CN109266686A - 一种基因组核苷酸定点替换的方法 - Google Patents

一种基因组核苷酸定点替换的方法 Download PDF

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CN109266686A
CN109266686A CN201811123556.4A CN201811123556A CN109266686A CN 109266686 A CN109266686 A CN 109266686A CN 201811123556 A CN201811123556 A CN 201811123556A CN 109266686 A CN109266686 A CN 109266686A
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杨进孝
杨永星
宋伟
宋金岭
李宏潮
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

本发明公开了一种基因组核苷酸定点替换的方法。本发明保护一种碱基编辑系统，包括融合蛋白和sgRNA；所述融合蛋白由切刻酶、胞嘧啶核苷脱氨酶PmCDA1和尿嘧啶DNA糖化酶抑制剂UGI组成；所述切刻酶如序列表的序列15自N端第1‑1423位氨基酸所示；所述sgRNA的核苷酸序列如序列表的序列3自5’端第441‑526位所示。本发明首次将高保真酶eSpCas9(1.1)n和优化的sgRNA运用到PmCDA1介导的碱基编辑系统中，大部分靶点提高了C·T碱基替换的替换效率并大幅度降低了脱靶效应。

Description

一种基因组核苷酸定点替换的方法
技术领域
本发明涉及一种基因组核苷酸定点替换的方法。
背景技术
随着CRISPR-Cas9(the clustered regularly interspaced short palindromicrepeats-CRISPR-associatedprotein 9)技术的出现和发展，目前已经成为强有力的基因组编辑手段，被广泛应用到很多组织和细胞中。CRISPR/Cas9protein–RNA复合物通过向导RNA(guide RNA)定位于靶点上，切割产生DNA双链断裂(DSB，dsDNAbreak)，而后，生物体会本能的启动DNA修复机制修复DSB。一般有两种修复机制，一种是非同源末端连接(NHEJ，non-homologous endjoining)，一种是同源重组(HDR，homology-directed repair)，通常情况下，NHEJ修复占大多数，因此，修复产生的随机的indels(insertions or deletions)比精确修复高很多。对于碱基精确替换，因为HDR效率低以及需要DNA模板，所以使用HDR实现碱基精确替换的应用受到很大的限制。
2016年，David Liu和Akihiko Kondo两个实验室分别独立报道了两个不同类型的胞嘧啶碱基编辑器(CBE，cytosine base editor)，原理都是通过使用胞苷脱氨酶实现直接对单个的C(胞嘧啶，Cytosine)碱基进行编辑，而不再通过产生DSB和启动HDR修复，大大提高了C替换为T(胸腺嘧啶，Thymine)的碱基编辑效率。PmCDA1(activation-inducedcytidine deaminase(AID)ortholog from sea lamprey)就是其中使用的一种胞苷脱氨酶。在所测试的PmCDA1编辑器中，SpCas9n(D10A)&PmCDA1&UGI(尿嘧啶DNA糖化酶抑制剂，uracil DNA glycosylase inhibitor)碱基编辑系统的平均突变率较高，一是因为UGI可以抑制UDG(尿嘧啶DNA糖化酶，uracil DNAglycosylase)催化清除DNA中U(尿嘧啶，Uracil)，二是因为SpCas9n(D10A)在非编辑链上产生切口，诱导真核错配修复机制或long-patchBER(base-excision repair)修复机制，促使U:G错配更多的偏好性修复成U:A。SpCas9n(D10A)连带着PmCDA1通过sgRNA定位到靶点，PmCDA1催化非配对的单链DNA上的C发生胞嘧啶脱氨反应变成U，通过DNA的修复使得U与A(腺嘌呤，Adenine)配对，又通过DNA复制，最终使得T与A配对，从而实现了C到T的转换。
植物中使用SpCas9进行基因组编辑时具有一定的脱靶效应，SpCas9n(D10A)与PmCDA融合使用进行碱基编辑，即使用SpCas9n(D10A)&PmCDA1&UGI碱基编辑系统时可能存在潜在的脱靶风险。虽然植物不同于动物，在后代可以通过遗传分离去除脱靶位点，但是由于一些潜在的脱靶位点可能是未知的，所以，后代中就很难有目的性的去除。因此，降低脱靶效应也是植物中一直以来的技术优化方向。
发明内容
本发明的目的是提供一种基因组核苷酸定点替换的方法。
本发明提供了一种碱基编辑系统，包括融合蛋白和sgRNA；
所述融合蛋白由切刻酶、胞嘧啶核苷脱氨酶PmCDA1和尿嘧啶DNA糖化酶抑制剂UGI组成；所述切刻酶如序列表的序列15自N端第1-1423位氨基酸所示；
所述sgRNA的核苷酸序列如序列表的序列3自5’端第441-526位所示。
所述胞嘧啶核苷脱氨酶PmCDA1如序列表的序列15自N端第1521-1728位氨基酸所示。
所述尿嘧啶DNA糖化酶抑制剂UGI如序列表的序列15自N端第1736-1833位氨基酸所示。
本发明还保护表达所述的碱基编辑系统的重组表达载体、表达盒、重组细胞或重组菌。表达所述融合蛋白的表达盒具体可为表达盒甲。当靶序列如表1所示时，表达所述sgRNA的表达盒具体可为表达盒乙。
本发明还保护一种用于基因组碱基替换的重组表达载体，包括表达盒甲和表达盒乙；所述表达盒甲表达所述融合蛋白；所述表达盒乙包括n个元件乙；所述元件乙包括所述sgRNA和靶序列；所述重组表达载体可靶向n个不同的靶序列进行碱基替换。
所述元件乙还包括pre-tRNA的核苷酸序列；所述pre-tRNA的核苷酸序列如序列表的序列3自5’端第344-420所示。
所述元件乙自5’端依次为pre-tRNA的核苷酸序列、靶点序列和sgRNA的核苷酸序列。
