CN108203714A - 一种棉花基因的编辑方法 - Google Patents

一种棉花基因的编辑方法 Download PDF

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CN108203714A
CN108203714A CN201611188402.4A CN201611188402A CN108203714A CN 108203714 A CN108203714 A CN 108203714A CN 201611188402 A CN201611188402 A CN 201611188402A CN 108203714 A CN108203714 A CN 108203714A
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金双侠
张献龙
张军
王鹏程
涂礼莉
梁思佳
孙琳
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Huazhong Agricultural University
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Abstract

本发明属于植物基因工程技术领域，具体涉及一种棉花基因的编辑方法。公开了用于棉花基因组编辑的CRISPR‑Cas9系统及其构建方法。本发明克隆得到两个陆地棉U6启动子pGhU6‑7和pGhU6‑9，并突变了Bsa Ⅰ酶切位点。利用优化后的启动子构建了在棉花中具有编辑能力的CRISPR‑Cas9载体的两个载体pRGEB32‑GhU6.7‑NPT Ⅱ、pRGEB32‑GhU6.9‑NPT Ⅱ。选取1个棉花内源基因GhCLA为目标基因，设计四个靶向该基因的sgRNA，引导Cas9蛋白在GhCLA的特异位点进行切割。通过棉花的白化表型、酶切图、测序验证，表明pGhU6‑7I和pGhU6‑9I驱动的CRISPR‑Cas9基因编辑系统在棉花内具有编辑能力。

Description

一种棉花基因的编辑方法
技术领域
本发明涉及基因工程技术领域，具体涉及一种陆地棉基因组的编辑方法。本发明涉及构建陆地棉的两个高效转化载体，利用这两个载体在陆地棉基因组中进行编辑的应用。
背景技术
基因突变是研究基因功能的常用方法之一，从EMS突变，辐射诱变，T-DNA插入，转座子插入等随机突变方法，到锌指核酸酶(ZFN)与类转录激活因子效应物核酸酶(TALEN)目标基因定点修饰技术，是生物技术领域的一次突破。CRISPR是指一段具有成簇的规律间隔的短回文重复序列，通过CRISPR系统将目标序列切断后，生物个体会启动自身的修复机制，包括非同源重组修复和同源重组修复，在修复后产生插入、缺失等突变，导致目标基因功能沉默。CRISPR系统可以靶向任何含5’-N20-NGG-3’(N＝A，T，G，C)或5’-CCN-N20-3’的DNA序列，其中NGG为Cas9识别所需的PAM，并且在PAM前3bp左右的位置精确的切割靶DNA双链。CRISPR/Cas9技术使基因定点突变变得十分简单快捷、方便且具有较高的突变效率，使其成为了科研工作者首选的基因定点修饰的技术之一，越来越广泛地应用于动植物研究中。
谢卡斌等(Xie,Minkenberg and Yang 2015)在水稻中已证实tRNA-gRNA重复序列在tRNA通过内源的RNaseP和RNaseZ修饰后释放多个gRNA。这一研究说明在植物中可以实现多基因多位点打靶，大大提高了CRISPR/Cas9系统用于基因组编辑的效率。
不同的物种，U6启动子的启动转录的效率不同，同一个物种中的U6转录效果也不一样，而棉花中含有多个GhU6启动子，所以克隆适用于棉花基因编辑的GhU6启动子对CRISPR-Cas9系统高效是非常重要的。
目前，已有许多关于CRISPR/Cas9系统应用在不同生物中的报道，但棉花作为重要的纤维作物，至今尚无报道。近几十年我国在转基因抗虫棉培育、现代分子育种体系的建立和棉花品种高效应用保障技术等方面取得成就的同时，也面临着许多新的难题。包括优异的种植资源匮乏；棉花枯、黄萎病逐年加重；抗虫棉的普及引起次生害虫的危害增加；盐渍化田地逐年增加等。因此，利用CRISPR基因编辑技术深入探讨基因功能，了解棉花抗逆、抗病、抗虫分子机理，对为棉花抗性育种提供理论依据和候选基因具有重要的意义。
发明内容
本发明的目的在于构建适合于陆地棉CRISPR/Cas9基因的编辑系统。克隆了棉花内源启动子pGhU6，所述的启动子包括pGhU6-7、pGhU6-9，并进行了BsaⅠ酶切位点优化。基于pRGEB32载体(Xie et al.2015)，构建了适合陆地棉转化并具有高效编辑的载体，所述的包括pRGEB32-GhU6.7-NPTⅡ和pRGEB32-GhU6.9-NPTⅡ。
本发明技术方案如下所述：
一种陆地棉高效转化载体pRGEB32-GhU6.7-NPTⅡ，该载体通过下列步骤制备获得：
(1)构建陆地棉内源RNA聚合酶三型U6启动子pGhU6-7I，该启动子的核苷酸序列如SEQ ID NO：1所示，具体地，该启动子通过以下步骤制备获得：
1)通过与拟南芥U6-26snRNA基因同源比对得到多个陆地棉U6启动子，用Primer5软件设计引物；
2)以陆地棉YZ1(Jin et al.2006)基因组DNA为模板，PCR扩增，然后用pGEM-TEasy克隆测序验证，将检测正确的启动子序列命名为pGhU6-7；
3)利用重叠延伸PCR(Urban,Neukirchen and Jaeger 1997)将步骤2)中pGhU6-7第319个碱基C人工突变为碱基G，删除pGhU6-7启动子所含有的BsaⅠ位点，得到pGhU6-7I启动子(其核苷酸序列如SEQ ID NO：1所示)；
(2)利用SbfⅠ、HindⅢ对pRGEB32进行酶切，与pGhU6-7I启动子进行连接，得到如SEQ ID NO：6所示的中间载体pRGEB32-GhU6.7；
(3)利用Primer5软件对pHellsgate4载体上的真核NPTⅡ序列(其核苷酸序列如SEQ ID NO：10所示)设计引物，然后以pHellsgate4质粒为模板进行PCR扩增；
(4)将pRGEB32载体(Xie et al.2015)用XmaⅠ、PspxⅠ酶切，然后将上述步骤(3)中的NPTⅡ序列(其核苷酸序列如SEQ ID NO：10所示)引入步骤(2)中的中间载体pRGEB32-GhU6.7，得到如SEQ ID NO：8所示的陆地棉高效转化载体pRGEB32-GhU6.7-NPTⅡ。
申请人同时制备了一种陆地棉高效转化载体pRGEB32-GhU6.9-NPTⅡ，该载体的制备步骤如下所述：
(1)构建一种陆地棉内源RNA聚合酶三型U6启动子pGhU6-9I，其核苷酸序列如SEQID NO：3所示，该启动子通过以下步骤制备获得：
1)通过与拟南芥U6-26snRNA基因同源比对得到多个陆地棉U6启动子，用Primer5软件进行设计引物；
2)以陆地棉YZ1(Jin et al.2006)基因组DNA为模板，PCR扩增，然后用pGEM-TEasy克隆测序验证，得到启动子pGhU6-9，所述的pGhU6-9启动子的核苷酸序列如SEQ IDNO：4所示；
3)利用重叠延伸PCR(Urban et al.1997)将步骤2)中pGhU6-9启动子第616个碱基C人工突变为碱基G，删除pGhU6-9启动子所含有的的BsaⅠ位点，得到启动子pGhU6-9I(核苷酸序列如SEQ ID NO：3所示)；
(2)利用SbfⅠ、HindⅢ对pRGEB32进行酶切，与启动子pGhU6-9I连接，得到如SEQ IDNO：7所示的中间载体pRGEB32-GhU6.9；
(3)利用Primer5软件对pHellsgate4载体上的真核NPTⅡ序列(其核苷酸序列如SEQ ID NO：10所示)设计引物，然后以pHellsgate4质粒为模板进行PCR扩增；
(4)将pRGEB32载体(Xie et al.2015)用XmaⅠ、PspxⅠ酶切，然后将上述(3)中的NPTⅡ序列(其核苷酸序列如SEQ ID NO：10所示)引入步骤(2)中的中间载体pRGEB32-GhU6.9中得到如SEQ ID NO：9所示核苷酸序列的陆地棉高效转化载体pRGEB32-GhU6.9-NPTⅡ。
附图说明
序列表SEQ ID NO：1是pGhU6-7I启动子的核苷酸序列。序列长度为988bp。
序列表SEQ ID NO：2是pGhU6-7启动子的核苷酸序列。序列长度为988bp。
序列表SEQ ID NO：3是pGhU6-9I启动子的核苷酸序列。序列长度为997bp。
序列表SEQ ID NO：4是pGhU6-9启动子的核苷酸序列。序列长度为997bp。
序列表SEQ ID NO：5是获得的起始载体pRGEB32的核苷酸序列。序列长度为15905bp。
序列表SEQ ID NO：6是中间载体pRGEB32-GhU6.7的核苷酸序列。序列长度为16516bp。
序列表SEQ ID NO：7是中间载体pRGEB32-GhU6.9的核苷酸序列。序列长度为16525bp。
序列表SEQ ID NO：8是高效转化载体pRGEB32-GhU6.7-NPTⅡ的核苷酸序列。序列长度为16241bp。
序列表SEQ ID NO：9是高效转化载体pRGEB32-GhU6.9-NPTⅡ的核苷酸序列。序列长度为16250bp。
序列表SEQ ID NO：10是NPTⅡ的核苷酸序列。序列长度为795bp。
图1：是本发明的起始载体pRGEB32改造路线图。
图2：是本发明的载体pRGEB32-GhU6.9-NPTⅡ与sgRNA构建流程图。
图3：是本发明的Cas9表达载体pRGEB32-GhU6.7-NPTⅡ的构建图。
图4：是本发明的Cas9表达载体pRGEB32-GhU6.9-NPTⅡ的构建图。
图5：是本发明的克隆pGhU6-7、pGhU6-9启动子的电泳图；附图标记说明：泳道1为5K的marker，泳道2、3为pGhU6-7启动子，泳道4、5为pGhU6-7启动子。
图6：是本发明的分段扩增pGhU6-7启动子、pGhU6-9启动子的电泳图；附图标记说明：泳道1为5K的marker，泳道2、3为pGhU6-9分段片段，泳道4、5为pGhU6-7分段片段；
图7：是本发明拼接后pGhU6-7I、pGhU6-9I的电泳图；附图标记说明：泳道1为5K的marker，泳道2、3为pGhU6-7I，泳道4、5为pGhU6-7I；
图8：是本发明的第一次PCR电泳图，两个片段的分别扩增。附图标记说明：泳道1为5K的marker，泳道2为第一个片段，泳道3为第二个片段。
图9：是本发明的第二次PCR电泳图，利用重叠延伸PCR将第一次PCR的两个片段拼接。
图10：是本发明的7NC1、9NC1对GhCLA基因编辑后酶切图。
图11：是本发明的7NC2、9NC2对GhCLA基因编辑后酶切图。
图12：是本发明的GhCLA基因编辑后植株白化表型图。
图13：是本发明的GhCLA基因sgRNA1、sgRNA2、sgRNA3、sgRNA4编辑序列图。
图14：是本发明的GhCLA基因sgRNA2、sgRNA4之间大片段编辑序列图。
具体实施方式
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
实施例1：pGhU6-7启动子、pGhU6-9启动子的克隆与优化
1.pGhU6启动子的Blast
通过与拟南芥U6-26snRNA基因同源比对，用Primer5软件进行设计引物。
2.启动子的克隆
以陆地棉YZ1(Jin et al.2006)基因组DNA为模板，利用以下PCR体系、程序进行扩增，得到两个pGhU6，最后用pGEM-T Easy Vector Systems克隆测序验证，将序列正确的启动子分别命名为pGhU6-7启动子、pGhU6-9启动子。扩增pGhU6-7启动子的引物序列分别为pGhU6-7F(5’-TTAATCTGATGCTCCACCTGCTTTTGAT-3’)和pGhU6-7R(5’-CATCTGATGCTTCTTTCGCTTATATAAACG-3’)，扩增pGhU6-9启动子的引物序列分别为pGhU6-9F(5’-TGATTGCTAGAATTTGCAGTAGGCTT-3’)和pGhU6-9R(5’-CATCTGATGCTTCTTTCGCTTATATAAACG-3’)。PCR体系见表1。PCR条件见表2。
表1 pGhU6-7、pGhU6-9克隆PCR体系
表2 pGhU6-7、pGhU6-9克隆的PCR条件
3.pGhU6-7启动子、pGhU6-9启动子序列的优化
利用重叠延伸PCR(Urban et al.1997)将pGhU6-7启动子第319个碱基C突变为G，pGhU6-9第616个碱基C突变为G，从而突变所含有的BSAⅠ位点，优化后的pGhU6-7启动子、pGhU6-9启动子序列分别命名为pGhU6-7I、pGhU6-9I(其序列如SEQ ID NO:1和3所示)。第一次PCR：pGhU6-7I使用引物pGhU6-7F(5’-TTAATCTGATGCTCCACCTGCTTTTGAT-3’)和pGhU6-71R(5’-ATCCGTCTCTATTCGTTGCTATTGA-3’，下划线碱基为突变碱基)扩增第一个片段，引物pGhU6-72F(5’-TCAATAGCAACGAATAGAGACGGAT-3’下划线碱基为突变碱基，)和pGhU6-7R(5’-CATCTGATGCTTCTTTCGCTTATATAAACG-3’)扩增第二个片段，pGhU6-9I使用引物pGhU6-9F(5’-TGATTGCTAGAATTTGCAGTAGGCTT-3’)和pGhU6-91R(5’-TAGCCGTCTCATTTCACATTCATTG-3’，其中：下划线碱基为突变碱基)扩增第一个片段，引物pGhU6-92F(5’-TCAATAGCAACGAATAGAGACGGAT-3’，下划线碱基为突变碱基)和pGhU6-9R(5’-CATCTGATGCTTCTTTCGCTTATATAAACG-3’)扩增第二个片段，第二次PCR，pGhU6-7I使用上述引物pGhU6-7F和pGhU6-7R，pGhU6-9I使用上述引物pGhU6-9F和pGhU6-9R进行拼接，然后用pGEM-T Easy Vector Systems(购自北京普洛麦格生物技术有限公司)克隆测序(测序委托武汉擎科生物科技有限公司完成)。
实施例2：pRGEB32-GhU6.7-NPTⅡ、pRGEB32-GhU6.9-NPTⅡ载体的构建
1.pRGEB32与pGhU6-7I、pGhU6-9I的连接
首先将pRGEB32载体酶切，酶切体系如表3所示，37℃放置5h，然后利用凝胶回收试剂盒(购自武汉华信阳光生物科技有限公司)将酶切产物纯化。
表3 50μL酶切体系
从pGhU6-7I、pGhU6-9I正确序列的质粒中扩增，通过重叠延伸PCR引入BSAⅠ、gRNA序列，然后利用凝胶回收试剂盒将PCR产物纯化，酶切后的PRGEB32载体与pGhU6-7I、pGhU6-9I片段通过ClonExpress II One Step Cloning Kit(Vazyme C112-02)进行连接，转化到大肠杆菌感受态，挑取阳性克隆进行测序，将测序正确的质粒命名为pRGEB32-GhU6.7、pRGEB32-GhU6.9。
表4 5μL连接体系
2.pRGEB32-pGhU6.7、pRGEB32-pGhU6.9与NPTⅡ的连接
利用Primer5软件对pHellsgate4载体上的真核NPTⅡ序列设计引物，并加上载体酶切位点两侧的同源序列，然后以pHellsgate4质粒为模板进行扩增。将pRGEB32载体(Xieet al.2015)酶切，37℃放置5h，然后利用凝胶回收试剂盒将酶切产物纯化。
扩增NPTⅡ序列的引物：inf-kan S：TTTCGCAGATCCCGGGATGATTGAACAAGATGGATTG
inf-kan A s：TATGGAGAAACTCGAGCTCAGAAGAACTCGTCAAGAAG
表5 50μL酶切体系
表6 5μL连接体系
将酶切后的载体与NPTⅡ片段通过ClonExpress II One Step Cloning Kit(Vazyme C112-02)进行连接，转化到大肠杆菌感受态，挑取阳性克隆进行测序，将测序正确的质粒命名为pRGEB32-pGhU6.7-NPTⅡ、pRGEB32-pGhU6.9-NPTⅡ(这两个载体的序列分别如SEQ ID NO:8和SEQ ID NO:9所示)。
实施例3：pRGEB32-GhU6.7-NPTⅡ-sgRNA、pRGEB32-GhU6.9-NPTⅡ-sgRNA载体的构建
1.GhCLA基因的sgRNA设计
在陆地棉1-脱氧木酮糖-5-磷酸合成酶(Cloroplasto alterados，CLA)Gh_A10G2292基因外显子区域中设计四个sgRNA，分别命名为sgRNA1、sgRNA2、sgRNA3、sgRNA4。
表7GhCLA基因sgRNA统计表
2.sgRNA与pRGEB32-GhU6.7-NPTⅡ、pRGEB32-GhU6.9-NPTⅡ载体连接
插入载体的序列为tRNA、gRNA的重复序列，采用重叠延伸PCR(Urban et al.1997)将重复序列拼接，靶标以引物接头加上，然后用一步克隆法与载体连接。以gRNA2与gRNA4为组合1，gRNA1与gRNA3为组合2，分别与pRGEB32-GhU6.7-NPTⅡ、pRGEB32-GhU6.9-NPTⅡ载体进行连接，构建成功的载体被命名为7NC1、7NC2、9NC1和9NC2。
3.以7NC1载体的构建为例进行说明，其他三个同类载体的构建参见本实施例的操作步骤(1)第一次PCR
pGTR4载体(Xie et al.2015)中含有gRNA+tRNA序列，第一次PCR引物序列如下：
pRGEB32-7s:AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG
CLA1AS:ATACCACCTGTGGGAGCTTTTGCACCAGCCGGGAAT下划线碱基是sgRNA序列
CLA 2S:AGAGCTGCTGACAAGTATCAGTTTTAGAGCTAGAAATA下划线碱基是sgRNA序列
CLA2AS:TGATACTTGTCAGCAGCTCTTGCACCAGCCGGGAAT下划线碱基是sgRNA序列
表8 PCR体系
表9 PCR条件
第二次PCR，将第一次PCR的两个小片段进行拼接及扩增，引物序列如下所示：
Inf CLA2AS:TTCTAGCTCTAAAACTGATACTTGTCAGCAGCTCT下划线碱基是sgRNA序列Inf pRGEB32-7S:AAGCATCAGATGGGCAAACAAA
表10 PCR体系
表11 PCR条件
pRGEB32-GhU6.7-NPTⅡ载体用BSAⅠ进行酶切，然后通过ClonExpress II OneStep Cloning Kit(Vazyme C112-02)将其与第二次PCR产物进行连接，热激大肠杆菌感受态后，挑取阳性克隆进行测序，测序正确的质粒，电转至EHA105农杆菌感受态中，挑取阳性农杆菌，加甘油至最终浓度10％，-70℃超低温冰箱保存。
实施例4：CRISPR-Cas9在棉花中的遗传转化
将上述构建好的载体通过农杆菌介导的转化方法导入棉花宿主细胞。具体转化步骤如下：
1.挑选饱满、健康的陆地棉YZ1种子，剥去种皮后，用0.1％的HgCl2浸泡10min，再用无菌水洗涤3次，将灭菌后的棉种。接种于无菌苗萌发培养基上，在黑暗条件下，置于28℃恒温箱中培养4-6d；
2.取无菌苗下胚轴，切成0.5-0.8cm小段接种于0.5OD的用农杆菌活化培养基悬浮的农杆菌菌液中，侵染10mim后，用无菌滤纸吸干下胚轴表面的菌液；
3.将下胚轴接种在含有共培培养基的培养皿中，21℃下共培养48h，将下胚轴放入含有头孢霉素500mg/L的无菌水中，冲洗3遍，吸干表面水份后，接种到选择培养基中，每1个月继代1次直到获得胚性愈伤组织，将胚性愈伤组织转入分化培养基，获得大量胚状体；
4.将获得的胚状体转移到胚萌发及成苗培养基，直到获得转基因植株幼苗；
转化所用的培养基组分及配比：
无菌苗萌发培养基：1/2MS大量元素，15g/L葡萄糖，2.5g/L的Phytagel；pH:6.1-6.2；
愈伤诱导培养基：MSB+24-D 0.1mg/L+KT 0.1mg/L+3％Glucose+0.3％Phytagel；pH:5.85-5.95；
农杆菌活化培养基：胰化蛋白胨5g/L+NaCl 5g/L+MgSO4.7H2O 0.1g/L+KH2PO4+0.25g/L+甘露醇5g/L+甘氨酸1.0g/L；pH:5.85-5.95；
共培养培养基：MSB+2，4-D 0.1mg/l+KT 0.1mg/l+50mg/l AS+3％Glucose+0.25％Phytagel，pH5.8；
选择培养基：MSB+2，4-D 0.1mg/L+KT 0.1mg/L+3％Glucose+0.3％Phytagel，卡那霉素50mg/L和头孢霉素400mg/L；pH:5.85-5.95；
分化培养基：分化培养基：MSB培养基中去掉NH4NO3,将KNO3用量加倍+Gln 1.0g/L+Asn 0.5g/L+IBA 0.5mg/L+KT 0.15mg/L+3％Glucose+0.25％Phytagel，pH:6.1-6.2；
生根培养基：1/2MS无机盐+B5有机物，15g/L葡萄糖，2.5g/L的Phytagel；pH:5.90-5.95；
MSB的成分如下：MS培养基+B5维生素；
实施例5：检测CRISPR-Cas9对GhCLA编辑效果
1.T7EⅠ酶切
取200ngDNA(转基因单株)做模板，使用以下体系及程序扩增GhCLA基因，取5μLPCR产物，与Buffer2混合在PCR中形成异源双链，加入T7EⅠ做酶切，酶切体系如下所示，然后用2％琼脂糖凝胶低压分离。酶切效果图如图11、12所示。
表12 GhCLA扩增PCR体系
表13 GhCLA扩增PCR条件
表14 异源双链形成PCR体系
表15 异源双链形成PCR条件
2.测序
取出白化苗，用植物基因组DNA抽提试剂盒【天根生化(北京)科技有限公司】提取总DNA，扩增GhCLA,做TA克隆测序，与完整序列进行比对，不同的编辑结果如图13所示，特别是图14，gRNA2、gRNA4两个靶标之间的大片段408bp的缺失。
3.白化表型
由图13，绿色的单株为转入空载体的对照，白化苗为基因编辑后的表型。敲除成功的棉花单株的酶切图、测序、白化表型三个方面结果，均证明本发明的pRGEB32-GhU6.7-NPTⅡ、pRGEB32-GhU6.9-NPTⅡ对棉花基因组具有编辑能力。
主要参考文献：
Jin,S.X.,S.G.Liang,X.L.Zhang,Y.C.Nie&X.P.Guo(2006)An efficientgrafting system for transgenic plant recovery in cotton(Gossypium hirsutumL.).Plant Cell Tissue and Organ Culture,85,181-185.
Urban,A.,S.Neukirchen&K.E.Jaeger(1997)A rapid and efficient methodfor site-directed mutagenesis using one-step overlap extension PCR.Nucleicacids research,25,2227-8.
Xie,K.,B.Minkenberg&Y.Yang(2015)Boosting CRISPR/Cas9multiplex editingcapability with the endogenous tRNA-processing system.Proceedings of theNational Academy of Sciences of the United States of America,112,3570-3575。
SEQUENCE LISTING
<110> 华中农业大学
<120> 一种棉花基因的编辑方法
<130> 2016-12-20
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 988
<212> DNA
<213> 人工序列
<400> 1
ttaatctgat gctccacctg cttttgattt tctttattgg aagagtcttt aagagatatg 60
ttaagtagca taacagtttc atcaaaaaca acatttctgt taatcacaac ttttctattt 120
tcaggatacc ataacttata cacttttaca ctagctttat aaccaagaaa aacacattta 180
atggtacaca attttaattt tccattatca gcatgagtat acgcaaaaca cccaaaaatc 240
tttaaatcag aatcgtcagc aggattacta aaccatactt cttatggagt ctttttctca 300
atagcaacga atagagacgg attgatcaaa aacatatagt tgacattgct ttggcccaaa 360
ataactttga taagttgcca tttgacaaca tacatcgaac attctccatg atcgttctat 420
tcattcgttc tacaatacct tttttaaaat gttcaggttc taaaatgaaa aacaatatga 480
attgcatgaa ttgcttatat gtcctatgaa ttataaagga atgcggttga aatattccca 540
tcgatacata catacatatt cgtgaagtat gttccaatat aatatcaata ttgggattta 600
cgttttataa agcaacatta ttgattggta atatacatta attccaaggc aaacccaaat 660
attttaaaat ttaacctaca actgtggtaa atcaaactta atagtaaccc gattgtaatg 720
tgaagtcaaa tatgaaagta acattggttt atatatatat ttttctctaa attctaataa 780
tcaagttggg ataagtgata aacactgagc ttgccacgtg tgttaacctc gttttcatca 840
tgtgccactc caaagacatc aggcctctat tcaagctggc atggtcagga cgtggtagca 900
tacttcaggg atctggttag aaaatatccc atatcgctaa agaactataa cacaggagcg 960
tttatataag cgaaagaagc atcagatg 988
<210> 2
<211> 988
<212> DNA
<213> 陆地棉（Gossypium hirsutum）
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ttaatctgat gctccacctg cttttgattt tctttattgg aagagtcttt aagagatatg 60
ttaagtagca taacagtttc atcaaaaaca acatttctgt taatcacaac ttttctattt 120
tcaggatacc ataacttata cacttttaca ctagctttat aaccaagaaa aacacattta 180
atggtacaca attttaattt tccattatca gcatgagtat acgcaaaaca cccaaaaatc 240
tttaaatcag aatcgtcagc aggattacta aaccatactt cttatggagt ctttttctca 300
atagcaacga atagagacgg attgatcaaa aacatatagt tgacattgct ttggcccaaa 360
ataactttga taagttgcca tttgacaaca tacatcgaac attctccatg atcgttctat 420
tcattcgttc tacaatacct tttttaaaat gttcaggttc taaaatgaaa aacaatatga 480
attgcatgaa ttgcttatat gtcctatgaa ttataaagga atgcggttga aatattccca 540
tcgatacata catacatatt cgtgaagtat gttccaatat aatatcaata ttgggattta 600
cgttttataa agcaacatta ttgattggta atatacatta attccaaggc aaacccaaat 660
attttaaaat ttaacctaca actgtggtaa atcaaactta atagtaaccc gattgtaatg 720
tgaagtcaaa tatgaaagta acattggttt atatatatat ttttctctaa attctaataa 780
tcaagttggg ataagtgata aacactgagc ttgccacgtg tgttaacctc gttttcatca 840
tgtgccactc caaagacatc aggcctctat tcaagctggc atggtcagga cgtggtagca 900
tacttcaggg atctggttag aaaatatccc atatcgctaa agaactataa cacaggagcg 960
tttatataag cgaaagaagc atcagatg 988
<210> 3
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tgattgctag aatttgcagt aggctttgca attaaataca ttattcgaaa ccattagatg 60
atgcttagta tttaatattt atcctcagcc gatgatggct gaggccctcg agatcataaa 120
ccagtgtaaa aattttgaga cgctcaatgc catttggatc aagcaatccc tcgagattgg 180
ttatttgaaa gcttcggaaa atgcggaaag gatggtgagc tcgagaataa gatcaaggct 240
aaggaaaagg aacttgccaa agtgagcaat aagctcgagg aggcagataa ctcattggca 300
gaccgggaga acaagatgca gaaaaaacta ggaaccttct gcgtgagagg tgttgactac 360
gattgctcgg tgatgtctct catcgaccta gcgaatggat gctactttat gcattagtta 420
ccatttatat ttcaattaga aatgcatcac ataattgaac tattgtgtta tatgattaaa 480
ttctctgtta aggaatatca ttcaaagggt tatctggaaa ctaacatatc tttgtcttcc 540
agaatacggt tccaaacaat tcgctacgac cgactgccgt agagcaaaag aaggccaatg 600
aatgtgaaat gagacggcta gtagttgacg atctggaggt ttgttcttcg tgttcttttc 660
attaatacca caataccata tgcataccct gtcatgaata ttgtataata gaatgttagt 720
ttgcataacc ttccaatatg ctgcaaatag tttccaacat tttactctgt gctacgattt 780
aattctctgt taaggaatat cgttcaaaga gtcatcgcga aagtaacatc ttttctcatc 840
caggctgatg aatatgtaat ggaactgcca gtgaccgaag attcggaggt ttgttcttcg 900
gggacatact tcagggccat ggtcaaaaac tatcccacat tgctaaagaa ccataaggaa 960
gaagcattta tataagcgaa agaagcagca cataact 997
