的线性化的sgrna空质粒；

（2）制备区分片段，所述区分片段两端具有与iis型内切酶酶切后对应的末端片段；

（3）制备重组sgrna骨架载体，将步骤（1）胶回收的所述线性化的sgrna空质粒与步骤（2）制备的区分片段连接，制备重组sgrna骨架载体；

（4）挑选重组sgrna骨架载体，将步骤（3）的sgrna骨架载体转化大肠杆菌，涂抗性平板，形成单克隆，挑单克隆用含有所述iis型内切酶酶切位点接头的引物进行菌落pcr鉴定；

其特征在于：

所述步骤（2）的区分片段包含sacb基因及其开放阅读框（orf）、启动子和终止子，所述sacb基因的核苷酸序列如seqidno.1所示；

所述步骤（4）还包括将pcr鉴定阳性的菌分别涂lb抗性板和含5%~10%蔗糖的lb抗性板，若在含5%~10%蔗糖的lb抗性板上无法生长而在lb抗性板上形成菌苔，则表面该菌含有构建成功的重组sgrna骨架载体。

进一步的，步骤（1）制备线性化的sgrna空质粒，若已有sgrna空质粒，则用相应的iis型内切酶直接对其进行酶切，酶切后胶回收得到线性化的sgrna空质粒；或者根据已构建入某sgrna的质粒，设计引物对此质粒进行全质粒pcr，以获取含有iis型内切酶酶切接头的sgrna空质粒，随后同样用iis型内切酶对其进行酶切，胶回收酶切后的线性化的sgrna空质粒。

进一步的，所述步骤（2）制备区分片段，根据pk18mobsacb载体图谱和序列设计一对含有上述iis型内切酶酶切位点接头的引物，该引物的扩增子应包含sacb基因的orf及其启动子和终止子，用该引物对pk18mobsacb载体进行pcr，获得目的片段，胶回收pcr产物，用iis型内切酶对其进行酶切，柱纯化后得到区分片段。

进一步的，步骤（3）制备重组sgrna骨架载体，将步骤（1）胶回收的所述线性化的sgrna空质粒与步骤（2）制备的区分片段混匀，加入t4连接酶以及t4连接酶buffer，置于16°金属浴过夜连接。

进一步的，步骤（4）挑选重组sgrna骨架载体，将步骤（3）的连接产物转化大肠杆菌，涂抗性平板，培养12~20小时至平板上形成单克隆，挑单克隆并用步骤（2）中所述引物进行菌落pcr鉴定，pcr鉴定阳性的菌落转入摇瓶培养过夜，随后进行质粒抽提，用所述iis型内切酶酶切质粒，将酶切鉴定正确的克隆摇过夜，稀释后分别涂lb抗性板和含5%-10%蔗糖的lb抗性板，若在含5%-10%蔗糖的lb抗性板上无法生长而在lb抗性板上形成菌苔，则表明菌中含有构建成功的重组sgrna骨架载体。

本发明的构建方法将长达两千多bp的sacb基因相关序列构建在传统sgrna载体的两个iis型内切酶识别位点之中，一者可以通过电泳将空质粒与线性化质粒分开；再者，蔗糖致死基因sacb可使大肠杆菌将环境中的蔗糖分解为葡萄糖和果糖，并催化后者形成果聚糖，导致菌死亡，即使回收产物之中依然残留的空质粒，其大肠杆菌转化子也无法在含蔗糖的抗性板上形成单菌落，因为平板上长出来的大多是sacb基因已被iis型内切酶切除、5%-10%蔗糖无法致死、构入了sgrna片段的克隆，经实验表明，采用本方法构建sgrna骨架载体的阳性率可以达到95%以上。

附图说明

图1为应用例1中px330-p53为出发载体制备线性化的px330空质粒pcr引物设计示意图。

图2为应用例1的步骤（3）的制备重组sgrna骨架载体的示意图。

图3为应用例1的步骤（4）的bbsi酶切鉴定的电泳图，

marker：dl10000marker；lane1:bbsi酶切后质粒约8500，酶切下来的sacb基因相关片段约2500。

图4为应用例1的px330-sacb质粒图谱。

具体实施方式

实施例1

cripsr/cas9系统中重组sgrna骨架载体的构建方法，其包括以下步骤：

（1）制备线性化的sgrna空质粒

采用的sgrna空质粒具有一对对称的iis型内切酶酶切位点，目的是为了sgrna短片段能够无缝插入到启动子与scaffold之间；若已有sgrna空质粒，直接用相应的iis型内切酶酶切处理即可；酶切完成后胶回收获得线性化的sgrna空质粒。