以上任一所述表达盒甲中由启动子甲启动所述切刻酶的编码基因、胞嘧啶核苷脱氨酶PmCDA1的编码基因和尿嘧啶DNA糖化酶抑制剂UGI的编码基因表达。所述表达盒甲自5’端依次包括启动子甲、切刻酶的编码基因、胞嘧啶核苷脱氨酶PmCDA1的编码基因、尿嘧啶DNA糖化酶抑制剂UGI的编码基因和终止子甲。所述启动子甲具体可为OsUbq3启动子。所述OsUbq3启动子的核苷酸序列如序列表的序列1自5’端第1290-3003位所示。所述终止子甲具体可为CaMV35S终止子。所述CaMV35S终止子的核苷酸序列如序列表的序列1自5’端第8518-8712位。所述切刻酶的编码基因如序列表的序列1自5’端第3010-7278位所示。所述胞嘧啶核苷脱氨酶PmCDA1的编码基因如序列表的序列1自5’端第7570-8193位所示。所述尿嘧啶DNA糖化酶抑制剂UGI编码基因如序列表的序列1自5’端第8215-8511位所示。所述表达盒甲具体可如序列表的序列1自5’端第1290-8712位所示。
以上任一所述表达盒乙中由启动子乙启动所述元件乙表达。所述表达盒乙自5’端依次为启动子乙、元件乙和终止子乙。所述启动子乙具体可为OsU3启动子。所述OsU3启动子的核苷酸序列如序列表的序列3自5’端第1-337位所示。所述终止子乙具体可为OsU3终止子。所述OsU3终止子的核苷酸序列如序列表的序列3自5’端第893-1183位所示。当靶序列如表1所示时，所述表达盒乙具体可如序列表的序列3所示。
当靶序列如表1所示时，以上任一所述重组表达载体具体可为将序列表的序列1自5’端第131位-1283位替换为序列表的序列3得到的环状质粒。
本发明还保护所述融合蛋白。
本发明还保护所述融合蛋白的编码基因。
所述融合蛋白的编码基因具体可如序列表的序列1自5’端第3010-8511位所示(其中，第3010-7278位为切刻酶的编码基因，第7570-8193位为胞嘧啶核苷脱氨酶PmCDA1的编码基因，第8215-8511位为尿嘧啶DNA糖化酶抑制剂UGI的编码基因)。
本发明还保护一种植物基因组碱基替换的方法，包括如下步骤：利用以上任一所述碱基编辑系统完成植物基因组碱基替换。
本发明还保护一种植物基因组碱基替换的方法，包括如下步骤：将以上任一所述的重组表达载体导入目的植物，实现植物基因组碱基替换。
本发明还保护所述碱基编辑系统，或，以上任一所述的重组表达载体、表达盒、重组细胞或重组菌，或，所述的融合蛋白，在植物基因组碱基替换中的应用。
以上任一所述碱基替换为将碱基C替换为T。
以上任一所述所述植物具体可为水稻，更具体可为日本晴水稻。
本发明首次将高保真酶eSpCas9(1.1)n和优化的sgRNA运用到PmCDA1介导的碱基编辑系统中，大部分靶点提高了C·T碱基替换的替换效率；sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI相对于sgRNA(wt)+SpCas9n&PmCDA1&UGI,大大降低了C·T碱基替换的替换效率；而sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI则能够增加C·T碱基替换的替换效率，使得C·T碱基替换的替换效率与sgRNA(wt)+SpCas9n&PmCDA1&UGI相当甚至更高；sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI脱靶效率几乎为零，相对于sgRNA(wt)+SpCas9n&PmCDA1&UGI，大幅度降低了脱靶效应。
附图说明
图1为sgRNA(wt)+SpCas9n&PmCDA1&UGI和sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI的C·T碱基替换效率。
图2为sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI和sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI的C·T碱基替换效率。
图3为脱靶效率检测结果。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
日本晴水稻：参考文献：梁卫红,王高华,杜京尧,等.硝普钠及其光解产物对日本晴水稻幼苗生长和5种激素标记基因表达的影响[J].河南师范大学学报(自然版),2017(2):48-52.；公众可以从北京市农林科学院获得。
下述实施例中靶标基因、靶点名称和序列如表1所示。
表1
靶标基因 靶点名称 靶点序列
OsALS CS650 cgcgtccatggagatccacc
OsCDC48 CS651 gaccagccagcgtctggcgc
OsNRT1.1B CS652 cggcgacggcgagcaagtgg
实施例1、sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI系统C·T碱基替换效率
一、基因组编辑载体的构建
1、sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI：人工合成序列表的序列1所示的环状质粒。
序列表的序列1包括如下三个表达盒：
序列1自5’端第131位-1283位为表达盒I，其中，第131-467位为OsU3启动子的核苷酸序列，第474-550位为pre-tRNA的核苷酸序列，第551-570位为CS650靶点的核苷酸序列，第571-646位为sgRNA(wt)的核苷酸序列，第647-723位为pre-tRNA的核苷酸序列，第724-743位为CS651靶点的核苷酸序列，第744-819位为sgRNA(wt)的核苷酸序列，第820-896位为pre-tRNA的核苷酸序列，第897-916位为CS652靶点的核苷酸序列，第917-992位为sgRNA(wt)的核苷酸序列，第993-1283位为OsU3终止子的核苷酸序列。
序列1自5’端第1290-8712位为表达盒Ⅱ，其中，第1290-3003位为OsUbq3启动子的核苷酸序列，第3010-7278位为eSpCas9(1.1)n的核苷酸序列(不含有终止密码子)，第7570-8193位为PmCDA1的核苷酸序列，第8215-8511位为UGI的核苷酸序列，第8518-8712位为CaMV35S终止子的核苷酸序列。