<210> 4
<211> 997
<212> DNA
<213> 陆地棉（Gossypium hirsutum）
<400> 4
tgattgctag aatttgcagt aggctttgca attaaataca ttattcgaaa ccattagatg 60
atgcttagta tttaatattt atcctcagcc gatgatggct gaggccctcg agatcataaa 120
ccagtgtaaa aattttgaga cgctcaatgc catttggatc aagcaatccc tcgagattgg 180
ttatttgaaa gcttcggaaa atgcggaaag gatggtgagc tcgagaataa gatcaaggct 240
aaggaaaagg aacttgccaa agtgagcaat aagctcgagg aggcagataa ctcattggca 300
gaccgggaga acaagatgca gaaaaaacta ggaaccttct gcgtgagagg tgttgactac 360
gattgctcgg tgatgtctct catcgaccta gcgaatggat gctactttat gcattagtta 420
ccatttatat ttcaattaga aatgcatcac ataattgaac tattgtgtta tatgattaaa 480
ttctctgtta aggaatatca ttcaaagggt tatctggaaa ctaacatatc tttgtcttcc 540
agaatacggt tccaaacaat tcgctacgac cgactgccgt agagcaaaag aaggccaatg 600
aatgtgaaat gagaccgcta gtagttgacg atctggaggt ttgttcttcg tgttcttttc 660
attaatacca caataccata tgcataccct gtcatgaata ttgtataata gaatgttagt 720
ttgcataacc ttccaatatg ctgcaaatag tttccaacat tttactctgt gctacgattt 780
aattctctgt taaggaatat cgttcaaaga gtcatcgcga aagtaacatc ttttctcatc 840
caggctgatg aatatgtaat ggaactgcca gtgaccgaag attcggaggt ttgttcttcg 900
gggacatact tcagggccat ggtcaaaaac tatcccacat tgctaaagaa ccataaggaa 960
gaagcattta tataagcgaa agaagcagca cataact 997
<210> 5
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<212> DNA
<213> 人工序列
<400> 5
cttgtacaaa gtggttgata acagcgacta caaggatgac gatgacaagg cttagagctc 60
gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc 120
cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa 180
catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata 240
catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc 300
ggtgtcatct atgttactag atcgggaatt cactggccgt cgttttacac tggccgtcgt 360
tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 420
tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 480
gttgcgcagc ctgaatggcg aatgctagag cagcttgagc ttggatcaga ttgtcgtttc 540
ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga 600
aaagagcgtt tattagaata acggatattt aaaagggcgt gaaaaggttt atccgttcgt 660
ccatttgtat gtgcatgcca accacagggt tcccctcggg atcaaagtac tttgatccaa 720
cccctccgct gctatagtgc agtcggcttc tgacgttcag tgcagccgtc ttctgaaaac 780
gacatgtcgc acaagtccta agttacgcga caggctgccg ccctgccctt ttcctggcgt 840
tttcttgtcg cgtgttttag tcgcataaag tagaatactt gcgactagaa ccggagacat 900
tacgccatga acaagagcgc cgccgctggc ctgctgggct atgcccgcgt cagcaccgac 960
gaccaggact tgaccaacca acgggccgaa ctgcacgcgg ccggctgcac caagctgttt 1020
tccgagaaga tcaccggcac caggcgcgac cgcccggagc tggccaggat gcttgaccac 1080
ctacgccctg gcgacgttgt gacagtgacc aggctagacc gcctggcccg cagcacccgc 1140
gacctactgg acattgccga gcgcatccag gaggccggcg cgggcctgcg tagcctggca 1200
gagccgtggg ccgacaccac cacgccggcc ggccgcatgg tgttgaccgt gttcgccggc 1260
attgccgagt tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg cgaggccgcc 1320
aaggcccgag gcgtgaagtt tggcccccgc cctaccctca ccccggcaca gatcgcgcac 1380
gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag aggcggctgc actgcttggc 1440
gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac gcccaccgag 1500
gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc cctggcggcc 1560
gccgagaatg aacgccaaga ggaacaagca tgaaaccgca ccaggacggc caggacgaac 1620
cgtttttcat taccgaagag atcgaggcgg agatgatcgc ggccgggtac gtgttcgagc 1680
cgcccgcgca cgtctcaacc gtgcggctgc atgaaatcct ggccggtttg tctgatgcca 1740
agctggcggc ctggccggcc agcttggccg ctgaagaaac cgagcgccgc cgtctaaaaa 1800
ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat atgatgcgat 1860
gagtaaataa acaaatacgc aaggggaacg catgaaggtt atcgctgtac ttaaccagaa 1920
aggcgggtca ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc aactcgccgg 1980
ggccgatgtt ctgttagtcg attccgatcc ccagggcagt gcccgcgatt gggcggccgt 2040
gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg accgcgacgt 2100
gaaggccatc ggccggcgcg acttcgtagt gatcgacgga gcgccccagg cggcggactt 2160
ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc caagccctta 2220
cgacatatgg gccaccgccg acctggtgga gctggttaag cagcgcattg aggtcacgga 2280
tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc gcatcggcgg 2340
tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt cttgagtccc gtatcacgca 2400
gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc gttcttgaat cagaacccga 2460
gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa aactcatttg 2520
agttaatgag gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc cggccgtccg 2580
agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc agccatgaag 2640
cgggtcaact ttcagttgcc ggcggaggat cacaccaagc tgaagatgta cgcggtacgc 2700
caaggcaaga ccattaccga gctgctatct gaatacatcg cgcagctacc agagtaaatg 2760
agcaaatgaa taaatgagta gatgaatttt agcggctaaa ggaggcggca tggaaaatca 2820
agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg gcggttggcc 2880
aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc actggaaccc ccaagcccga 2940
ggaatcggcg tgacggtcgc aaaccatccg gcccggtaca aatcggcgcg gcgctgggtg 3000
atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc atcgaggcag 3060
aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa gaatcccggc 3120
aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac gagcaaccag 3180
attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc atcatggacg 3240
tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc cgctacgagc 3300
ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt gtgtgggatt 3360
acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga taccgggaag 3420
ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta ctcaagttct 3480
gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc attcggttaa 3540
acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc ctggtgacgg 3600
tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa accgggcggc 3660
cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca gaaggcaaga 3720
acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc atcggccgtt 3780
ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg ttgttcaaga 3840
cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc accgtgcgca 3900
agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg gggcaggctg 3960
gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc gccggttcct 4020
aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt cgaaaagcac 4080
tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg aaccggaacc 4140
cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa gtgactgata 4200
taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt aaaactctta 4260
aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag ctgcaaaaag 4320
cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg cctatcgcgg 4380
ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg cgcggacaag 4440
ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg caccctgcct cgcgcgtttc 4500
ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 4560
taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 4620
cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg 4680
cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 4740
gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc 4800
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4860
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4920
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 4980
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5040
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5100
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 5160
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5220
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5280
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5340
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5400
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5460
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5520
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5580
ggaacgaaaa ctcacgttaa gggattttgg tcatgcattc taggtactaa aacaattcat 5640
ccagtaaaat ataatatttt attttctccc aatcaggctt gatccccagt aagtcaaaaa 5700
atagctcgac atactgttct tccccgatat cctccctgat cgaccggacg cagaaggcaa 5760
tgtcatacca cttgtccgcc ctgccgcttc tcccaagatc aataaagcca cttactttgc 5820
catctttcac aaagatgttg ctgtctccca ggtcgccgtg ggaaaagaca agttcctctt 5880
cgggcttttc cgtctttaaa aaatcataca gctcgcgcgg atctttaaat ggagtgtctt 5940
cttcccagtt ttcgcaatcc acatcggcca gatcgttatt cagtaagtaa tccaattcgg 6000
ctaagcggct gtctaagcta ttcgtatagg gacaatccga tatgtcgatg gagtgaaaga 6060
gcctgatgca ctccgcatac agctcgataa tcttttcagg gctttgttca tcttcatact 6120
cttccgagca aaggacgcca tcggcctcac tcatgagcag attgctccag ccatcatgcc 6180
gttcaaagtg caggaccttt ggaacaggca gctttccttc cagccatagc atcatgtcct 6240
tttcccgttc cacatcatag gtggtccctt tataccggct gtccgtcatt tttaaatata 6300
ggttttcatt ttctcccacc agcttatata ccttagcagg agacattcct tccgtatctt 6360
ttacgcagcg gtatttttcg atcagttttt tcaattccgg tgatattctc attttagcca 6420
tttattattt ccttcctctt ttctacagta tttaaagata ccccaagaag ctaattataa 6480
caagacgaac tccaattcac tgttccttgc attctaaaac cttaaatacc agaaaacagc 6540
tttttcaaag ttgttttcaa agttggcgta taacatagta tcgacggagc cgattttgaa 6600
accgcggtga tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg 6660
cgagatcatc cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt 6720
aacatgagca aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat 6780
gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg 6840
gctggtggca ggatatattg tggtgtaaac aaattgacgc ttagacaact taataacaca 6900
ttgcggacgt ttttaatgta ctgaattaac gccgaattaa ttcgggggat ctggatttta 6960
gtactggatt ttggttttag gaattagaaa ttttattgat agaagtattt tacaaataca 7020
aatacatact aagggtttct tatatgctca acacatgagc gaaaccctat aggaacccta 7080
attcccttat ctgggaacta ctcacacatt attatggaga aactcgagct tgtcgatcga 7140
cagatccggt cggcatctac tctatttctt tgccctcgga cgagtgctgg ggcgtcggtt 7200
tccactatcg gcgagtactt ctacacagcc atcggtccag acggccgcgc ttctgcgggc 7260
gatttgtgta cgcccgacag tcccggctcc ggatcggacg attgcgtcgc atcgaccctg 7320
cgcccaagct gcatcatcga aattgccgtc aaccaagctc tgatagagtt ggtcaagacc 7380
aatgcggagc atatacgccc ggagtcgtgg cgatcctgca agctccggat gcctccgctc 7440
gaagtagcgc gtctgctgct ccatacaagc caaccacggc ctccagaaga agatgttggc 7500
gacctcgtat tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg ctgttatgcg 7560
gccattgtcc gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt gccggacttc 7620
ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg acgcactgac 7680
ggtgtcgtcc atcacagttt gccagtgata cacatgggga tcagcaatcg cgcatatgaa 7740
atcacgccat gtagtgtatt gaccgattcc ttgcggtccg aatgggccga acccgctcgt 7800
ctggctaaga tcggccgcag cgatcgcatc catagcctcc gcgaccggtt gtagaacagc 7860
gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc gggagatgca 7920
ataggtcagg ctctcgctaa actccccaat gtcaagcact tccggaatcg ggagcgcggc 7980
cgatgcaaag tgccgataaa cataacgatc tttgtagaaa ccatcggcgc agctatttac 8040
ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa gcacgagatt cttcgccctc 8100
cgagagctgc atcaggtcgg agacgctgtc gaacttttcg atcagaaact tctcgacaga 8160
cgtcgcggtg agttcaggct ttttcatatc tcattgcccc ccgggatctg cgaaagctcg 8220
agagagatag atttgtagag agagactggt gatttcagcg tgtcctctcc aaatgaaatg 8280
aacttcctta tatagaggaa ggtcttgcga aggatagtgg gattgtgcgt catcccttac 8340
gtcagtggag atatcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt 8400
ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg cagaggcatc 8460
ttgaacgata gcctttcctt tatcgcaatg atggcatttg taggtgccac cttccttttc 8520
tactgtcctt ttgatgaagt gacagatagc tgggcaatgg aatccgagga ggtttcccga 8580
tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg tatctttgat 8640
attcttggag tagacgagag tgtcgtgctc caccatgtta tcacatcaat ccacttgctt 8700
tgaagacgtg gttggaacgt cttctttttc cacgatgctc ctcgtgggtg ggggtccatc 8760
tttgggacca ctgtcggcag aggcatcttg aacgatagcc tttcctttat cgcaatgatg 8820
gcatttgtag gtgccacctt ccttttctac tgtccttttg atgaagtgac agatagctgg 8880
gcaatggaat ccgaggaggt ttcccgatat taccctttgt tgaaaagtct caatagccct 8940
ttggtcttct gagactgtat ctttgatatt cttggagtag acgagagtgt cgtgctccac 9000
catgttggca agctgctcta gccaatacgc aaaccgcctc tccccgcgcg ttggccgatt 9060
cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca 9120
attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct 9180
cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat 9240
gattacgcca agcttaagga atctttaaac atacgaacag atcacttaaa gttcttctga 9300
agcaacttaa agttatcagg catgcatgga tcttggagga atcagatgtg cagtcaggga 9360
ccatagcaca agacaggcgt cttctactgg tgctaccagc aaatgctgga agccgggaac 9420
actgggtacg ttggaaacca cgtgatgtga agaagtaaga taaactgtag gagaaaagca 9480
tttcgtagtg ggccatgaag cctttcagga catgtattgc agtatgggcc ggcccattac 9540
gcaattggac gacaacaaag actagtatta gtaccacctc ggctatccac atagatcaaa 9600
gctgatttaa aagagttgtg cagatgatcc gtggcaggag accgaggtct cggttttaga 9660
gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga 9720
gtcggtgctt ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt 9780
tagcgcgtgc atgcctgcag gtccacaaat tcgggtcaag gcggaagcca gcgcgccacc 9840
ccacgtcagc aaatacggag gcgcggggtt gacggcgtca cccggtccta acggcgacca 9900
acaaaccagc cagaagaaat tacagtaaaa aaaaagtaaa ttgcactttg atccaccttt 9960
tattacctaa gtctcaattt ggatcaccct taaacctatc ttttcaattt gggccgggtt 10020
gtggtttgga ctaccatgaa caacttttcg tcatgtctaa cttccctttc agcaaacata 10080
tgaaccatat atagaggaga tcggccgtat actagagctg atgtgtttaa ggtcgttgat 10140
tgcacgagaa aaaaaaatcc aaatcgcaac aatagcaaat ttatctggtt caaagtgaaa 10200
agatatgttt aaaggtagtc caaagtaaaa cttatagata ataaaatgtg gtccaaagcg 10260
taattcactc aaaaaaaatc aacgagacgt gtaccaaacg gagacaaacg gcatcttctc 10320
gaaatttccc aaccgctcgc tcgcccgcct cgtcttcccg gaaaccgcgg tggtttcagc 10380
gtggcggatt ctccaagcag acggagacgt cacggcacgg gactcctccc accacccaac 10440
cgccataaat accagccccc tcatctcctc tcctcgcatc agctccaccc ccgaaaaatt 10500
tctccccaat ctcgcgaggc tctcgtcgtc gaatcgaatc ctctcgcgtc ctcaaggtac 10560
gctgcttctc ctctcctcgc ttcgtttcga ttcgatttcg gacgggtgag gttgttttgt 10620
tgctagatcc gattggtggt tagggttgtc gatgtgatta tcgtgagatg tttaggggtt 10680
gtagatctga tggttgtgat ttgggcacgg ttggttcgat aggtggaatc gtggttaggt 10740
tttgggattg gatgttggtt ctgatgattg gggggaattt ttacggttag atgaattgtt 10800
ggatgattcg attggggaaa tcggtgtaga tctgttgggg aattgtggaa ctagtcatgc 10860
ctgagtgatt ggtgcgattt gtagcgtgtt ccatcttgta ggccttgttg cgagcatgtt 10920