若无sgrna空质粒，只有已构建入某sgrna的质粒，则设计引物对此质粒进行全质粒pcr，以消除质粒上的sgrna，并获得含有iis型内切酶酶切接头的sgrna空质粒；所用初始sgrna质粒不能有设计在引物之上的iis型内切酶识别位点；pcr完成后胶回收获得线性化的含有iis型内切酶酶切接头的sgrna空质粒；随后用相应的iis型内切酶酶切处理；酶切完成后柱纯化酶切产物，获得线性化的sgrna空质粒。

酶切体系：质粒1ug，10x内切酶buffer5ul，内切酶1ul(neb)，用双蒸水补至50ul。

酶切时间：37°1小时。

胶回收：将酶切产物全部点入1%琼脂糖胶，120v电泳半小时。

全质粒pcr：

pcr体系：5xq5reactionbuffer5ul，10mmdntps0.5ul，10um上游引物1.25ul，10um下游引物1.25ul，初始sgrna质粒200ng，q5高保真酶0.5ul，用双蒸水补至50ul；

pcr程序：预变性98°c30s；扩增阶段：98°30s，50-72°c10-30s，

72°c20-30s/kb；最终延伸72°c2min。

（2）制备区分片段

区分片段包含sacb基因及其开放阅读框（orf）、启动子和终止子，sacb基因的核苷酸序列如seqidno.1所示，区分片段两端具有iis型内切酶酶切后对应的末端片段；具体的，可以以pk18mobsacb质粒为供体对sacb基因及其启动子和终止子进行亚克隆；sacb基因相关片段也可以从其他来源处取得，如直接制备；引物的设计要确保获得的片段两端含有相应的iis型内切酶酶切接头；pcr完成后胶回收获得含有iis型内切酶酶切接头的sacb基因相关片段；随后用相应的iis型内切酶酶切处理；酶切完成后柱纯化酶切产物，获得区分片段。

pcr体系、pcr程序同上以及酶切体系、酶切时间参考步骤（1）。

（3）制备重组sgrna骨架载体

将前两步得到的酶切后线性化的sgrna空质粒与柱纯化的区分片段进行连接反应，获得重组sgrna骨架载体。

反应体系：1ult4dna连接酶（neb），2ul10xt4连接酶buffer，线性化的sgrna空质粒与区分片段的摩尔浓度1比5，双蒸水补至20ul；反应体系置于16°金属浴过夜。

（4）挑选重组sgrna骨架载体

将步骤（3）的sgrna骨架载体转化大肠杆菌，涂抗性（载体自身抗性基因）平板，培养12~20小时至平板上形成单克隆，挑单克隆用步骤2中引物进行菌落pcr鉴定；pcr鉴定阳性的菌落转入摇瓶培养过夜，随后进行质粒抽提，用上述iis型内切酶酶切质粒，酶切体系同上；酶切产物点于2%琼脂糖胶中，120v电泳1小时；将酶切鉴定正确的克隆摇过夜，稀释一百倍分别涂100ul至lb抗性板（与后者区别仅在于不含蔗糖）和含5%-10%蔗糖的lb抗性板；若在含5%-10%蔗糖的lb抗性板上无法生长而在lb抗性板上形成菌苔，则表明菌中含有构建成功的重组sgrna骨架载体。

菌落pcr鉴定：

pcr体系：预染taqmix5ul，10um上游引物0.5ul，10um下游引物0.5ul，适量菌体，4ul双蒸水；pcr程序：预变性95°c3min。

扩增阶段：95°c30s，52-58°c30s，72°c1min/kb；最终延伸72°c2min。

完成后将菌落pcr产物分别点入1%琼脂糖胶，120v电泳半小时后看胶。

应用例1

（1）制备线性化的px330sgrna空质粒：

px330是公开的最常用的哺乳动物细胞sgrna载体之一（addgene#42230），其核苷酸序列如seqidno.3所示，其空载体含有一对对称的bbsi酶切位点，bbsi酶切后的载体具有粘性末端，用以加入sgrna短片段双链。若已有px330空质粒，直接用bbsi酶切处理，酶切完成后胶回收获得线性化的px330空质粒，其核苷酸序列如seqidno.4所示。