序列1自5’端第8787-12064位为表达盒Ⅲ，其中，第8787-10779为ZmUbi1启动子的核苷酸序列，第10786-11811位为潮霉素的核苷酸序列，第11812-12064位为Nos终止子的核苷酸序列。
2、sgRNA(wt)+SpCas9n&PmCDA1&UGI：将序列表的序列1自5’端第3010-7278位替换为序列表的序列2得到的环状质粒。
sgRNA(wt)+SpCas9n&PmCDA1&UGI与sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI的区别仅在于采用SpCas9n替换了eSpCas9(1.1)n。
3、sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI：将序列表的序列1自5’端第131位-1283位(表达盒I)替换为序列表的序列3得到的环状质粒。
序列表的序列3中，自5’端第1-337位为OsU3启动子的核苷酸序列，第344-420位为pre-tRNA的核苷酸序列，第421-440位为CS650靶点的核苷酸序列，第441-526位为sgRNA(modified)的核苷酸序列，第527-603位为pre-tRNA的核苷酸序列，第604-623位为CS651靶点的核苷酸序列，第624-709位为sgRNA(modified)的核苷酸序列，第710-786位为pre-tRNA的核苷酸序列，第787-806位为CS652靶点的核苷酸序列，第807-892位为sgRNA(modified)的核苷酸序列，第893-1183位为OsU3终止子的核苷酸序列。
sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI和sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI的区别仅在于采用sgRNA(modified)替换了sgRNA(wt)。
二、在水稻愈伤组织中进行基因编辑
将步骤一构建的三个载体分别按照如下步骤1-5进行操作：
1、将载体导入农杆菌LBA4404(唯地生物，上海，CAT#:AC1030)，得到重组菌，采用YEP培养基培养重组菌，得到菌液OD600nm为0.2的侵染液。
2、选取日本晴水稻种子，剥去种皮，灭菌洗涤后，均匀的点入在N6培养基中，28℃暗培养4-6周以诱导愈伤组织的产生。
3、将步骤2得到的水稻愈伤组织浸泡在步骤1得到的侵染液中浸泡10min，取愈伤组织接种于含有两层滤纸的培养皿上，25℃黑暗下培养3天(培养基为含有100mg/L的特美汀的N6培养基)，然后愈伤组织在筛选培养基中(含有50mg/L潮霉素的N6培养基，pH5.7)28℃黑暗下筛选培养2周，转入新配置的筛选培养基中再次进行筛选培养2周，获得抗性愈伤。
4、提取步骤3得到的抗性愈伤的基因组DNA，采用引物F(5’-attatgtagcttgtgcgtttcg-3’)和引物R(5’-gatgaagagcttatcgacgt-3’)组成的引物对进行PCR扩增；将获得的扩增产物进行琼脂糖凝胶电泳，如电泳图显示有1150bp大小的条带，说明对应的愈伤为阳性抗性愈伤。
5、对步骤4得到的阳性抗性愈伤DNA(每个载体选择15块)，分别采用CS650靶点引物(CS650-F和CS650-R)、CS651靶点引物(CS651-F和CS651-R)和CS652靶点引物(CS652-F和CS652-R)进行PCR扩增，然后将扩增产物测序。
CS650-F:5’-taagaaccaccagcgacacc-3’；
CS650-R:5’-ggtaattgtgcttggtgatggag-3’；
CS651-F:5’-acatcgagatggagaagcgg-3’；
CS651-R:5’-ccatgctccaatcgatgaatac-3’；
CS652-F:5’-ttacgaactttataactttgtcgg-3’；
CS652-R:5’-atggaggcgatgaggaagac-3’。
对于CS650靶点，发生C·T碱基替换的位点对应为：cgTgtccatggagatccacc；
对于CS651靶点，发生C·T碱基替换的位点对应为：gaTTagccagcgtctggcgc；
对于CS652靶点，发生C·T碱基替换的位点对应为：cggTgacggcgagcaagtgg。
三个靶点分别统计C·T碱基替换的阳性抗性愈伤数，碱基替换效率为15块阳性抗性愈伤中发生C·T碱基替换的阳性抗性愈伤所占的比例。
结果如图1和图2所示。实验结果表明，相对于sgRNA(wt)+SpCas9n&PmCDA1&UGI，sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI对靶点的C·T碱基替换的替换效率显著下降(图1)；而sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI对靶点的C·T碱基替换效率相对于sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI显著增加(图2)，增至与sgRNA(wt)+SpCas9n&PmCDA1&UGI相当甚至更高(图1和图2)。
实施例2、sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI系统脱靶效应
一、基因编辑载体的构建
sgRNA(wt)+SpCas9n&PmCDA1&UGI-T1：将sgRNA(wt)+SpCas9n&PmCDA1&UGI载体中自5’端第551-916位替换为序列表的序列4得到的环状质粒。
sgRNA(wt)+SpCas9n&PmCDA1&UGI-T2：将sgRNA(wt)+SpCas9n&PmCDA1&UGI载体中自5’端第551-916位替换为序列表的序列5得到的环状质粒。
sgRNA(wt)+SpCas9n&PmCDA1&UGI-T3：将sgRNA(wt)+SpCas9n&PmCDA1&UGI载体中自5’端第551-916位替换为序列表的序列6得到的环状质粒。
sgRNA(wt)+SpCas9n&PmCDA1&UGI-T4：将sgRNA(wt)+SpCas9n&PmCDA1&UGI载体中自5’端第551-916位替换为序列表的序列7得到的环状质粒。