cagatctact gttccgctct tgattgagtt attggtgcca tgggttggtg caaacacagg 10980
ctttaatatg ttatatctgt tttgtgtttg atgtagatct gtagggtagt tcttcttaga 11040
catggttcaa ttatgtagct tgtgcgtttc gatttgattt catatgttca cagattagat 11100
aatgatgaac tcttttaatt aattgtcaat ggtaaatagg aagtcttgtc gctatatctg 11160
tcataatgat ctcatgttac tatctgccag taatttatgc taagaactat attagaatat 11220
catgttacaa tctgtagtaa tatcatgtta caatctgtag ttcatctata taatctattg 11280
tggtaatttc tttttactat ctgtgtgaag attattgcca ctagttcatt ctacttattt 11340
ctgaagttca ggatacgtgt gctgttacta cctatctgaa tacatgtgtg atgtgcctgt 11400
tactatcttt ttgaatacat gtatgttctg ttggaatatg tttgctgttt gatccgttgt 11460
tgtgtcctta atcttgtgct agttcttacc ctatctgttt ggtgattatt tcttgcagat 11520
agttatcaac aagtttgtac aaaaaagcag gcttcgaagg agatagaacc aattctctaa 11580
ggaaatactt aaccatggac tataaggacc acgacggaga ctacaaggat catgatattg 11640
attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg 11700
gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg 11760
gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca 11820
acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg 11880
aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga 11940
accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct 12000
tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc 12060
ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc 12120
acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg 12180
ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg 12240
acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg 12300
aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga 12360
gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc 12420
tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg 12480
acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca 12540
acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt 12600
ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc 12660
tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag 12720
ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga 12780
acggctacgc cggctacatt gacggcggag ccagccagga agagttctac aagttcatca 12840
agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg 12900
acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg 12960
gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc 13020
gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca 13080
ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga 13140
acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca 13200
acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt 13260
acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc 13320
ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc 13380
ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact 13440
ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc 13500
tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg 13560
aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga 13620
aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca 13680
ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca 13740
agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga 13800
tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg 13860
gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca 13920
tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg 13980
agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca 14040
gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga 14100
aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc 14160
agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg 14220
atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc 14280
tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga 14340
agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt 14400
tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca 14460
tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact 14520
cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca 14580
ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg 14640
agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc 14700
tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg 14760
acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact 14820
tcttctacag caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga 14880
tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg 14940
gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa 15000
agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg 15060
ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc 15120
ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac 15180
tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga 15240
atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca 15300
agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg 15360
ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt 15420
acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc 15480
tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct 15540
ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc 15600
accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca 15660
atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca 15720
ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg 15780
agacacggat cgacctgtct cagctgggag gcgacaaaag gccggcggcc acgaaaaagg 15840
ccggccaggc aaaaaagaaa aagtaagaat tcgcggccgc actcgagata tctagaccca 15900
gcttt 15905
<210> 6
<211> 16516
<212> DNA
<213> 人工序列
<400> 6
cttgtacaaa gtggttgata acagcgacta caaggatgac gatgacaagg cttagagctc 60
gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc 120
cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa 180
catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata 240
catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc 300
ggtgtcatct atgttactag atcgggaatt cactggccgt cgttttacac tggccgtcgt 360
tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 420
tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 480
gttgcgcagc ctgaatggcg aatgctagag cagcttgagc ttggatcaga ttgtcgtttc 540
ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga 600
aaagagcgtt tattagaata acggatattt aaaagggcgt gaaaaggttt atccgttcgt 660
ccatttgtat gtgcatgcca accacagggt tcccctcggg atcaaagtac tttgatccaa 720
cccctccgct gctatagtgc agtcggcttc tgacgttcag tgcagccgtc ttctgaaaac 780
gacatgtcgc acaagtccta agttacgcga caggctgccg ccctgccctt ttcctggcgt 840
tttcttgtcg cgtgttttag tcgcataaag tagaatactt gcgactagaa ccggagacat 900
tacgccatga acaagagcgc cgccgctggc ctgctgggct atgcccgcgt cagcaccgac 960
gaccaggact tgaccaacca acgggccgaa ctgcacgcgg ccggctgcac caagctgttt 1020
tccgagaaga tcaccggcac caggcgcgac cgcccggagc tggccaggat gcttgaccac 1080
ctacgccctg gcgacgttgt gacagtgacc aggctagacc gcctggcccg cagcacccgc 1140
gacctactgg acattgccga gcgcatccag gaggccggcg cgggcctgcg tagcctggca 1200
gagccgtggg ccgacaccac cacgccggcc ggccgcatgg tgttgaccgt gttcgccggc 1260
attgccgagt tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg cgaggccgcc 1320
aaggcccgag gcgtgaagtt tggcccccgc cctaccctca ccccggcaca gatcgcgcac 1380
gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag aggcggctgc actgcttggc 1440
gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac gcccaccgag 1500
gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc cctggcggcc 1560
gccgagaatg aacgccaaga ggaacaagca tgaaaccgca ccaggacggc caggacgaac 1620
cgtttttcat taccgaagag atcgaggcgg agatgatcgc ggccgggtac gtgttcgagc 1680
cgcccgcgca cgtctcaacc gtgcggctgc atgaaatcct ggccggtttg tctgatgcca 1740
agctggcggc ctggccggcc agcttggccg ctgaagaaac cgagcgccgc cgtctaaaaa 1800
ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat atgatgcgat 1860
gagtaaataa acaaatacgc aaggggaacg catgaaggtt atcgctgtac ttaaccagaa 1920
aggcgggtca ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc aactcgccgg 1980
ggccgatgtt ctgttagtcg attccgatcc ccagggcagt gcccgcgatt gggcggccgt 2040
gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg accgcgacgt 2100
gaaggccatc ggccggcgcg acttcgtagt gatcgacgga gcgccccagg cggcggactt 2160
ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc caagccctta 2220
cgacatatgg gccaccgccg acctggtgga gctggttaag cagcgcattg aggtcacgga 2280
tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc gcatcggcgg 2340
tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt cttgagtccc gtatcacgca 2400
gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc gttcttgaat cagaacccga 2460
gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa aactcatttg 2520
agttaatgag gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc cggccgtccg 2580
agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc agccatgaag 2640
cgggtcaact ttcagttgcc ggcggaggat cacaccaagc tgaagatgta cgcggtacgc 2700
caaggcaaga ccattaccga gctgctatct gaatacatcg cgcagctacc agagtaaatg 2760
agcaaatgaa taaatgagta gatgaatttt agcggctaaa ggaggcggca tggaaaatca 2820
agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg gcggttggcc 2880
aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc actggaaccc ccaagcccga 2940
ggaatcggcg tgacggtcgc aaaccatccg gcccggtaca aatcggcgcg gcgctgggtg 3000
atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc atcgaggcag 3060
aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa gaatcccggc 3120
aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac gagcaaccag 3180
attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc atcatggacg 3240
tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc cgctacgagc 3300
ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt gtgtgggatt 3360
acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga taccgggaag 3420
ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta ctcaagttct 3480
gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc attcggttaa 3540
acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc ctggtgacgg 3600
tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa accgggcggc 3660
cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca gaaggcaaga 3720
acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc atcggccgtt 3780
ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg ttgttcaaga 3840
cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc accgtgcgca 3900
agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg gggcaggctg 3960
gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc gccggttcct 4020
aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt cgaaaagcac 4080
tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg aaccggaacc 4140
cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa gtgactgata 4200
taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt aaaactctta 4260
aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag ctgcaaaaag 4320
cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg cctatcgcgg 4380
ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg cgcggacaag 4440
ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg caccctgcct cgcgcgtttc 4500
ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 4560
taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 4620
cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg 4680
cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 4740
gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc 4800
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4860
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4920
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 4980
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5040
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5100
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 5160
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5220
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5280
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5340
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5400
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5460
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5520
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5580
ggaacgaaaa ctcacgttaa gggattttgg tcatgcattc taggtactaa aacaattcat 5640
ccagtaaaat ataatatttt attttctccc aatcaggctt gatccccagt aagtcaaaaa 5700
atagctcgac atactgttct tccccgatat cctccctgat cgaccggacg cagaaggcaa 5760
tgtcatacca cttgtccgcc ctgccgcttc tcccaagatc aataaagcca cttactttgc 5820
catctttcac aaagatgttg ctgtctccca ggtcgccgtg ggaaaagaca agttcctctt 5880
cgggcttttc cgtctttaaa aaatcataca gctcgcgcgg atctttaaat ggagtgtctt 5940
cttcccagtt ttcgcaatcc acatcggcca gatcgttatt cagtaagtaa tccaattcgg 6000
ctaagcggct gtctaagcta ttcgtatagg gacaatccga tatgtcgatg gagtgaaaga 6060
gcctgatgca ctccgcatac agctcgataa tcttttcagg gctttgttca tcttcatact 6120
cttccgagca aaggacgcca tcggcctcac tcatgagcag attgctccag ccatcatgcc 6180
gttcaaagtg caggaccttt ggaacaggca gctttccttc cagccatagc atcatgtcct 6240
tttcccgttc cacatcatag gtggtccctt tataccggct gtccgtcatt tttaaatata 6300
ggttttcatt ttctcccacc agcttatata ccttagcagg agacattcct tccgtatctt 6360
ttacgcagcg gtatttttcg atcagttttt tcaattccgg tgatattctc attttagcca 6420
tttattattt ccttcctctt ttctacagta tttaaagata ccccaagaag ctaattataa 6480
caagacgaac tccaattcac tgttccttgc attctaaaac cttaaatacc agaaaacagc 6540
tttttcaaag ttgttttcaa agttggcgta taacatagta tcgacggagc cgattttgaa 6600
accgcggtga tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg 6660
cgagatcatc cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt 6720
aacatgagca aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat 6780
gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg 6840
gctggtggca ggatatattg tggtgtaaac aaattgacgc ttagacaact taataacaca 6900
ttgcggacgt ttttaatgta ctgaattaac gccgaattaa ttcgggggat ctggatttta 6960
gtactggatt ttggttttag gaattagaaa ttttattgat agaagtattt tacaaataca 7020
aatacatact aagggtttct tatatgctca acacatgagc gaaaccctat aggaacccta 7080
attcccttat ctgggaacta ctcacacatt attatggaga aactcgagct tgtcgatcga 7140
cagatccggt cggcatctac tctatttctt tgccctcgga cgagtgctgg ggcgtcggtt 7200
tccactatcg gcgagtactt ctacacagcc atcggtccag acggccgcgc ttctgcgggc 7260
gatttgtgta cgcccgacag tcccggctcc ggatcggacg attgcgtcgc atcgaccctg 7320
cgcccaagct gcatcatcga aattgccgtc aaccaagctc tgatagagtt ggtcaagacc 7380
aatgcggagc atatacgccc ggagtcgtgg cgatcctgca agctccggat gcctccgctc 7440