酶切体系：px330质粒1ug，10x内切酶buffer5ul、bbsi1ul(neb)，用双蒸水补至50ul。

酶切时间：37°1小时。

胶回收：将酶切产物全部点入1%琼脂糖胶，120v电泳半小时。

若无px330空质粒，以已插入sgrna的如px330-p53（addgene#59910）为出发质粒，其核苷酸序列如seqidno.5所示，设计引物对此质粒进行全质粒pcr，以消除质粒上靶向p53的sgrna，并获得含有bbsi酶切位点接头的sgrna空质粒；pcr完成后胶回收获得线性化的含bbsi酶切接头的px330空质粒；随后用bbsi酶切处理；酶切完成后柱纯化酶切产物，获得线性化的px330空质粒。

引物设计示意图见图1。

px330上游引物：gagaagacgggttttagagctagaaatagcaagtt

px330下游引物：gagaagacggggtgtttcgtcctttccacaagata

（下划线为bbsi识别位点）

该引物扩增子（约8500bp）经过bbsi酶切之后所得线性化质粒与px330空载体经bbsi酶切后的线性化产物完全一致。

pcr体系：5xq5reactionbuffer5ul，10mmdntps0.5ul，10um上游引物1.25ul，10um下游引物1.25ul，px330p53质粒200ng，q5高保真酶0.5ul，用双蒸水补至50ul。

pcr程序：预变性98°c30s；扩增阶段：98°c30s，62°c20s，72°c4min；最终延伸72°c2min。

酶切体系、时间以及胶回收参数、时间同上。

（2）制备区分片段

根据pk18mobsacb全质粒序列设计含bbsi酶切接头的上下游引物，对pk18mobsacb上sacb基因及其启动子和终止子相关片段进行pcr扩增，扩增片的核苷酸序列如seqidno.2；pcr完成后胶回收获得含有bbsi酶切位点接头的sacb基因相关片段；随后用bbsi酶切处理；酶切完成后柱纯化酶切产物，。

sacb上游引物：ggcaccgggtcttcgcgatgcctgcttgccgaat

sacb下游引物：ggaaacaggtcttcccagacgaacgaagagcgattgag

（下划线为bbsi识别位点）

pcr体系及pcr程序与步骤1基本相同，不同之处仅在于延伸时间缩短至60s。

酶切体系和酶切时间与步骤1相同。

（3）制备重组sgrna骨架载体

如图2所示，将前两步得到的经bbsi线性化的px330质粒与sacb基因酶切纯化片段进行连接反应。

反应体系：1ult4dna连接酶（neb），2ul10xt4连接酶buffer，线性化的px330质粒与sacb基因酶切纯化片段的摩尔浓度1比5，双蒸水补至20ul。

反应体系置于16°金属浴过夜连接。

（4）挑选重组sgrna骨架载体

将步骤（3）连接获得的sgrna骨架载体转化大肠杆菌，涂氨苄抗性lb平板（px330质粒自身具有氨苄抗性基因），置于37°c培养箱中培养，12小时后平板上形成单克隆，用步骤2中引物进行菌落pcr鉴定；pcr鉴定阳性的菌落（有2400bp特异条带）转入摇瓶培养过夜，随后进行质粒抽提，用bbsi酶切质粒，酶切产物点于2%琼脂糖胶中，120v电泳1小时后看胶；将酶切鉴定正确（有且只有2400bp以及8500bp两条带，其电泳图如图3所示）的克隆摇过夜，稀释一百倍分别涂100ul至lb氨苄板和含5%蔗糖的lb氨苄板，若在含5%蔗糖的lb氨苄板上无法生长而在lb氨苄板上形成菌苔，则px330-sacb骨架载体构建成功。px330-sacb质粒图谱如图4所示，利用该质粒及对应的蔗糖致死筛选进行传统的sgrna构建，可使阳性率达到百分之九十以上。

菌落pcr鉴定：

pcr体系：预染taqmix5ul，10um上游引物0.5ul，10um下游引物0.5ul，适量菌体，4ul双蒸水。

pcr程序：预变性95°c3min；扩增阶段：95°c30s，55°c30s，72°c3min；最终延伸72°c2min。

完成后将菌落pcr产物分别点入1%琼脂糖胶，120v电泳半小时后看胶。

酶切体系和酶切时间与步骤1相同。

序列表

<110>江南大学

<120>cripsr/cas9系统中重组sgrna骨架载体的构建方法

<130>2017

<160>5

<170>siposequencelisting1.0

<210>1

<211>1422

<212>dna

<213>人工序列(artificialsequence)