sgRNA(wt)+SpCas9n&PmCDA1&UGI-T5：将sgRNA(wt)+SpCas9n&PmCDA1&UGI载体中自5’端第551-916位替换为序列表的序列8得到的环状质粒。
sgRNA(wt)+SpCas9n&PmCDA1&UGI-T6：将sgRNA(wt)+SpCas9n&PmCDA1&UGI载体中自5’端第551-916位替换为序列表的序列9得到的环状质粒。
sgRNA(wt)+SpCas9n&PmCDA1&UGI-T7：将sgRNA(wt)+SpCas9n&PmCDA1&UGI载体中自5’端第551-916位替换为序列表的序列10得到的环状质粒。
sgRNA(wt)+SpCas9n&PmCDA1&UGI-T8：将sgRNA(wt)+SpCas9n&PmCDA1&UGI载体中自5’端第551-916位替换为序列表的序列11得到的环状质粒。
sgRNA(wt)+SpCas9n&PmCDA1&UGI-T9：将sgRNA(wt)+SpCas9n&PmCDA1&UGI载体中自5’端第551-916位替换为序列表的序列12得到的环状质粒。
sgRNA(wt)+SpCas9n&PmCDA1&UGI-T10：将sgRNA(wt)+SpCas9n&PmCDA1&UGI载体中自5’端第551-916位替换为序列表的序列13得到的环状质粒。
sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI-T1：将序列表的序列1自5’端第551-916位替换为序列表的序列4得到的环状质粒。
sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI-T2：将序列表的序列1自5’端第551-916位替换为序列表的序列5得到的环状质粒。
sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI-T3：将序列表的序列1自5’端第551-916位替换为序列表的序列6得到的环状质粒。
sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI-T4：将序列表的序列1自5’端第551-916位替换为序列表的序列7得到的环状质粒。
sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI-T5：将序列表的序列1自5’端第551-916位替换为序列表的序列8得到的环状质粒。
sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI-T6：将序列表的序列1自5’端第551-916位替换为序列表的序列9得到的环状质粒。
sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI-T7：将序列表的序列1自5’端第551-916位替换为序列表的序列10得到的环状质粒。
sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI-T8：将序列表的序列1自5’端第551-916位替换为序列表的序列11得到的环状质粒。
sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI-T9：将序列表的序列1自5’端第551-916位替换为序列表的序列12得到的环状质粒。
sgRNA(wt)+eSpCas9(1.1)n&PmCDA1&UGI-T10：将序列表的序列1自5’端第551-916位替换为序列表的序列13得到的环状质粒。
sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T1：将sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI载体的表达盒I替换为序列表的序列14得到的环状质粒。序列表的序列14中，自5’端第421-440位为靶点序列。
sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T2：将sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T1载体的靶点序列替换为序列表的序列5得到的环状质粒。
sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T3：将sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T1载体的靶点序列替换为序列表的序列6得到的环状质粒。
sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T4：将sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T1载体的靶点序列替换为序列表的序列7得到的环状质粒。
sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T5：将sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T1载体的靶点序列替换为序列表的序列8得到的环状质粒。
sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T6：将sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T1载体的靶点序列替换为序列表的序列9得到的环状质粒。
sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T7：将sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T1载体的靶点序列替换为序列表的序列10得到的环状质粒。
sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T8：将sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T1载体的靶点序列替换为序列表的序列11得到的环状质粒。
sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T9：将sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T1载体的靶点序列替换为序列表的序列12得到的环状质粒。
sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T10：将sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI-T1载体的靶点序列替换为序列表的序列13得到的环状质粒。
二、在水稻愈伤组织中进行基因编辑
1、将步骤一得到的载体分别按照实施例1中步骤二的1-4进行操作，得到阳性抗性愈伤。
2、每个载体从步骤1得到的阳性抗性愈伤中随机选取8块，将其DNA进行混合，分别采用CS652靶点引物(CS652-F和CS652-R)进行PCR扩增，以第一轮PCR产物为模板，将正向和反向条码加入PCR产物末端进行文库构建，使用Illumina HiSeq2500高通量测序平台测序，测序深度为10000X(石家庄博瑞迪生物技术有限公司)，目标靶点区检测C·T碱基替换和indels，靶点区任何C·T碱基替换和indels均计入脱靶突变，脱靶效率为检测到的有突变的reads数占总reads数的比例。
结果如图3所示。实验结果表明，sgRNA(modified)+eSpCas9(1.1)n&PmCDA1&UGI脱靶效率几乎为零。
序列表
<110> 北京市农林科学院
<120> 一种基因组核苷酸定点替换的方法
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18476
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggtggcagga tatattgtgg tgtaaacatg gcactagcct caccgtcttc gcagacgagg 60
ccgctaagtc gcagctacgc tctcaacggc actgactagg tagtttaaac gtgcacttaa 120
ttaaggtacc gaagcaactt aaagttatca ggcatgcatg gatcttggag gaatcagatg 180
tgcagtcagg gaccatagca caagacaggc gtcttctact ggtgctacca gcaaatgctg 240
gaagccggga acactgggta cgttggaaac cacgtgatgt gaagaagtaa gataaactgt 300
aggagaaaag catttcgtag tgggccatga agcctttcag gacatgtatt gcagtatggg 360
ccggcccatt acgcaattgg acgacaacaa agactagtat tagtaccacc tcggctatcc 420
acatagatca aagctgattt aaaagagttg tgcagatgat ccgtggcgga tccaacaaag 480
caccagtggt ctagtggtag aatagtaccc tgccacggta cagacccggg ttcgattccc 540
ggctggtgca cgcgtccatg gagatccacc gttttagagc tagaaatagc aagttaaaat 600
aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcaaca aagcaccagt 660
ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt cccggctggt 720
gcagaccagc cagcgtctgg cgcgttttag agctagaaat agcaagttaa aataaggcta 780
gtccgttatc aacttgaaaa agtggcaccg agtcggtgca acaaagcacc agtggtctag 840
tggtagaata gtaccctgcc acggtacaga cccgggttcg attcccggct ggtgcacggc 900
gacggcgagc aagtgggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 960
atcaacttga aaaagtggca ccgagtcggt gctttttttt ttcgttttgc attgagtttt 1020
ctccgtcgca tgtttgcagt tttattttcc gttttgcatt gaaatttctc cgtctcatgt 1080
ttgcagcgtg ttcaaaaagt acgcagctgt atttcactta tttacggcgc cacattttca 1140
tgccgtttgt gccaactatc ccgagctagt gaatacagct tggcttcaca caacactggt 1200
gacccgctga cctgctcgta cctcgtaccg tcgtacggca cagcatttgg aattaaaggg 1260
tgtgatcgat actgcttgct gctaagctta caaattcggg tcaaggcgga agccagcgcg 1320
ccaccccacg tcagcaaata cggaggcgcg gggttgacgg cgtcacccgg tcctaacggc 1380
gaccaacaaa ccagccagaa gaaattacag taaaaaaaaa gtaaattgca ctttgatcca 1440
ccttttatta cctaagtctc aatttggatc acccttaaac ctatcttttc aatttgggcc 1500
gggttgtggt ttggactacc atgaacaact tttcgtcatg tctaacttcc ctttcagcaa 1560