gaagtagcgc gtctgctgct ccatacaagc caaccacggc ctccagaaga agatgttggc 7500
gacctcgtat tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg ctgttatgcg 7560
gccattgtcc gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt gccggacttc 7620
ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg acgcactgac 7680
ggtgtcgtcc atcacagttt gccagtgata cacatgggga tcagcaatcg cgcatatgaa 7740
atcacgccat gtagtgtatt gaccgattcc ttgcggtccg aatgggccga acccgctcgt 7800
ctggctaaga tcggccgcag cgatcgcatc catagcctcc gcgaccggtt gtagaacagc 7860
gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc gggagatgca 7920
ataggtcagg ctctcgctaa actccccaat gtcaagcact tccggaatcg ggagcgcggc 7980
cgatgcaaag tgccgataaa cataacgatc tttgtagaaa ccatcggcgc agctatttac 8040
ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa gcacgagatt cttcgccctc 8100
cgagagctgc atcaggtcgg agacgctgtc gaacttttcg atcagaaact tctcgacaga 8160
cgtcgcggtg agttcaggct ttttcatatc tcattgcccc ccgggatctg cgaaagctcg 8220
agagagatag atttgtagag agagactggt gatttcagcg tgtcctctcc aaatgaaatg 8280
aacttcctta tatagaggaa ggtcttgcga aggatagtgg gattgtgcgt catcccttac 8340
gtcagtggag atatcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt 8400
ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg cagaggcatc 8460
ttgaacgata gcctttcctt tatcgcaatg atggcatttg taggtgccac cttccttttc 8520
tactgtcctt ttgatgaagt gacagatagc tgggcaatgg aatccgagga ggtttcccga 8580
tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg tatctttgat 8640
attcttggag tagacgagag tgtcgtgctc caccatgtta tcacatcaat ccacttgctt 8700
tgaagacgtg gttggaacgt cttctttttc cacgatgctc ctcgtgggtg ggggtccatc 8760
tttgggacca ctgtcggcag aggcatcttg aacgatagcc tttcctttat cgcaatgatg 8820
gcatttgtag gtgccacctt ccttttctac tgtccttttg atgaagtgac agatagctgg 8880
gcaatggaat ccgaggaggt ttcccgatat taccctttgt tgaaaagtct caatagccct 8940
ttggtcttct gagactgtat ctttgatatt cttggagtag acgagagtgt cgtgctccac 9000
catgttggca agctgctcta gccaatacgc aaaccgcctc tccccgcgcg ttggccgatt 9060
cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca 9120
attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct 9180
cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat 9240
gattacgcca agcttttaat ctgatgctcc acctgctttt gattttcttt attggaagag 9300
tctttaagag atatgttaag tagcataaca gtttcatcaa aaacaacatt tctgttaatc 9360
acaacttttc tattttcagg ataccataac ttatacactt ttacactagc tttataacca 9420
agaaaaacac atttaatggt acacaatttt aattttccat tatcagcatg agtatacgca 9480
aaacacccaa aaatctttaa atcagaatcg tcagcaggat tactaaacca tacttcttat 9540
ggagtctttt tctcaatagc aacgaataga gacggattga tcaaaaacat atagttgaca 9600
ttgctttggc ccaaaataac tttgataagt tgccatttga caacatacat cgaacattct 9660
ccatgatcgt tctattcatt cgttctacaa tacctttttt aaaatgttca ggttctaaaa 9720
tgaaaaacaa tatgaattgc atgaattgct tatatgtcct atgaattata aaggaatgcg 9780
gttgaaatat tcccatcgat acatacatac atattcgtga agtatgttcc aatataatat 9840
caatattggg atttacgttt tataaagcaa cattattgat tggtaatata cattaattcc 9900
aaggcaaacc caaatatttt aaaatttaac ctacaactgt ggtaaatcaa acttaatagt 9960
aacccgattg taatgtgaag tcaaatatga aagtaacatt ggtttatata tatatttttc 10020
tctaaattct aataatcaag ttgggataag tgataaacac tgagcttgcc acgtgtgtta 10080
acctcgtttt catcatgtgc cactccaaag acatcaggcc tctattcaag ctggcatggt 10140
caggacgtgg tagcatactt cagggatctg gttagaaaat atcccatatc gctaaagaac 10200
tataacacag gagcgtttat ataagcgaaa gaagcatcag atgggcagga gaccgaggtc 10260
tcggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 10320
agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc 10380
tagtccgttt ttagcgcgtg catgcctgca ggtccacaaa ttcgggtcaa ggcggaagcc 10440
agcgcgccac cccacgtcag caaatacgga ggcgcggggt tgacggcgtc acccggtcct 10500
aacggcgacc aacaaaccag ccagaagaaa ttacagtaaa aaaaaagtaa attgcacttt 10560
gatccacctt ttattaccta agtctcaatt tggatcaccc ttaaacctat cttttcaatt 10620
tgggccgggt tgtggtttgg actaccatga acaacttttc gtcatgtcta acttcccttt 10680
cagcaaacat atgaaccata tatagaggag atcggccgta tactagagct gatgtgttta 10740
aggtcgttga ttgcacgaga aaaaaaaatc caaatcgcaa caatagcaaa tttatctggt 10800
tcaaagtgaa aagatatgtt taaaggtagt ccaaagtaaa acttatagat aataaaatgt 10860
ggtccaaagc gtaattcact caaaaaaaat caacgagacg tgtaccaaac ggagacaaac 10920
ggcatcttct cgaaatttcc caaccgctcg ctcgcccgcc tcgtcttccc ggaaaccgcg 10980
gtggtttcag cgtggcggat tctccaagca gacggagacg tcacggcacg ggactcctcc 11040
caccacccaa ccgccataaa taccagcccc ctcatctcct ctcctcgcat cagctccacc 11100
cccgaaaaat ttctccccaa tctcgcgagg ctctcgtcgt cgaatcgaat cctctcgcgt 11160
cctcaaggta cgctgcttct cctctcctcg cttcgtttcg attcgatttc ggacgggtga 11220
ggttgttttg ttgctagatc cgattggtgg ttagggttgt cgatgtgatt atcgtgagat 11280
gtttaggggt tgtagatctg atggttgtga tttgggcacg gttggttcga taggtggaat 11340
cgtggttagg ttttgggatt ggatgttggt tctgatgatt ggggggaatt tttacggtta 11400
gatgaattgt tggatgattc gattggggaa atcggtgtag atctgttggg gaattgtgga 11460
actagtcatg cctgagtgat tggtgcgatt tgtagcgtgt tccatcttgt aggccttgtt 11520
gcgagcatgt tcagatctac tgttccgctc ttgattgagt tattggtgcc atgggttggt 11580
gcaaacacag gctttaatat gttatatctg ttttgtgttt gatgtagatc tgtagggtag 11640
ttcttcttag acatggttca attatgtagc ttgtgcgttt cgatttgatt tcatatgttc 11700
acagattaga taatgatgaa ctcttttaat taattgtcaa tggtaaatag gaagtcttgt 11760
cgctatatct gtcataatga tctcatgtta ctatctgcca gtaatttatg ctaagaacta 11820
tattagaata tcatgttaca atctgtagta atatcatgtt acaatctgta gttcatctat 11880
ataatctatt gtggtaattt ctttttacta tctgtgtgaa gattattgcc actagttcat 11940
tctacttatt tctgaagttc aggatacgtg tgctgttact acctatctga atacatgtgt 12000
gatgtgcctg ttactatctt tttgaataca tgtatgttct gttggaatat gtttgctgtt 12060
tgatccgttg ttgtgtcctt aatcttgtgc tagttcttac cctatctgtt tggtgattat 12120
ttcttgcaga tagttatcaa caagtttgta caaaaaagca ggcttcgaag gagatagaac 12180
caattctcta aggaaatact taaccatgga ctataaggac cacgacggag actacaagga 12240
tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga agcggaaggt 12300
cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg acatcggcac 12360
caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa 12420
ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt 12480
cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac 12540
cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt 12600
ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca 12660
cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc 12720
caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct 12780
gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga 12840
cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa 12900
ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc 12960
tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa 13020
gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa 13080
gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga 13140
cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc 13200
caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac 13260
caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac 13320
cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga 13380
ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta 13440
caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct 13500
gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca 13560
gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct 13620
gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg 13680
ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat 13740
caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga 13800
gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct 13860
gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg 13920
aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt 13980
caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga 14040
gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac 14100
ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga 14160
ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga 14220
ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg 14280
gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa 14340
gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt 14400
catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt 14460
gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat 14520
taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg 14580
gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg 14640
acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag 14700
ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct 14760
gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct 14820
gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga 14880
caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga 14940
agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac 15000
ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa 15060
ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca 15120
gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt 15180
gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta 15240
caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt 15300
gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta 15360
caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac 15420
cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc 15480
caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt 15540
gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa 15600
tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa 15660
gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg 15720
cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa 15780
gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag 15840
cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga 15900
cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat 15960
gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt 16020
gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga 16080
gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat 16140
cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc 16200
ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt 16260
taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg 16320
gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac 16380
cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc 16440
cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa ttcgcggccg cactcgagat 16500
atctagaccc agcttt 16516
<210> 7
<211> 16525
<212> DNA
<213> 人工序列
<400> 7
cttgtacaaa gtggttgata acagcgacta caaggatgac gatgacaagg cttagagctc 60
gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc 120
cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa 180
catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata 240
catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc 300
ggtgtcatct atgttactag atcgggaatt cactggccgt cgttttacac tggccgtcgt 360
tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 420
tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 480
gttgcgcagc ctgaatggcg aatgctagag cagcttgagc ttggatcaga ttgtcgtttc 540
ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga 600
aaagagcgtt tattagaata acggatattt aaaagggcgt gaaaaggttt atccgttcgt 660
ccatttgtat gtgcatgcca accacagggt tcccctcggg atcaaagtac tttgatccaa 720
cccctccgct gctatagtgc agtcggcttc tgacgttcag tgcagccgtc ttctgaaaac 780
gacatgtcgc acaagtccta agttacgcga caggctgccg ccctgccctt ttcctggcgt 840
tttcttgtcg cgtgttttag tcgcataaag tagaatactt gcgactagaa ccggagacat 900
tacgccatga acaagagcgc cgccgctggc ctgctgggct atgcccgcgt cagcaccgac 960
gaccaggact tgaccaacca acgggccgaa ctgcacgcgg ccggctgcac caagctgttt 1020
tccgagaaga tcaccggcac caggcgcgac cgcccggagc tggccaggat gcttgaccac 1080
ctacgccctg gcgacgttgt gacagtgacc aggctagacc gcctggcccg cagcacccgc 1140
gacctactgg acattgccga gcgcatccag gaggccggcg cgggcctgcg tagcctggca 1200
gagccgtggg ccgacaccac cacgccggcc ggccgcatgg tgttgaccgt gttcgccggc 1260
attgccgagt tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg cgaggccgcc 1320
aaggcccgag gcgtgaagtt tggcccccgc cctaccctca ccccggcaca gatcgcgcac 1380
gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag aggcggctgc actgcttggc 1440
gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac gcccaccgag 1500
gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc cctggcggcc 1560
gccgagaatg aacgccaaga ggaacaagca tgaaaccgca ccaggacggc caggacgaac 1620
cgtttttcat taccgaagag atcgaggcgg agatgatcgc ggccgggtac gtgttcgagc 1680
cgcccgcgca cgtctcaacc gtgcggctgc atgaaatcct ggccggtttg tctgatgcca 1740
agctggcggc ctggccggcc agcttggccg ctgaagaaac cgagcgccgc cgtctaaaaa 1800
ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat atgatgcgat 1860
gagtaaataa acaaatacgc aaggggaacg catgaaggtt atcgctgtac ttaaccagaa 1920
aggcgggtca ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc aactcgccgg 1980
ggccgatgtt ctgttagtcg attccgatcc ccagggcagt gcccgcgatt gggcggccgt 2040
gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg accgcgacgt 2100
gaaggccatc ggccggcgcg acttcgtagt gatcgacgga gcgccccagg cggcggactt 2160
ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc caagccctta 2220
cgacatatgg gccaccgccg acctggtgga gctggttaag cagcgcattg aggtcacgga 2280
tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc gcatcggcgg 2340
tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt cttgagtccc gtatcacgca 2400
gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc gttcttgaat cagaacccga 2460
gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa aactcatttg 2520
agttaatgag gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc cggccgtccg 2580
agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc agccatgaag 2640
cgggtcaact ttcagttgcc ggcggaggat cacaccaagc tgaagatgta cgcggtacgc 2700
caaggcaaga ccattaccga gctgctatct gaatacatcg cgcagctacc agagtaaatg 2760
agcaaatgaa taaatgagta gatgaatttt agcggctaaa ggaggcggca tggaaaatca 2820
agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg gcggttggcc 2880
aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc actggaaccc ccaagcccga 2940
ggaatcggcg tgacggtcgc aaaccatccg gcccggtaca aatcggcgcg gcgctgggtg 3000
atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc atcgaggcag 3060
aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa gaatcccggc 3120
aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac gagcaaccag 3180
attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc atcatggacg 3240
tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc cgctacgagc 3300
ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt gtgtgggatt 3360
acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga taccgggaag 3420
ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta ctcaagttct 3480
gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc attcggttaa 3540
acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc ctggtgacgg 3600
tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa accgggcggc 3660
cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca gaaggcaaga 3720
acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc atcggccgtt 3780
ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg ttgttcaaga 3840
cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc accgtgcgca 3900
agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg gggcaggctg 3960
gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc gccggttcct 4020
aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt cgaaaagcac 4080
tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg aaccggaacc 4140
cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa gtgactgata 4200
taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt aaaactctta 4260
aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag ctgcaaaaag 4320
cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg cctatcgcgg 4380
ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg cgcggacaag 4440
ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg caccctgcct cgcgcgtttc 4500
ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 4560
taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 4620
cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg 4680
cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 4740
gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc 4800
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4860
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4920
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 4980
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5040
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5100
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 5160
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5220
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5280
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5340
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5400
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5460
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5520
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5580
ggaacgaaaa ctcacgttaa gggattttgg tcatgcattc taggtactaa aacaattcat 5640
ccagtaaaat ataatatttt attttctccc aatcaggctt gatccccagt aagtcaaaaa 5700
atagctcgac atactgttct tccccgatat cctccctgat cgaccggacg cagaaggcaa 5760
tgtcatacca cttgtccgcc ctgccgcttc tcccaagatc aataaagcca cttactttgc 5820
catctttcac aaagatgttg ctgtctccca ggtcgccgtg ggaaaagaca agttcctctt 5880
cgggcttttc cgtctttaaa aaatcataca gctcgcgcgg atctttaaat ggagtgtctt 5940
cttcccagtt ttcgcaatcc acatcggcca gatcgttatt cagtaagtaa tccaattcgg 6000
ctaagcggct gtctaagcta ttcgtatagg gacaatccga tatgtcgatg gagtgaaaga 6060
gcctgatgca ctccgcatac agctcgataa tcttttcagg gctttgttca tcttcatact 6120
cttccgagca aaggacgcca tcggcctcac tcatgagcag attgctccag ccatcatgcc 6180
gttcaaagtg caggaccttt ggaacaggca gctttccttc cagccatagc atcatgtcct 6240
tttcccgttc cacatcatag gtggtccctt tataccggct gtccgtcatt tttaaatata 6300
ggttttcatt ttctcccacc agcttatata ccttagcagg agacattcct tccgtatctt 6360
ttacgcagcg gtatttttcg atcagttttt tcaattccgg tgatattctc attttagcca 6420
tttattattt ccttcctctt ttctacagta tttaaagata ccccaagaag ctaattataa 6480
caagacgaac tccaattcac tgttccttgc attctaaaac cttaaatacc agaaaacagc 6540
tttttcaaag ttgttttcaa agttggcgta taacatagta tcgacggagc cgattttgaa 6600
accgcggtga tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg 6660
cgagatcatc cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt 6720
aacatgagca aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat 6780
gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg 6840
gctggtggca ggatatattg tggtgtaaac aaattgacgc ttagacaact taataacaca 6900
ttgcggacgt ttttaatgta ctgaattaac gccgaattaa ttcgggggat ctggatttta 6960
gtactggatt ttggttttag gaattagaaa ttttattgat agaagtattt tacaaataca 7020
aatacatact aagggtttct tatatgctca acacatgagc gaaaccctat aggaacccta 7080
attcccttat ctgggaacta ctcacacatt attatggaga aactcgagct tgtcgatcga 7140
cagatccggt cggcatctac tctatttctt tgccctcgga cgagtgctgg ggcgtcggtt 7200
tccactatcg gcgagtactt ctacacagcc atcggtccag acggccgcgc ttctgcgggc 7260
gatttgtgta cgcccgacag tcccggctcc ggatcggacg attgcgtcgc atcgaccctg 7320
cgcccaagct gcatcatcga aattgccgtc aaccaagctc tgatagagtt ggtcaagacc 7380
aatgcggagc atatacgccc ggagtcgtgg cgatcctgca agctccggat gcctccgctc 7440
gaagtagcgc gtctgctgct ccatacaagc caaccacggc ctccagaaga agatgttggc 7500
gacctcgtat tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg ctgttatgcg 7560
gccattgtcc gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt gccggacttc 7620
ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg acgcactgac 7680
ggtgtcgtcc atcacagttt gccagtgata cacatgggga tcagcaatcg cgcatatgaa 7740
atcacgccat gtagtgtatt gaccgattcc ttgcggtccg aatgggccga acccgctcgt 7800
ctggctaaga tcggccgcag cgatcgcatc catagcctcc gcgaccggtt gtagaacagc 7860
gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc gggagatgca 7920
ataggtcagg ctctcgctaa actccccaat gtcaagcact tccggaatcg ggagcgcggc 7980
cgatgcaaag tgccgataaa cataacgatc tttgtagaaa ccatcggcgc agctatttac 8040
ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa gcacgagatt cttcgccctc 8100
cgagagctgc atcaggtcgg agacgctgtc gaacttttcg atcagaaact tctcgacaga 8160
cgtcgcggtg agttcaggct ttttcatatc tcattgcccc ccgggatctg cgaaagctcg 8220
agagagatag atttgtagag agagactggt gatttcagcg tgtcctctcc aaatgaaatg 8280
aacttcctta tatagaggaa ggtcttgcga aggatagtgg gattgtgcgt catcccttac 8340
gtcagtggag atatcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt 8400
ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg cagaggcatc 8460
ttgaacgata gcctttcctt tatcgcaatg atggcatttg taggtgccac cttccttttc 8520
tactgtcctt ttgatgaagt gacagatagc tgggcaatgg aatccgagga ggtttcccga 8580
tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg tatctttgat 8640
attcttggag tagacgagag tgtcgtgctc caccatgtta tcacatcaat ccacttgctt 8700
tgaagacgtg gttggaacgt cttctttttc cacgatgctc ctcgtgggtg ggggtccatc 8760
tttgggacca ctgtcggcag aggcatcttg aacgatagcc tttcctttat cgcaatgatg 8820
gcatttgtag gtgccacctt ccttttctac tgtccttttg atgaagtgac agatagctgg 8880
gcaatggaat ccgaggaggt ttcccgatat taccctttgt tgaaaagtct caatagccct 8940
ttggtcttct gagactgtat ctttgatatt cttggagtag acgagagtgt cgtgctccac 9000
catgttggca agctgctcta gccaatacgc aaaccgcctc tccccgcgcg ttggccgatt 9060
cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca 9120
attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct 9180
cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat 9240
gattacgcca agctttgatt gctagaattt gcagtaggct ttgcaattaa atacattatt 9300
cgaaaccatt agatgatgct tagtatttaa tatttatcct cagccgatga tggctgaggc 9360
cctcgagatc ataaaccagt gtaaaaattt tgagacgctc aatgccattt ggatcaagca 9420
atccctcgag attggttatt tgaaagcttc ggaaaatgcg gaaaggatgg tgagctcgag 9480
aataagatca aggctaagga aaaggaactt gccaaagtga gcaataagct cgaggaggca 9540
gataactcat tggcagaccg ggagaacaag atgcagaaaa aactaggaac cttctgcgtg 9600
agaggtgttg actacgattg ctcggtgatg tctctcatcg acctagcgaa tggatgctac 9660
tttatgcatt agttaccatt tatatttcaa ttagaaatgc atcacataat tgaactattg 9720
tgttatatga ttaaattctc tgttaaggaa tatcattcaa agggttatct ggaaactaac 9780
atatctttgt cttccagaat acggttccaa acaattcgct acgaccgact gccgtagagc 9840
aaaagaaggc caatgaatgt gaaatgagac ggctagtagt tgacgatctg gaggtttgtt 9900
cttcgtgttc ttttcattaa taccacaata ccatatgcat accctgtcat gaatattgta 9960
taatagaatg ttagtttgca taaccttcca atatgctgca aatagtttcc aacattttac 10020
tctgtgctac gatttaattc tctgttaagg aatatcgttc aaagagtcat cgcgaaagta 10080
acatcttttc tcatccaggc tgatgaatat gtaatggaac tgccagtgac cgaagattcg 10140
gaggtttgtt cttcggggac atacttcagg gccatggtca aaaactatcc cacattgcta 10200
aagaaccata aggaagaagc atttatataa gcgaaagaag cagcacataa ctggcaggag 10260
accgaggtct cggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca 10320
acttgaaaaa gtggcaccga gtcggtgctt ttttgtttta gagctagaaa tagcaagtta 10380
aaataaggct agtccgtttt tagcgcgtgc atgcctgcag gtccacaaat tcgggtcaag 10440
gcggaagcca gcgcgccacc ccacgtcagc aaatacggag gcgcggggtt gacggcgtca 10500
cccggtccta acggcgacca acaaaccagc cagaagaaat tacagtaaaa aaaaagtaaa 10560
ttgcactttg atccaccttt tattacctaa gtctcaattt ggatcaccct taaacctatc 10620
ttttcaattt gggccgggtt gtggtttgga ctaccatgaa caacttttcg tcatgtctaa 10680
cttccctttc agcaaacata tgaaccatat atagaggaga tcggccgtat actagagctg 10740
atgtgtttaa ggtcgttgat tgcacgagaa aaaaaaatcc aaatcgcaac aatagcaaat 10800
ttatctggtt caaagtgaaa agatatgttt aaaggtagtc caaagtaaaa cttatagata 10860
ataaaatgtg gtccaaagcg taattcactc aaaaaaaatc aacgagacgt gtaccaaacg 10920
gagacaaacg gcatcttctc gaaatttccc aaccgctcgc tcgcccgcct cgtcttcccg 10980
gaaaccgcgg tggtttcagc gtggcggatt ctccaagcag acggagacgt cacggcacgg 11040
gactcctccc accacccaac cgccataaat accagccccc tcatctcctc tcctcgcatc 11100
agctccaccc ccgaaaaatt tctccccaat ctcgcgaggc tctcgtcgtc gaatcgaatc 11160
ctctcgcgtc ctcaaggtac gctgcttctc ctctcctcgc ttcgtttcga ttcgatttcg 11220
gacgggtgag gttgttttgt tgctagatcc gattggtggt tagggttgtc gatgtgatta 11280
tcgtgagatg tttaggggtt gtagatctga tggttgtgat ttgggcacgg ttggttcgat 11340
aggtggaatc gtggttaggt tttgggattg gatgttggtt ctgatgattg gggggaattt 11400
ttacggttag atgaattgtt ggatgattcg attggggaaa tcggtgtaga tctgttgggg 11460
aattgtggaa ctagtcatgc ctgagtgatt ggtgcgattt gtagcgtgtt ccatcttgta 11520
ggccttgttg cgagcatgtt cagatctact gttccgctct tgattgagtt attggtgcca 11580
tgggttggtg caaacacagg ctttaatatg ttatatctgt tttgtgtttg atgtagatct 11640
gtagggtagt tcttcttaga catggttcaa ttatgtagct tgtgcgtttc gatttgattt 11700
catatgttca cagattagat aatgatgaac tcttttaatt aattgtcaat ggtaaatagg 11760
aagtcttgtc gctatatctg tcataatgat ctcatgttac tatctgccag taatttatgc 11820
taagaactat attagaatat catgttacaa tctgtagtaa tatcatgtta caatctgtag 11880
ttcatctata taatctattg tggtaatttc tttttactat ctgtgtgaag attattgcca 11940
ctagttcatt ctacttattt ctgaagttca ggatacgtgt gctgttacta cctatctgaa 12000
tacatgtgtg atgtgcctgt tactatcttt ttgaatacat gtatgttctg ttggaatatg 12060
tttgctgttt gatccgttgt tgtgtcctta atcttgtgct agttcttacc ctatctgttt 12120
ggtgattatt tcttgcagat agttatcaac aagtttgtac aaaaaagcag gcttcgaagg 12180
agatagaacc aattctctaa ggaaatactt aaccatggac tataaggacc acgacggaga 12240
ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc caaagaagaa 12300
gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca tcggcctgga 12360
catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa 12420
gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc 12480
cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag 12540
aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat 12600
ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga 12660
taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga 12720
gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga 12780
cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat 12840
cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca 12900
gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc 12960
catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc 13020
cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc 13080
caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac 13140
ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt 13200
tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac 13260
cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca 13320
ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat 13380
tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga 13440
agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct 13500
cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat 13560
cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta 13620
cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta 13680
ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga 13740
ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag 13800
cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa 13860
gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt 13920
gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga 13980
cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa 14040
gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc 14100
cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga 14160
ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga 14220
gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca 14280
gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat 14340
ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa 14400
cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa 14460
agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag 14520
ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt 14580
gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac 14640
ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga 14700
gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa 14760
gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat 14820
caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga 14880
ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt 14940
gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa 15000
gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga 15060
actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca 15120
cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat 15180
ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt 15240
ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa 15300
cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta 15360
cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg 15420
caaggctacc gccaagtact tcttctacag caacatcatg aactttttca agaccgagat 15480
taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg 15540
ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc 15600
ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat 15660
cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa 15720
gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga 15780
aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga 15840
aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt 15900
gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg 15960
gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc 16020
caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga 16080
ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat 16140
cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt 16200
gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat 16260
ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac 16320
catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca 16380
gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaaaag 16440
gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagtaagaat tcgcggccgc 16500
actcgagata tctagaccca gcttt 16525
<210> 8
<211> 16241
<212> DNA
<213> 人工序列
<400> 8
cttgtacaaa gtggttgata acagcgacta caaggatgac gatgacaagg cttagagctc 60
gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc 120
cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa 180
catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata 240
catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc 300
ggtgtcatct atgttactag atcgggaatt cactggccgt cgttttacac tggccgtcgt 360
tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 420
tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 480
gttgcgcagc ctgaatggcg aatgctagag cagcttgagc ttggatcaga ttgtcgtttc 540
ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga 600
aaagagcgtt tattagaata acggatattt aaaagggcgt gaaaaggttt atccgttcgt 660
ccatttgtat gtgcatgcca accacagggt tcccctcggg atcaaagtac tttgatccaa 720
cccctccgct gctatagtgc agtcggcttc tgacgttcag tgcagccgtc ttctgaaaac 780
gacatgtcgc acaagtccta agttacgcga caggctgccg ccctgccctt ttcctggcgt 840
tttcttgtcg cgtgttttag tcgcataaag tagaatactt gcgactagaa ccggagacat 900
tacgccatga acaagagcgc cgccgctggc ctgctgggct atgcccgcgt cagcaccgac 960
gaccaggact tgaccaacca acgggccgaa ctgcacgcgg ccggctgcac caagctgttt 1020
tccgagaaga tcaccggcac caggcgcgac cgcccggagc tggccaggat gcttgaccac 1080
ctacgccctg gcgacgttgt gacagtgacc aggctagacc gcctggcccg cagcacccgc 1140
gacctactgg acattgccga gcgcatccag gaggccggcg cgggcctgcg tagcctggca 1200
gagccgtggg ccgacaccac cacgccggcc ggccgcatgg tgttgaccgt gttcgccggc 1260
attgccgagt tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg cgaggccgcc 1320
aaggcccgag gcgtgaagtt tggcccccgc cctaccctca ccccggcaca gatcgcgcac 1380
gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag aggcggctgc actgcttggc 1440
gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac gcccaccgag 1500
gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc cctggcggcc 1560
gccgagaatg aacgccaaga ggaacaagca tgaaaccgca ccaggacggc caggacgaac 1620
cgtttttcat taccgaagag atcgaggcgg agatgatcgc ggccgggtac gtgttcgagc 1680
cgcccgcgca cgtctcaacc gtgcggctgc atgaaatcct ggccggtttg tctgatgcca 1740
agctggcggc ctggccggcc agcttggccg ctgaagaaac cgagcgccgc cgtctaaaaa 1800
ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat atgatgcgat 1860
gagtaaataa acaaatacgc aaggggaacg catgaaggtt atcgctgtac ttaaccagaa 1920
aggcgggtca ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc aactcgccgg 1980
ggccgatgtt ctgttagtcg attccgatcc ccagggcagt gcccgcgatt gggcggccgt 2040
gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg accgcgacgt 2100
gaaggccatc ggccggcgcg acttcgtagt gatcgacgga gcgccccagg cggcggactt 2160
ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc caagccctta 2220
cgacatatgg gccaccgccg acctggtgga gctggttaag cagcgcattg aggtcacgga 2280
tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc gcatcggcgg 2340
tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt cttgagtccc gtatcacgca 2400
gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc gttcttgaat cagaacccga 2460
gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa aactcatttg 2520
agttaatgag gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc cggccgtccg 2580
agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc agccatgaag 2640
cgggtcaact ttcagttgcc ggcggaggat cacaccaagc tgaagatgta cgcggtacgc 2700
caaggcaaga ccattaccga gctgctatct gaatacatcg cgcagctacc agagtaaatg 2760
agcaaatgaa taaatgagta gatgaatttt agcggctaaa ggaggcggca tggaaaatca 2820
agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg gcggttggcc 2880
aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc actggaaccc ccaagcccga 2940
ggaatcggcg tgacggtcgc aaaccatccg gcccggtaca aatcggcgcg gcgctgggtg 3000
atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc atcgaggcag 3060
aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa gaatcccggc 3120
aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac gagcaaccag 3180
attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc atcatggacg 3240
tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc cgctacgagc 3300
ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt gtgtgggatt 3360
acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga taccgggaag 3420
ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta ctcaagttct 3480
gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc attcggttaa 3540
acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc ctggtgacgg 3600
tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa accgggcggc 3660
cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca gaaggcaaga 3720
acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc atcggccgtt 3780
ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg ttgttcaaga 3840
cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc accgtgcgca 3900
agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg gggcaggctg 3960
gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc gccggttcct 4020
aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt cgaaaagcac 4080
tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg aaccggaacc 4140
cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa gtgactgata 4200
taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt aaaactctta 4260
aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag ctgcaaaaag 4320
cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg cctatcgcgg 4380
ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg cgcggacaag 4440
ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg caccctgcct cgcgcgtttc 4500
ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 4560
taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 4620
cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg 4680
cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 4740
gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc 4800
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4860
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4920
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 4980
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5040
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5100
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 5160
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5220
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5280
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5340
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5400
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5460
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5520
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5580
ggaacgaaaa ctcacgttaa gggattttgg tcatgcattc taggtactaa aacaattcat 5640
ccagtaaaat ataatatttt attttctccc aatcaggctt gatccccagt aagtcaaaaa 5700
atagctcgac atactgttct tccccgatat cctccctgat cgaccggacg cagaaggcaa 5760
tgtcatacca cttgtccgcc ctgccgcttc tcccaagatc aataaagcca cttactttgc 5820
catctttcac aaagatgttg ctgtctccca ggtcgccgtg ggaaaagaca agttcctctt 5880
cgggcttttc cgtctttaaa aaatcataca gctcgcgcgg atctttaaat ggagtgtctt 5940
cttcccagtt ttcgcaatcc acatcggcca gatcgttatt cagtaagtaa tccaattcgg 6000
ctaagcggct gtctaagcta ttcgtatagg gacaatccga tatgtcgatg gagtgaaaga 6060
gcctgatgca ctccgcatac agctcgataa tcttttcagg gctttgttca tcttcatact 6120
cttccgagca aaggacgcca tcggcctcac tcatgagcag attgctccag ccatcatgcc 6180
gttcaaagtg caggaccttt ggaacaggca gctttccttc cagccatagc atcatgtcct 6240
tttcccgttc cacatcatag gtggtccctt tataccggct gtccgtcatt tttaaatata 6300
ggttttcatt ttctcccacc agcttatata ccttagcagg agacattcct tccgtatctt 6360
ttacgcagcg gtatttttcg atcagttttt tcaattccgg tgatattctc attttagcca 6420
tttattattt ccttcctctt ttctacagta tttaaagata ccccaagaag ctaattataa 6480
caagacgaac tccaattcac tgttccttgc attctaaaac cttaaatacc agaaaacagc 6540
tttttcaaag ttgttttcaa agttggcgta taacatagta tcgacggagc cgattttgaa 6600
accgcggtga tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg 6660
cgagatcatc cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt 6720
aacatgagca aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat 6780
gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg 6840
gctggtggca ggatatattg tggtgtaaac aaattgacgc ttagacaact taataacaca 6900
ttgcggacgt ttttaatgta ctgaattaac gccgaattaa ttcgggggat ctggatttta 6960
gtactggatt ttggttttag gaattagaaa ttttattgat agaagtattt tacaaataca 7020
aatacatact aagggtttct tatatgctca acacatgagc gaaaccctat aggaacccta 7080
attcccttat ctgggaacta ctcacacatt attatggaga aactcgagct cagaagaact 7140
cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca 7200
cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg 7260
ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc 7320
ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcat 7380
cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat 7440
gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct 7500
cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc 7560
gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga 7620
gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt 7680
cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt 7740
cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct 7800
gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat 7860
agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa 7920
tcatcccggg atctgcgaaa gctcgagaga gatagatttg tagagagaga ctggtgattt 7980
cagcgtgtcc tctccaaatg aaatgaactt ccttatatag aggaaggtct tgcgaaggat 8040
agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg 8100
aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt 8160
tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc 8220
atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc 8280
aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt 8340
ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca 8400
tgttatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga 8460
tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga 8520
tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc 8580
ttttgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc 8640
tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg 8700
agtagacgag agtgtcgtgc tccaccatgt tggcaagctg ctctagccaa tacgcaaacc 8760
gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg 8820
gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca 8880
ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt 8940
tcacacagga aacagctatg accatgatta cgccaagctt ttaatctgat gctccacctg 9000
cttttgattt tctttattgg aagagtcttt aagagatatg ttaagtagca taacagtttc 9060
atcaaaaaca acatttctgt taatcacaac ttttctattt tcaggatacc ataacttata 9120
cacttttaca ctagctttat aaccaagaaa aacacattta atggtacaca attttaattt 9180
tccattatca gcatgagtat acgcaaaaca cccaaaaatc tttaaatcag aatcgtcagc 9240
aggattacta aaccatactt cttatggagt ctttttctca atagcaacga atagagacgg 9300
attgatcaaa aacatatagt tgacattgct ttggcccaaa ataactttga taagttgcca 9360
tttgacaaca tacatcgaac attctccatg atcgttctat tcattcgttc tacaatacct 9420
tttttaaaat gttcaggttc taaaatgaaa aacaatatga attgcatgaa ttgcttatat 9480
gtcctatgaa ttataaagga atgcggttga aatattccca tcgatacata catacatatt 9540
cgtgaagtat gttccaatat aatatcaata ttgggattta cgttttataa agcaacatta 9600
ttgattggta atatacatta attccaaggc aaacccaaat attttaaaat ttaacctaca 9660
actgtggtaa atcaaactta atagtaaccc gattgtaatg tgaagtcaaa tatgaaagta 9720
acattggttt atatatatat ttttctctaa attctaataa tcaagttggg ataagtgata 9780
aacactgagc ttgccacgtg tgttaacctc gttttcatca tgtgccactc caaagacatc 9840
aggcctctat tcaagctggc atggtcagga cgtggtagca tacttcaggg atctggttag 9900
aaaatatccc atatcgctaa agaactataa cacaggagcg tttatataag cgaaagaagc 9960
atcagatggg caggagaccg aggtctcggt tttagagcta gaaatagcaa gttaaaataa 10020
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gttttagagc 10080
tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcatgc ctgcaggtcc 10140
acaaattcgg gtcaaggcgg aagccagcgc gccaccccac gtcagcaaat acggaggcgc 10200
ggggttgacg gcgtcacccg gtcctaacgg cgaccaacaa accagccaga agaaattaca 10260
gtaaaaaaaa agtaaattgc actttgatcc accttttatt acctaagtct caatttggat 10320
cacccttaaa cctatctttt caatttgggc cgggttgtgg tttggactac catgaacaac 10380
ttttcgtcat gtctaacttc cctttcagca aacatatgaa ccatatatag aggagatcgg 10440
ccgtatacta gagctgatgt gtttaaggtc gttgattgca cgagaaaaaa aaatccaaat 10500
cgcaacaata gcaaatttat ctggttcaaa gtgaaaagat atgtttaaag gtagtccaaa 10560
gtaaaactta tagataataa aatgtggtcc aaagcgtaat tcactcaaaa aaaatcaacg 10620
agacgtgtac caaacggaga caaacggcat cttctcgaaa tttcccaacc gctcgctcgc 10680
ccgcctcgtc ttcccggaaa ccgcggtggt ttcagcgtgg cggattctcc aagcagacgg 10740
agacgtcacg gcacgggact cctcccacca cccaaccgcc ataaatacca gccccctcat 10800
ctcctctcct cgcatcagct ccacccccga aaaatttctc cccaatctcg cgaggctctc 10860
gtcgtcgaat cgaatcctct cgcgtcctca aggtacgctg cttctcctct cctcgcttcg 10920
tttcgattcg atttcggacg ggtgaggttg ttttgttgct agatccgatt ggtggttagg 10980
gttgtcgatg tgattatcgt gagatgttta ggggttgtag atctgatggt tgtgatttgg 11040
gcacggttgg ttcgataggt ggaatcgtgg ttaggttttg ggattggatg ttggttctga 11100
tgattggggg gaatttttac ggttagatga attgttggat gattcgattg gggaaatcgg 11160
tgtagatctg ttggggaatt gtggaactag tcatgcctga gtgattggtg cgatttgtag 11220
cgtgttccat cttgtaggcc ttgttgcgag catgttcaga tctactgttc cgctcttgat 11280
tgagttattg gtgccatggg ttggtgcaaa cacaggcttt aatatgttat atctgttttg 11340
tgtttgatgt agatctgtag ggtagttctt cttagacatg gttcaattat gtagcttgtg 11400