<400>1

atgaacatcaaaaagtttgcaaaacaagcaacagtattaacctttactaccgcactgctg60

gcaggaggcgcaactcaagcgtttgcgaaagaaacgaaccaaaagccatataaggaaaca120

tacggcatttcccatattacacgccatgatatgctgcaaatccctgaacagcaaaaaaat180

gaaaaatatcaagtttctgaatttgattcgtccacaattaaaaatatctcttctgcaaaa240

ggcctggacgtttgggacagctggccattacaaaacgctgacggcactgtcgcaaactat300

cacggctaccacatcgtctttgcattagccggagatcctaaaaatgcggatgacacatcg360

atttacatgttctatcaaaaagtcggcgaaacttctattgacagctggaaaaacgctggc420

cgcgtctttaaagacagcgacaaattcgatgcaaatgattctatcctaaaagaccaaaca480

caagaatggtcaggttcagccacatttacatctgacggaaaaatccgtttattctacact540

gatttctccggtaaacattacggcaaacaaacactgacaactgcacaagttaacgtatca600

gcatcagacagctctttgaacatcaacggtgtagaggattataaatcaatctttgacggt660

gacggaaaaacgtatcaaaatgtacagcagttcatcgatgaaggcaactacagctcaggc720

gacaaccatacgctgagagatcctcactacgtagaagataaaggccacaaatacttagta780

tttgaagcaaacactggaactgaagatggctaccaaggcgaagaatctttatttaacaaa840

gcatactatggcaaaagcacatcattcttccgtcaagaaagtcaaaaacttctgcaaagc900

gataaaaaacgcacggctgagttagcaaacggcgctctcggtatgattgagctaaacgat960

gattacacactgaaaaaagtgatgaaaccgctgattgcatctaacacagtaacagatgaa1020

attgaacgcgcgaacgtctttaaaatgaacggcaaatggtacctgttcactgactcccgc1080

ggatcaaaaatgacgattgacggcattacgtctaacgatatttacatgcttggttatgtt1140

tctaattctttaactggcccatacaagccgctgaacaaaactggccttgtgttaaaaatg1200

gatcttgatcctaacgatgtaacctttacttactcacacttcgctgtacctcaagcgaaa1260

ggaaacaatgtcgtgattacaagctatatgacaaacagaggattctacgcagacaaacaa1320

tcaacgtttgcgccgagcttcctgctgaacatcaaaggcaagaaaacatctgttgtcaaa1380

gacagcatccttgaacaaggacaattaacagttaacaaataa1422

<210>2

<211>2403

<212>dna

<213>人工序列(artificialsequence)