acatatgaac catatataga ggagatcggc cgtatactag agctgatgtg tttaaggtcg 1620
ttgattgcac gagaaaaaaa aatccaaatc gcaacaatag caaatttatc tggttcaaag 1680
tgaaaagata tgtttaaagg tagtccaaag taaaacttat agataataaa atgtggtcca 1740
aagcgtaatt cactcaaaaa aaatcaacga gacgtgtacc aaacggagac aaacggcatc 1800
ttctcgaaat ttcccaaccg ctcgctcgcc cgcctcgtct tcccggaaac cgcggtggtt 1860
tcagcgtggc ggattctcca agcagacgga gacgtcacgg cacgggactc ctcccaccac 1920
ccaaccgcca taaataccag ccccctcatc tcctctcctc gcatcagctc cacccccgaa 1980
aaatttctcc ccaatctcgc gaggctctcg tcgtcgaatc gaatcctctc gcgtcctcaa 2040
ggtacgctgc ttctcctctc ctcgcttcgt ttcgattcga tttcggacgg gtgaggttgt 2100
tttgttgcta gatccgattg gtggttaggg ttgtcgatgt gattatcgtg agatgtttag 2160
gggttgtaga tctgatggtt gtgatttggg cacggttggt tcgataggtg gaatcgtggt 2220
taggttttgg gattggatgt tggttctgat gattgggggg aatttttacg gttagatgaa 2280
ttgttggatg attcgattgg ggaaatcggt gtagatctgt tggggaattg tggaactagt 2340
catgcctgag tgattggtgc gatttgtagc gtgttccatc ttgtaggcct tgttgcgagc 2400
atgttcagat ctactgttcc gctcttgatt gagttattgg tgccatgggt tggtgcaaac 2460
acaggcttta atatgttata tctgttttgt gtttgatgta gatctgtagg gtagttcttc 2520
ttagacatgg ttcaattatg tagcttgtgc gtttcgattt gatttcatat gttcacagat 2580
tagataatga tgaactcttt taattaattg tcaatggtaa ataggaagtc ttgtcgctat 2640
atctgtcata atgatctcat gttactatct gccagtaatt tatgctaaga actatattag 2700
aatatcatgt tacaatctgt agtaatatca tgttacaatc tgtagttcat ctatataatc 2760
tattgtggta atttcttttt actatctgtg tgaagattat tgccactagt tcattctact 2820
tatttctgaa gttcaggata cgtgtgctgt tactacctat ctgaatacat gtgtgatgtg 2880
cctgttacta tctttttgaa tacatgtatg ttctgttgga atatgtttgc tgtttgatcc 2940
gttgttgtgt ccttaatctt gtgctagttc ttaccctatc tgtttggtga ttatttcttg 3000
cagtacgtaa tggactacaa ggaccacgac ggggattaca aagaccacga catagactac 3060
aaggatgacg atgacaaaat ggcaccgaag aaaaaaagga aggtcggaat ccatggcgtt 3120
ccagctgccg ataagaaata ttccatcgga ctcgccattg gcacgaatag cgtcggatgg 3180
gctgttatta ctgatgagta caaagttccg tctaagaagt tcaaggtgct gggcaacaca 3240
gaccgccaca gcataaagaa aaatctcatc ggtgcactcc ttttcgatag tggggagact 3300
gcagaagcga caagattgaa aaggactgcg agaaggcgct atacacggcg taagaataga 3360
atctgctacc ttcaggagat tttctctaac gaaatggcta aggtcgatga cagtttcttt 3420
catagacttg aggaatcgtt cttggttgag gaggataaga aacatgagag gcacccgata 3480
tttggaaaca tcgtggatga ggtcgcatat catgaaaagt accccacaat ctaccacctg 3540
agaaagaaac tcgttgattc caccgacaaa gcggatttga gactcatcta cctcgctctt 3600
gcccatatga taaagttccg cggacacttt ctgatcgagg gcgacctcaa ccctgataat 3660
agcgacgtcg ataagctctt catccagttg gttcaaacct acaatcagct ctttgaggaa 3720
aacccaatta atgctagtgg agtggatgca aaagcgatac tgtcggccag actctccaag 3780
agcagaaggt tggagaacct gatcgctcaa cttcctggag aaaagaaaaa cggtcttttt 3840