cgtttcgatt tgatttcata tgttcacaga ttagataatg atgaactctt ttaattaatt 11460
gtcaatggta aataggaagt cttgtcgcta tatctgtcat aatgatctca tgttactatc 11520
tgccagtaat ttatgctaag aactatatta gaatatcatg ttacaatctg tagtaatatc 11580
atgttacaat ctgtagttca tctatataat ctattgtggt aatttctttt tactatctgt 11640
gtgaagatta ttgccactag ttcattctac ttatttctga agttcaggat acgtgtgctg 11700
ttactaccta tctgaataca tgtgtgatgt gcctgttact atctttttga atacatgtat 11760
gttctgttgg aatatgtttg ctgtttgatc cgttgttgtg tccttaatct tgtgctagtt 11820
cttaccctat ctgtttggtg attatttctt gcagatagtt atcaacaagt ttgtacaaaa 11880
aagcaggctt cgaaggagat agaaccaatt ctctaaggaa atacttaacc atggactata 11940
aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 12000
tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 12060
acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 12120
acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 12180
agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 12240
agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 12300
tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 12360
tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 12420
aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 12480
gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 12540
ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 12600
tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 12660
gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 12720
tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 12780
gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 12840
agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 12900
agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 12960
tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 13020
acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 13080
agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 13140
gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 13200
gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 13260
tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 13320
ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 13380
ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 13440
tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 13500
gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 13560
agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 13620
ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 13680
aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 13740
aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 13800
atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 13860
acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 13920
tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 13980
acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 14040
agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 14100
ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 14160
aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 14220
ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 14280
acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 14340
gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 14400
aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 14460
agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 14520
accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 14580
gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 14640
gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 14700
agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 14760
gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 14820
ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 14880
agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 14940
atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 15000
acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 15060
aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 15120
gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 15180
ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 15240
caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 15300
aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 15360
tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 15420
actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 15480
tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 15540
ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 15600
agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 15660
agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 15720
aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 15780
tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 15840
acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 15900
ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 15960
aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 16020
agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 16080
ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 16140
tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt 16200
aagaattcgc ggccgcactc gagatatcta gacccagctt t 16241
<210> 9
<211> 16250
<212> DNA
<213> 人工序列
<400> 9
cttgtacaaa gtggttgata acagcgacta caaggatgac gatgacaagg cttagagctc 60
gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc 120
cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa 180
catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata 240
catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc 300
ggtgtcatct atgttactag atcgggaatt cactggccgt cgttttacac tggccgtcgt 360
tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 420
tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 480
gttgcgcagc ctgaatggcg aatgctagag cagcttgagc ttggatcaga ttgtcgtttc 540
ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga 600
aaagagcgtt tattagaata acggatattt aaaagggcgt gaaaaggttt atccgttcgt 660
ccatttgtat gtgcatgcca accacagggt tcccctcggg atcaaagtac tttgatccaa 720
cccctccgct gctatagtgc agtcggcttc tgacgttcag tgcagccgtc ttctgaaaac 780
gacatgtcgc acaagtccta agttacgcga caggctgccg ccctgccctt ttcctggcgt 840
tttcttgtcg cgtgttttag tcgcataaag tagaatactt gcgactagaa ccggagacat 900
tacgccatga acaagagcgc cgccgctggc ctgctgggct atgcccgcgt cagcaccgac 960
gaccaggact tgaccaacca acgggccgaa ctgcacgcgg ccggctgcac caagctgttt 1020
tccgagaaga tcaccggcac caggcgcgac cgcccggagc tggccaggat gcttgaccac 1080
ctacgccctg gcgacgttgt gacagtgacc aggctagacc gcctggcccg cagcacccgc 1140
gacctactgg acattgccga gcgcatccag gaggccggcg cgggcctgcg tagcctggca 1200
gagccgtggg ccgacaccac cacgccggcc ggccgcatgg tgttgaccgt gttcgccggc 1260
attgccgagt tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg cgaggccgcc 1320
aaggcccgag gcgtgaagtt tggcccccgc cctaccctca ccccggcaca gatcgcgcac 1380
gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag aggcggctgc actgcttggc 1440
gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac gcccaccgag 1500
gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc cctggcggcc 1560
gccgagaatg aacgccaaga ggaacaagca tgaaaccgca ccaggacggc caggacgaac 1620
cgtttttcat taccgaagag atcgaggcgg agatgatcgc ggccgggtac gtgttcgagc 1680
cgcccgcgca cgtctcaacc gtgcggctgc atgaaatcct ggccggtttg tctgatgcca 1740
agctggcggc ctggccggcc agcttggccg ctgaagaaac cgagcgccgc cgtctaaaaa 1800
ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat atgatgcgat 1860
gagtaaataa acaaatacgc aaggggaacg catgaaggtt atcgctgtac ttaaccagaa 1920
aggcgggtca ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc aactcgccgg 1980
ggccgatgtt ctgttagtcg attccgatcc ccagggcagt gcccgcgatt gggcggccgt 2040
gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg accgcgacgt 2100
gaaggccatc ggccggcgcg acttcgtagt gatcgacgga gcgccccagg cggcggactt 2160
ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc caagccctta 2220
cgacatatgg gccaccgccg acctggtgga gctggttaag cagcgcattg aggtcacgga 2280
tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc gcatcggcgg 2340
tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt cttgagtccc gtatcacgca 2400
gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc gttcttgaat cagaacccga 2460
gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa aactcatttg 2520
agttaatgag gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc cggccgtccg 2580
agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc agccatgaag 2640
cgggtcaact ttcagttgcc ggcggaggat cacaccaagc tgaagatgta cgcggtacgc 2700
caaggcaaga ccattaccga gctgctatct gaatacatcg cgcagctacc agagtaaatg 2760
agcaaatgaa taaatgagta gatgaatttt agcggctaaa ggaggcggca tggaaaatca 2820
agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg gcggttggcc 2880
aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc actggaaccc ccaagcccga 2940
ggaatcggcg tgacggtcgc aaaccatccg gcccggtaca aatcggcgcg gcgctgggtg 3000
atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc atcgaggcag 3060
aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa gaatcccggc 3120
aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac gagcaaccag 3180
attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc atcatggacg 3240
tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc cgctacgagc 3300
ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt gtgtgggatt 3360
acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga taccgggaag 3420
ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta ctcaagttct 3480
gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc attcggttaa 3540
acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc ctggtgacgg 3600
tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa accgggcggc 3660
cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca gaaggcaaga 3720
acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc atcggccgtt 3780
ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg ttgttcaaga 3840
cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc accgtgcgca 3900
agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg gggcaggctg 3960
gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc gccggttcct 4020
aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt cgaaaagcac 4080
tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg aaccggaacc 4140
cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa gtgactgata 4200
taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt aaaactctta 4260
aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag ctgcaaaaag 4320
cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg cctatcgcgg 4380
ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg cgcggacaag 4440
ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg caccctgcct cgcgcgtttc 4500
ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 4560
taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 4620
cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg 4680
cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 4740
gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc 4800
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4860
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4920
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 4980
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5040
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5100
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 5160
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5220
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5280
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5340
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5400
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5460
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5520
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5580
ggaacgaaaa ctcacgttaa gggattttgg tcatgcattc taggtactaa aacaattcat 5640
ccagtaaaat ataatatttt attttctccc aatcaggctt gatccccagt aagtcaaaaa 5700
atagctcgac atactgttct tccccgatat cctccctgat cgaccggacg cagaaggcaa 5760
tgtcatacca cttgtccgcc ctgccgcttc tcccaagatc aataaagcca cttactttgc 5820
catctttcac aaagatgttg ctgtctccca ggtcgccgtg ggaaaagaca agttcctctt 5880
cgggcttttc cgtctttaaa aaatcataca gctcgcgcgg atctttaaat ggagtgtctt 5940
cttcccagtt ttcgcaatcc acatcggcca gatcgttatt cagtaagtaa tccaattcgg 6000
ctaagcggct gtctaagcta ttcgtatagg gacaatccga tatgtcgatg gagtgaaaga 6060
gcctgatgca ctccgcatac agctcgataa tcttttcagg gctttgttca tcttcatact 6120
cttccgagca aaggacgcca tcggcctcac tcatgagcag attgctccag ccatcatgcc 6180
gttcaaagtg caggaccttt ggaacaggca gctttccttc cagccatagc atcatgtcct 6240
tttcccgttc cacatcatag gtggtccctt tataccggct gtccgtcatt tttaaatata 6300
ggttttcatt ttctcccacc agcttatata ccttagcagg agacattcct tccgtatctt 6360
ttacgcagcg gtatttttcg atcagttttt tcaattccgg tgatattctc attttagcca 6420
tttattattt ccttcctctt ttctacagta tttaaagata ccccaagaag ctaattataa 6480
caagacgaac tccaattcac tgttccttgc attctaaaac cttaaatacc agaaaacagc 6540
tttttcaaag ttgttttcaa agttggcgta taacatagta tcgacggagc cgattttgaa 6600
accgcggtga tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg 6660
cgagatcatc cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt 6720
aacatgagca aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat 6780
gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg 6840
gctggtggca ggatatattg tggtgtaaac aaattgacgc ttagacaact taataacaca 6900
ttgcggacgt ttttaatgta ctgaattaac gccgaattaa ttcgggggat ctggatttta 6960
gtactggatt ttggttttag gaattagaaa ttttattgat agaagtattt tacaaataca 7020
aatacatact aagggtttct tatatgctca acacatgagc gaaaccctat aggaacccta 7080
attcccttat ctgggaacta ctcacacatt attatggaga aactcgagct cagaagaact 7140
cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca 7200
cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg 7260
ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc 7320
ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcat 7380
cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat 7440
gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct 7500
cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc 7560
gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga 7620
gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt 7680
cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt 7740
cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct 7800
gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat 7860
agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa 7920
tcatcccggg atctgcgaaa gctcgagaga gatagatttg tagagagaga ctggtgattt 7980
cagcgtgtcc tctccaaatg aaatgaactt ccttatatag aggaaggtct tgcgaaggat 8040
agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg 8100
aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt 8160
tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc 8220
atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc 8280
aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt 8340
ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca 8400
tgttatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga 8460
tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga 8520
tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc 8580
ttttgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc 8640
tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg 8700
agtagacgag agtgtcgtgc tccaccatgt tggcaagctg ctctagccaa tacgcaaacc 8760
gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg 8820
gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca 8880
ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt 8940
tcacacagga aacagctatg accatgatta cgccaagctt tgattgctag aatttgcagt 9000
aggctttgca attaaataca ttattcgaaa ccattagatg atgcttagta tttaatattt 9060
atcctcagcc gatgatggct gaggccctcg agatcataaa ccagtgtaaa aattttgaga 9120
cgctcaatgc catttggatc aagcaatccc tcgagattgg ttatttgaaa gcttcggaaa 9180
atgcggaaag gatggtgagc tcgagaataa gatcaaggct aaggaaaagg aacttgccaa 9240
agtgagcaat aagctcgagg aggcagataa ctcattggca gaccgggaga acaagatgca 9300
gaaaaaacta ggaaccttct gcgtgagagg tgttgactac gattgctcgg tgatgtctct 9360
catcgaccta gcgaatggat gctactttat gcattagtta ccatttatat ttcaattaga 9420
aatgcatcac ataattgaac tattgtgtta tatgattaaa ttctctgtta aggaatatca 9480
ttcaaagggt tatctggaaa ctaacatatc tttgtcttcc agaatacggt tccaaacaat 9540
tcgctacgac cgactgccgt agagcaaaag aaggccaatg aatgtgaaat gagacggcta 9600
gtagttgacg atctggaggt ttgttcttcg tgttcttttc attaatacca caataccata 9660
tgcataccct gtcatgaata ttgtataata gaatgttagt ttgcataacc ttccaatatg 9720
ctgcaaatag tttccaacat tttactctgt gctacgattt aattctctgt taaggaatat 9780
cgttcaaaga gtcatcgcga aagtaacatc ttttctcatc caggctgatg aatatgtaat 9840
ggaactgcca gtgaccgaag attcggaggt ttgttcttcg gggacatact tcagggccat 9900
ggtcaaaaac tatcccacat tgctaaagaa ccataaggaa gaagcattta tataagcgaa 9960
agaagcagca cataactggc aggagaccga ggtctcggtt ttagagctag aaatagcaag 10020
ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg 10080
ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcatgcc 10140
tgcaggtcca caaattcggg tcaaggcgga agccagcgcg ccaccccacg tcagcaaata 10200
cggaggcgcg gggttgacgg cgtcacccgg tcctaacggc gaccaacaaa ccagccagaa 10260
gaaattacag taaaaaaaaa gtaaattgca ctttgatcca ccttttatta cctaagtctc 10320
aatttggatc acccttaaac ctatcttttc aatttgggcc gggttgtggt ttggactacc 10380
atgaacaact tttcgtcatg tctaacttcc ctttcagcaa acatatgaac catatataga 10440
ggagatcggc cgtatactag agctgatgtg tttaaggtcg ttgattgcac gagaaaaaaa 10500
aatccaaatc gcaacaatag caaatttatc tggttcaaag tgaaaagata tgtttaaagg 10560
tagtccaaag taaaacttat agataataaa atgtggtcca aagcgtaatt cactcaaaaa 10620
aaatcaacga gacgtgtacc aaacggagac aaacggcatc ttctcgaaat ttcccaaccg 10680
ctcgctcgcc cgcctcgtct tcccggaaac cgcggtggtt tcagcgtggc ggattctcca 10740
agcagacgga gacgtcacgg cacgggactc ctcccaccac ccaaccgcca taaataccag 10800
ccccctcatc tcctctcctc gcatcagctc cacccccgaa aaatttctcc ccaatctcgc 10860
gaggctctcg tcgtcgaatc gaatcctctc gcgtcctcaa ggtacgctgc ttctcctctc 10920
ctcgcttcgt ttcgattcga tttcggacgg gtgaggttgt tttgttgcta gatccgattg 10980
gtggttaggg ttgtcgatgt gattatcgtg agatgtttag gggttgtaga tctgatggtt 11040
gtgatttggg cacggttggt tcgataggtg gaatcgtggt taggttttgg gattggatgt 11100
tggttctgat gattgggggg aatttttacg gttagatgaa ttgttggatg attcgattgg 11160
ggaaatcggt gtagatctgt tggggaattg tggaactagt catgcctgag tgattggtgc 11220
gatttgtagc gtgttccatc ttgtaggcct tgttgcgagc atgttcagat ctactgttcc 11280
gctcttgatt gagttattgg tgccatgggt tggtgcaaac acaggcttta atatgttata 11340
tctgttttgt gtttgatgta gatctgtagg gtagttcttc ttagacatgg ttcaattatg 11400
tagcttgtgc gtttcgattt gatttcatat gttcacagat tagataatga tgaactcttt 11460
taattaattg tcaatggtaa ataggaagtc ttgtcgctat atctgtcata atgatctcat 11520
gttactatct gccagtaatt tatgctaaga actatattag aatatcatgt tacaatctgt 11580
agtaatatca tgttacaatc tgtagttcat ctatataatc tattgtggta atttcttttt 11640
actatctgtg tgaagattat tgccactagt tcattctact tatttctgaa gttcaggata 11700
cgtgtgctgt tactacctat ctgaatacat gtgtgatgtg cctgttacta tctttttgaa 11760
tacatgtatg ttctgttgga atatgtttgc tgtttgatcc gttgttgtgt ccttaatctt 11820
gtgctagttc ttaccctatc tgtttggtga ttatttcttg cagatagtta tcaacaagtt 11880
tgtacaaaaa agcaggcttc gaaggagata gaaccaattc tctaaggaaa tacttaacca 11940
tggactataa ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg 12000
acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg 12060
acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca 12120
ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca 12180
gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca 12240
cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc 12300
tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg 12360
aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca 12420
tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac 12480
tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga 12540
tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg 12600
acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca 12660
acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc 12720
tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga 12780
ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg 12840
ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga 12900
tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc 12960
tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga 13020
tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc 13080
agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct 13140
acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa 13200
agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc 13260
agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca 13320
ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga 13380
agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat 13440
tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg 13500
tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc 13560
tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata 13620
acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg 13680
gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga 13740
agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg 13800
gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca 13860
aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga 13920
ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc 13980
tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc 14040
tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt 14100
tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc 14160
tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg 14220
agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga 14280
aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg 14340
aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga 14400
agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg 14460
aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata 14520
tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg 14580
tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca 14640
agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact 14700
actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca 14760
aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg 14820
tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta 14880
agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc 14940
tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc 15000
accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc 15060
ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga 15120
tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca 15180
tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc 15240
tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca 15300
ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga 15360
caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca 15420
gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt 15480
ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag 15540
agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc 15600
tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact 15660
ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga 15720
agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact 15780
atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc 15840
acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc 15900
tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca 15960
tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg 16020
ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg 16080
tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc 16140
tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa 16200
agaaaaagta agaattcgcg gccgcactcg agatatctag acccagcttt 16250
<210> 10
<211> 795
<212> DNA
<213> 大肠埃希氏菌（Escherichia coli）
<400> 10
tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat 60
accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg 120
ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa 180
tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac 240
gacgagatca tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc 300
gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt 360
acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag 420
cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg 480
agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc 540
agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg 600
cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac 660
cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg 720
tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc 780
atcttgttca atcat 795

Claims (6)

1.一种陆地棉高效转化载体pRGEB32-GhU6.7-NPTⅡ，其特征在于，该载体通过下列步骤制备获得：
(1)构建陆地棉内源RNA聚合酶三型U6启动子pGhU6-7I，该启动子的核苷酸序列如SEQID NO：1所示，通过以下步骤制备得道：
1)通过与拟南芥U6-26snRNA基因同源比对得到陆地棉U6启动子，用Primer5软件设计引物；
2)以陆地棉YZ1基因组DNA为模板，PCR扩增，然后用载体pGEM-T Easy克隆测序验证，得到启动子pGhU6-7，其核苷酸序列如SEQ ID NO：2所示；
3)利用重叠延伸PCR将步骤2)中所述的pGhU6-7启动子的第319位的碱基C突变为碱基G，删除pGhU6-7启动子所含有的BsaⅠ位点，得到pGhU6-7I启动子，其核苷酸序列如SEQ IDNO：1所示；
(2)利用SbfⅠ、HindⅢ对载体pRGEB32进行酶切，与pGhU6-7I启动子进行连接，得到如SEQ ID NO：6所示序列的中间载体pRGEB32-GhU6.7；
(3)利用Primer5软件对pHellsgate4载体上的真核NPTⅡ序列设计引物，所述的真核NPTⅡ序列核苷酸序列如SEQ ID NO：10所示，然后以pHellsgate4质粒为模板进行PCR扩增；
(4)将pRGEB32载体用XmalⅠ、PspxⅠ酶切，然后将步骤(3)中的NPTⅡ序列引入步骤(2)中的中间载体pRGEB32-GhU6.7，得到如SEQ ID NO：8所示的表达载体pRGEB32-GhU6.7-NPTⅡ。
2.一种陆地棉高效转化的载体pRGEB32-GhU6.9-NPTⅡ，其特征在于，该载体通过下列步骤制备获得：
(1)构建陆地棉内源RNA聚合酶三型U6启动子pGhU6-9I，其核苷酸序列如SEQ ID NO：3所示，通过以下步骤制备得到：
1)通过与拟南芥U6-26snRNA基因同源比对得到多个陆地棉U6启动子，用Primer5软件设计引物；
2)以陆地棉YZ1基因组DNA为模板，PCR扩增，然后用载体pGEM-T Easy克隆测序验证，得到pGhU6-9启动子，其序列如SEQ ID NO：4所示；
3)利用重叠延伸PCR将步骤2)中的载体pGhU6-9第616位的碱基C突变为碱基G，删除pGhU6-9启动子所含有的的BsaⅠ位点，得到启动子pGhU6-9I，其核苷酸序列如SEQ ID NO：3所示；
(2)利用SbfⅠ、HindⅢ对pRGEB32进行酶切，与启动子pGhU6-9I连接，得到如SEQ ID NO：7所示的中间载体pRGEB32-GhU6.9；
(3)利用Primer5对pHellsgate4载体上的真核NPTⅡ序列设计引物，所述的真核NPTⅡ序列核苷酸序列如SEQ ID NO：10所示，然后以pHellsgate4质粒为模板进行PCR扩增；
(4)将PRGEB32载体用XmaⅠ、PspxⅠ酶切，然后将步骤(3)中的NPTⅡ序列(其核苷酸序列如SEQ ID NO：10所示)引入步骤2)中的中间载体pRGEB32-GhU6.9，得到如SEQ ID NO：9所示的表达载体pRGEB32-GhU6.9-NPTⅡ。
3.权利要求1所述的高效转化载体pRGEB32-GhU6.7-NPTⅡ在陆地棉基因组编辑中的应用。
4.权利要求2所述的高效转化载体pRGEB32-GhU6.9-NPTⅡ的在陆地棉基因组编辑中的应用。
5.一种分离的陆地棉内源RNA聚合酶三型U6启动子pGhU6-7I，其特征在于，该启动子的核苷酸序列如SEQ ID NO：1所示。
6.一种分离的陆地棉内源RNA聚合酶三型U6启动子pGhU6-9I，其核苷酸序列如SEQ IDNO：3所示。
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109517846A (zh) * 2018-11-21 2019-03-26 华中农业大学 基于CRISPR/Cas9系统高通量构建棉花突变体库的方法
CN109593781A (zh) * 2018-12-20 2019-04-09 华中农业大学 陆地棉基因组的精准高效编辑方法
CN111378684A (zh) * 2020-03-15 2020-07-07 华中农业大学 一种热诱导的基因编辑系统CRISPR-Cas12b在陆地棉中的应用
CN111575311A (zh) * 2020-04-15 2020-08-25 南京农业大学 一种基于基因枪介导的棉花基因编辑方法及应用
CN113122571A (zh) * 2021-05-27 2021-07-16 华中农业大学 一种新型CRISPR/Cas9基因编辑载体的载体制备方法
CN113122570A (zh) * 2021-05-27 2021-07-16 华中农业大学 一种新型CRISPR/Cas9基因编辑载体在棉花中的使用方法
CN113174401A (zh) * 2021-06-01 2021-07-27 华中农业大学 基因编辑介导的外源基因敲入到棉花基因组的方法及载体
CN113278647A (zh) * 2021-05-25 2021-08-20 华中农业大学 一种陆地棉基因组的高效定向基因调控的编辑方法
CN113337539A (zh) * 2021-05-27 2021-09-03 华中农业大学 一种适用于陆地棉的基因精准高效编辑的方法
CN113832180A (zh) * 2021-08-03 2021-12-24 华中农业大学 CRISPR/Cas13b介导的棉花RNA转录调控方法
CN116463375A (zh) * 2023-06-09 2023-07-21 新疆农垦科学院 一种利用基因编辑技术创制棉花同时矮化和黄化材料的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1936003A (zh) * 2006-10-13 2007-03-28 南京农业大学 陆地棉纤维特异性表达基因GhMYB9启动子
WO2015131101A1 (en) * 2014-02-27 2015-09-03 Monsanto Technology Llc Compositions and methods for site directed genomic modification
CN105779448A (zh) * 2015-12-04 2016-07-20 新疆农业大学 一种棉花启动子GbU6-7PS及应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1936003A (zh) * 2006-10-13 2007-03-28 南京农业大学 陆地棉纤维特异性表达基因GhMYB9启动子
WO2015131101A1 (en) * 2014-02-27 2015-09-03 Monsanto Technology Llc Compositions and methods for site directed genomic modification
CN105779448A (zh) * 2015-12-04 2016-07-20 新疆农业大学 一种棉花启动子GbU6-7PS及应用

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN109593781A (zh) * 2018-12-20 2019-04-09 华中农业大学 陆地棉基因组的精准高效编辑方法
CN109593781B (zh) * 2018-12-20 2021-02-23 华中农业大学 陆地棉基因组的精准高效编辑方法
CN111378684A (zh) * 2020-03-15 2020-07-07 华中农业大学 一种热诱导的基因编辑系统CRISPR-Cas12b在陆地棉中的应用
CN111378684B (zh) * 2020-03-15 2023-06-27 华中农业大学 一种热诱导的基因编辑系统CRISPR-Cas12b在陆地棉中的应用
CN111575311A (zh) * 2020-04-15 2020-08-25 南京农业大学 一种基于基因枪介导的棉花基因编辑方法及应用
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CN113122570A (zh) * 2021-05-27 2021-07-16 华中农业大学 一种新型CRISPR/Cas9基因编辑载体在棉花中的使用方法
CN113337539A (zh) * 2021-05-27 2021-09-03 华中农业大学 一种适用于陆地棉的基因精准高效编辑的方法
CN113122571A (zh) * 2021-05-27 2021-07-16 华中农业大学 一种新型CRISPR/Cas9基因编辑载体的载体制备方法
CN113174401A (zh) * 2021-06-01 2021-07-27 华中农业大学 基因编辑介导的外源基因敲入到棉花基因组的方法及载体
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