<400>2

gcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgact60

gtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattg120

ctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctc180

ccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactct240

ggggttcgctagaggatcgatcctttttaacccatcacatatacctgccgttcactatta300

tttagtgaaatgagatattatgatattttctgaattgtgattaaaaaggcaactttatgc360

ccatgcaacagaaactataaaaaatacagagaatgaaaagaaacagatagattttttagt420

tctttaggcccgtagtctgcaaatccttttatgattttctatcaaacaaaagaggaaaat480

agaccagttgcaatccaaacgagagtctaatagaatgaggtcgaaaagtaaatcgcgcgg540

gtttgttactgataaagcaggcaagacctaaaatgtgtaaagggcaaagtgtatactttg600

gcgtcaccccttacatattttaggtctttttttattgtgcgtaactaacttgccatcttc660

aaacaggagggctggaagaagcagaccgctaacacagtacataaaaaaggagacatgaac720

gatgaacatcaaaaagtttgcaaaacaagcaacagtattaacctttactaccgcactgct780

ggcaggaggcgcaactcaagcgtttgcgaaagaaacgaaccaaaagccatataaggaaac840

atacggcatttcccatattacacgccatgatatgctgcaaatccctgaacagcaaaaaaa900

tgaaaaatatcaagtttctgaatttgattcgtccacaattaaaaatatctcttctgcaaa960

aggcctggacgtttgggacagctggccattacaaaacgctgacggcactgtcgcaaacta1020

tcacggctaccacatcgtctttgcattagccggagatcctaaaaatgcggatgacacatc1080

gatttacatgttctatcaaaaagtcggcgaaacttctattgacagctggaaaaacgctgg1140

ccgcgtctttaaagacagcgacaaattcgatgcaaatgattctatcctaaaagaccaaac1200

acaagaatggtcaggttcagccacatttacatctgacggaaaaatccgtttattctacac1260

tgatttctccggtaaacattacggcaaacaaacactgacaactgcacaagttaacgtatc1320

agcatcagacagctctttgaacatcaacggtgtagaggattataaatcaatctttgacgg1380

tgacggaaaaacgtatcaaaatgtacagcagttcatcgatgaaggcaactacagctcagg1440

cgacaaccatacgctgagagatcctcactacgtagaagataaaggccacaaatacttagt1500

atttgaagcaaacactggaactgaagatggctaccaaggcgaagaatctttatttaacaa1560

agcatactatggcaaaagcacatcattcttccgtcaagaaagtcaaaaacttctgcaaag1620

cgataaaaaacgcacggctgagttagcaaacggcgctctcggtatgattgagctaaacga1680

tgattacacactgaaaaaagtgatgaaaccgctgattgcatctaacacagtaacagatga1740

aattgaacgcgcgaacgtctttaaaatgaacggcaaatggtacctgttcactgactcccg1800

cggatcaaaaatgacgattgacggcattacgtctaacgatatttacatgcttggttatgt1860

ttctaattctttaactggcccatacaagccgctgaacaaaactggccttgtgttaaaaat1920

ggatcttgatcctaacgatgtaacctttacttactcacacttcgctgtacctcaagcgaa1980

aggaaacaatgtcgtgattacaagctatatgacaaacagaggattctacgcagacaaaca2040

atcaacgtttgcgccgagcttcctgctgaacatcaaaggcaagaaaacatctgttgtcaa2100

agacagcatccttgaacaaggacaattaacagttaacaaataaaaacgcaaaagaaaatg2160

ccgatggtaccgagcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttc2220

gattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccctc2280

gcggacgtgctcatagtccacgacgcccgtgattttgtagccctggccgacggccagcag2340

gtaggccgacaggctcatgccggccgccgccgccttttcctcaatcgctcttcgttcgtc2400

tgg2403

<210>3

<211>8506

<212>dna

<213>人工序列(artificialsequence)