gggaatttga ttgccttgtc tctgggcctc acaccaaact tcaagtcaaa ttttgacctc 3900
gctgaggatg ccaaacttca gttgtctaag gatacctatg atgacgatct tgacaatttg 3960
ctggcacaaa ttggcgacca gtacgcggat ctgttcctcg cagcgaagaa tctgagtgat 4020
gctattctcc tttcggacat actcagggtt aacactgaga tcacaaaagc acctttgagt 4080
gcgtcgatga ttaagcgcta tgatgaacat caccaagacc tcactttgct gaaggccctt 4140
gtgcggcagc aattgccaga gaagtacaaa gaaatcttct ttgaccaatc taagaacgga 4200
tacgctggct atattgatgg aggagcttct caggaggaat tctataagtt tatcaaacct 4260
atacttgaga agatggatgg tacagaggaa ctccttgtta aattgaacag agaagatttg 4320
ctgcgcaagc aacggacctt tgacaacgga tcaattccgc atcagataca cctcggcgag 4380
cttcatgcca tccttcgccg gcaggaagat ttctacccct ttttgaagga caaccgcgag 4440
aagatagaaa aaatccttac gttccggatt ccttactatg tgggtccatt ggcaaggggg 4500
aattcccgct ttgcgtggat gactcggaaa agcgaggaaa ctatcacacc gtggaacttc 4560
gaggaagttg tggacaaggg agcttctgcc caatcattca ttgagaggat gactaacttc 4620
gataagaacc tgccgaacga gaaagttctc cccaagcact ccctccttta cgagtatttc 4680
accgtgtata acgaacttac gaaggttaaa tacgtgactg agggtatgag gaagccagca 4740
ttcttgagcg gggaacaaaa gaaagcgatt gttgatttgc tgtttaaaac taatcgcaag 4800
gtgacagtca agcagctcaa agaggattat ttcaagaaaa ttgaatgttt cgactctgtg 4860
gagatatcag gagtcgaaga taggtttaac gcttcccttg gcacatacca tgacctcctt 4920
aagatcatta aggacaaaga tttcctggat aacgaggaaa atgaggacat cctcgaagat 4980
attgttctta ccttgacgct gtttgaggat cgcgaaatga tcgaggaacg gcttaagacg 5040
tatgctcact tgttcgacga taaggttatg aagcagctca agcgtagaag gtacactgga 5100
tggggccgtc tgtctagaaa gctcatcaac ggaatacgtg ataaacaaag tggcaagaca 5160
attttggatt ttctgaagtc ggacggattc gccaacagaa attttatgca gctgattcat 5220
gacgatagtc tcaccttcaa agaggacata cagaaggctc aagtgagtgg tcaaggggat 5280
tcgctgcatg aacacatcgc aaacctcgcg ggttcaccgg ccataaagaa aggaatcctt 5340
caaactgtta aggtcgttga tgagttggtt aaagtgatgg gtaggcacaa gcccgaaaac 5400
atagtgatcg agatggctcg cgaaaatcag actacacaaa aagggcagaa gaactctcgc 5460
gagcggatga aaaggattga ggaaggaatc aaggaactgg gctcacagat tctcaaagag 5520
catccagtcg aaaacacaca gctgcaaaat gagaagctct atctttacta tctccaaaat 5580
ggccgggaca tgtatgttga tcaggagctt gacatcaacc gtttgtccga ctatgatgtg 5640
gaccacattg tcccgcaatc tttccttgca gacgattcaa tcgataataa ggtgttgacc 5700
cggagcgata aaaaccgtgg aaagtctgac aatgtccctt cagaggaagt ggttaagaag 5760
atgaagaact actggagaca attgctgaat gcaaaactga tcacacagag aaagttcgac 5820
aacctcacca aagcagagag aggtgggctc agtgaacttg ataaagcggg cttcattaag 5880
cgtcagctcg ttgagactag acagatcacg aagcatgtcg cgcagatttt ggattcgcgg 5940
atgaacacga agtacgacga gaatgataaa ctgatacgtg aagtcaaggt tatcactctt 6000
aagtccaaat tggtgagcga tttcagaaag gacttccaat tctataaggt cagggagatc 6060
aacaattatc atcacgctca cgatgcctac cttaatgctg ttgtggggac cgcccttatt 6120