<400>3

gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagag60

ataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtaga120

aagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcat180

atgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaagga240

cgaaacaccgggtcttcgagaagacctgttttagagctagaaatagcaagttaaaataag300

gctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct360

agaaatagcaagttaaaataaggctagtccgtttttagcgcgtgcgccaattctgcagac420

aaatggctctagaggtacccgttacataacttacggtaaatggcccgcctggctgaccgc480

ccaacgacccccgcccattgacgtcaatagtaacgccaatagggactttccattgacgtc540

aatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgc600

caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagt660

acatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctatta720

ccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccac780

ccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcgggggggg840

ggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcgga900

gaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggc960

ggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgacgc1020

tgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactg1080

accgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattag1140

ctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattac1200

ctggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatggactata1260

aggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataaga1320

tggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagccgacaagaagt1380

acagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagt1440

acaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaaga1500

agaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctga1560

agagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagaga1620

tcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtcct1680

tcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacg1740

aggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggaca1800

gcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttcc1860

ggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgt1920

tcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcg1980

gcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatc2040

tgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctga2100

gcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgc2160

agctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgacc2220

agtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgaca2280

tcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagat2340

acgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctg2400

agaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacg2460

gcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacg2520

gcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggacct2580

tcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggc2640

ggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctga2700

ccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctgga2760

tgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagg2820

gcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacg2880

agaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctga2940

ccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcaga3000

aaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctga3060

aagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaag3120

atcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaagg3180

acttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacac3240

tgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacg3300

acaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccgga3360

agctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagt3420

ccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgaccttta3480

aagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattg3540

ccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtgg3600

acgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggcca3660

gagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcg3720

aagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacaccc3780

agctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtgg3840

accaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcaga3900

gctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggg3960

gcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggc4020

agctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgaga4080

gaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaaccc4140

ggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacg4200

agaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccg4260

atttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgccc4320

acgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctgg4380

aaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaaga4440

gcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaact4500

ttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgaga4560

caaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcgga4620

aagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggct4680

tcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaagg4740

actgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctgg4800

tggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctgg4860

ggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagcca4920

agggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcg4980

agctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacg5040

aactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagc5100

tgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcact5160

acctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacg5220

ctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagc5280

aggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttca5340

agtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacg5400

ccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagc5460

tgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagt5520

aagaattcctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgt5580

tgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttc5640

ctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctgggggg5700

tggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctgggga5760

gcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgct5820

cactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagt5880

gagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgca5940

tctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggc6000

gcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcc6060

ctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccc6120

cgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctc6180

gaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacg6240

gtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaact6300

ggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatt6360

tcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaa6420

atattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatag6480

ttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctc6540

ccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttt6600

tcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttatag6660

gttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtg6720

cgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaga6780

caataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacat6840

ttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcaccca6900

gaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatc6960

gaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttcca7020

atgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccggg7080

caagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcacca7140

gtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccata7200

accatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggag7260

ctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccg7320

gagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggca7380

acaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaatta7440

atagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggct7500

ggctggtttattgctgataaatctggagccggtgagcgtggaagccgcggtatcattgca7560

gcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcag7620

gcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcat7680

tggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttt7740

taatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaa7800

cgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttga7860

gatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcg7920

gtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagc7980

agagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaag8040

aactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcc8100

agtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcg8160

cagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctac8220

accgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggaga8280

aaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagctt8340

ccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgag8400

cgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcg8460

gcctttttacggttcctggccttttgctggccttttgctcacatgt8506

<210>4

<211>8508

<212>dna

<213>人工序列(artificialsequence)

<400>4

gagaagacgggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaac60

ttgaaaaagtggcaccgagtcggtgcttttttgttttagagctagaaatagcaagttaaa120

ataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagaggta180

cccgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgccca240

ttgacgtcaatagtaacgccaatagggactttccattgacgtcaatgggtggagtattta300

cggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctatt360

gacgtcaatgacggtaaatggcccgcctggcattgtgcccagtacatgaccttatgggac420

tttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagc480

cccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtattta540

tttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgcca600

ggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagcc660

aatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccc720

tataaaaagcgaagcgcgcggcgggcgggagtcgctgcgacgctgccttcgccccgtgcc780

ccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccaca840

ggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagg900

gtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctga960

aatcactttttttcaggttggaccggtgccaccatggactataaggaccacgacggagac1020

tacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaag1080

cggaaggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggac1140

atcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaag1200

aaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagcc1260

ctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaaga1320

agatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatg1380

gccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggat1440

aagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgag1500

aagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgac1560

ctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatc1620

gagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcag1680

acctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggcc1740

atcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgccc1800

ggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgaccccc1860

aacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacc1920

tacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgttt1980

ctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacacc2040

gagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccag2100

gacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagatt2160

ttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaa2220

gagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctc2280

gtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatc2340

ccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttac2400

ccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctac2460

tacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgag2520

gaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagc2580

ttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaag2640

cacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtg2700

accgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggac2760

ctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaag2820

aaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctcc2880

ctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgag2940

gaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagag3000

atgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcag3060

ctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatc3120

cgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaac3180

agaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaa3240

gcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagc3300

cccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtg3360

atgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacc3420

cagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagag3480

ctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaag3540

ctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatc3600

aaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgac3660

tccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtg3720

ccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaag3780

ctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaa3840

ctggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcac3900

gtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatc3960

cgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttc4020

cagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaac4080

gccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtac4140

ggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggc4200

aaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagatt4260

accctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggg4320

gagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccc4380

caagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatc4440

ctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaag4500

tacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaa4560

aagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaa4620

agaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtg4680

aaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccgg4740

aagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctcc4800

aaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgag4860

gataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatc4920

gagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtg4980

ctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatc5040

cacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccacc5100

atcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccag5160

agcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgacaaaagg5220

ccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaagaattcctagagctc5280

gctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccg5340

tgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaa5400

ttgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggaca5460

gcaagggggaggattgggaagagaatagcaggcatgctggggagcggccgcaggaacccc5520

tagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgac5580

caaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgca5640

gctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcac5700

accgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcggg5760

tgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctccttt5820

cgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcg5880

ggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttga5940

tttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgac6000

gttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccc6060

tatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaa6120

aaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaat6180

tttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgaca6240

cccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacag6300

acaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaa6360

acgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataat6420

aatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttg6480

tttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaat6540

gcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttat6600

tcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagt6660

aaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacag6720

cggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaa6780

agttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcg6840

ccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatct6900

tacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacac6960

tgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgca7020

caacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccat7080

accaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaact7140

attaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggc7200

ggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctga7260

taaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatgg7320

taagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacg7380

aaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagacca7440

agtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatcta7500

ggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttcca7560

ctgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcg7620

cgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgga7680

tcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaa7740

tactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcc7800

tacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtg7860

tcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaac7920

ggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacct7980

acagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatcc8040

ggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctg8100

gtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatg8160

ctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcct8220

ggccttttgctggccttttgctcacatgtgagggcctatttcccatgattccttcatatt8280

tgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaa8340

agatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttt8400

taaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatt8460

tcttggctttatatatcttgtggaaaggacgaaacaccccgtcttctc8508

<210>5

<211>8487

<212>dna

<213>人工序列(artificialsequence)

<400>5

gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagag60

ataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtaga120

aagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcat180

atgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaagga240

cgaaacaccgcctcgagctccctctgagccgttttagagctagaaatagcaagttaaaat300

aaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttaga360

gctagaaatagcaagttaaaataaggctagtccgtttttagcgcgtgcgccaattctgca420

gacaaatggctctagaggtacccgttacataacttacggtaaatggcccgcctggctgac480

cgcccaacgacccccgcccattgacgtcaatagtaacgccaatagggactttccattgac540

gtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcata600

tgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgccc660

agtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgcta720

ttaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccc780

cacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggg840

gggggggggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagcca900

atcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccct960

ataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgcccc1020

gctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacagg1080

tgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggt1140

ttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaa1200

tcactttttttcaggttggaccggtgccaccatggactataaggaccacgacggagacta1260

caaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcg1320

gaaggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacat1380

cggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaa1440

attcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccct1500

gctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaag1560

atacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggc1620

caaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataa1680

gaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaa1740

gtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacct1800

gcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcga1860

gggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagac1920

ctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccat1980

cctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccgg2040

cgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaa2100

cttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacaccta2160

cgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttct2220

ggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccga2280

gatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccagga2340

cctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagatttt2400

cttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaaga2460

gttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgt2520

gaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatccc2580

ccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttaccc2640

attcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctacta2700

cgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgagga2760

aaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagctt2820

catcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagca2880

cagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgac2940

cgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacct3000

gctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaa3060

aatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccct3120

gggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgagga3180

aaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagat3240

gatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagct3300

gaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccg3360

ggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacag3420

aaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagc3480

ccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccc3540

cgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgat3600

gggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccaccca3660

gaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagct3720

gggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagct3780

gtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaa3840

ccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactc3900

catcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgcc3960

ctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagct4020

gattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaact4080

ggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgt4140

ggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccg4200

ggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttcca4260

gttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgc4320

cgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacgg4380

cgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaa4440

ggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattac4500

cctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccgggga4560

gatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgcccca4620

agtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcct4680

gcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagta4740

cggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaa4800

gggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaag4860

aagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaa4920

aaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaa4980

gagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaa5040

atatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgagga5100

taatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcga5160

gcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgct5220

gtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatcca5280

cctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccat5340

cgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagag5400

catcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgacaaaaggcc5460

ggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaagaattcctagagctcgc5520

tgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtg5580

ccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaatt5640

gcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagc5700

aagggggaggattgggaagagaatagcaggcatgctggggagcggccgcaggaaccccta5760

gtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgacca5820

aaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagc5880

tgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacac5940

cgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtg6000

tggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcg6060

ctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcggg6120

ggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatt6180

tgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgt6240

tggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaactcta6300

tctcgggctattcttttgatttataagggattttgccgatttcggtctattggttaaaaa6360

atgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattt6420

tatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacc6480

cgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagac6540

aagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaac6600

gcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataa6660

tggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtt6720

tatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgc6780

ttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattc6840

ccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaa6900

aagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcg6960

gtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaag7020

ttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgcc7080

gcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatctta7140

cggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactg7200

cggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcaca7260

acatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccatac7320

caaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactat7380

taactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcgg7440

ataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgata7500

aatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggta7560

agccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaa7620

atagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaag7680

tttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctagg7740

tgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccact7800

gagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcg7860

taatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatc7920

aagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaata7980

ctgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgccta8040

catacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtc8100

ttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacgg8160

ggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctac8220

agcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccgg8280

taagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggt8340

atctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgct8400

cgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctgg8460

ccttttgctggccttttgctcacatgt8487