aagaaatacc ctgcattgga gtctgaattc gtttacgggg attataaggt ctacgacgtt 6180
aggaaaatga tagctaagag tgagcaggag atcggtaaag caactgcgaa gtatttcttt 6240
tactcgaaca tcatgaattt ctttaagacc gagataacgc tggcaaatgg cgaaattaga 6300
aaggcacctc tcatagagac taacggtgag acaggggaaa tcgtctggga taagggtagg 6360
gactttgcga cagtgcgcaa ggtcctctct atgccgcaag ttaatattgt gaagaaaacc 6420
gaggtgcaga cgggaggctt ctccaaggaa agcatacttc ccaaacggaa ctctgataag 6480
ttgatcgctc gtaagaaaga ttgggaccct aagaaatatg gtgggttcga ttccccaact 6540
gttgcttaca gcgtgctggt cgttgccaag gtcgagaagg gtaaatccaa gaaactcaaa 6600
agcgttaagg aactccttgg gattactatc atggagagat cttcattcga aaagaatcct 6660
atcgactttc ttgaggccaa aggatataag gaagttaaga aagatctgat aatcaaactc 6720
ccaaagtact cattgtttga gctggaaaac ggcaggaagc gcatgcttgc ttccgccgga 6780
gagttgcaga aagggaacga gttggctctg ccttctaagt atgttaactt cctctatctt 6840
gcctctcatt acgagaagct caaaggctca ccagaggaca acgaacagaa acaacttttt 6900
gtcgagcaac ataagcacta tttggatgag attatagaac agatcagtga attctcgaaa 6960
agggttatcc ttgcagatgc gaatcttgac aaggtgttgt ctgcatacaa caaacataga 7020
gataagccga tcagggagca agcggaaaat atcattcacc tcttcactct tacaaacttg 7080
ggtgctcccg ctgccttcaa gtattttgat accacgattg accggaaacg ttacacctca 7140
acgaaggagg tgctggatgc caccctcatc caccaatcta ttaccggact ctacgagact 7200
agaatcgatc tctcacagct cggcggggat aaaagaccag cagcgacgaa aaaggcagga 7260
caggctaaga agaagaaaga gctcggagga ggaggcacgg gaggaggagg ctccgccgag 7320
tatgtgcgcg cgctcttcga cttcaacggc aatgacgagg aggatctccc tttcaagaag 7380
ggcgacatcc tccgcatccg cgataagccg gaggagcagt ggtggaacgc agaggactcc 7440
gagggcaagc ggggcatgat cctggtgcca tacgtcgaga agtacagcgg cgattacaag 7500
gaccacgatg gcgactacaa ggatcatgac atcgattaca aggacgatga cgataagtcc 7560
ggcgtcgaca tgacggacgc ggagtatgtg cgcatccacg agaagctcga tatctacacc 7620
ttcaagaagc agttcttcaa caataagaag tcggtgtccc atcggtgcta cgtcctcttc 7680
gagctgaagc gcaggggaga gcgccgcgcc tgcttctggg gctacgcggt gaataagccg 7740
cagtcaggca cagagcgcgg catccacgcc gagatcttct cgatccggaa ggtcgaggag 7800
tacctccgcg acaacccagg ccagttcacg atcaattggt actccagctg gtccccttgc 7860
gcagattgcg cagagaagat cctcgagtgg tacaaccagg agctgagggg caatggccat 7920
accctcaaga tctgggcctg caagctgtac tacgagaaga acgcgaggaa tcagatcggc 7980
ctctggaacc tgcgggataa tggcgtgggc ctcaacgtga tggtgtccga gcactaccag 8040
tgctgccgca agatcttcat ccagtcctcc cacaatcagc tgaacgagaa taggtggctc 8100
gaaaagaccc tgaagcgcgc cgagaagtgg aggagcgagc tgtctatcat gatccaggtc 8160
aagatcctgc acaccacaaa gtcaccggcg gtgggcggcg gcggcagcga attctccggc 8220
ggcagcacga acctcagcga catcatcgag aaggagacag gcaagcagct cgtgatccag 8280
gagtctatcc tcatgctgcc tgaggaggtg gaggaggtca tcggcaacaa gccggagtcc 8340
gatatcctcg tgcacaccgc ctacgacgag tcgacagatg agaatgtcat gctcctgacc 8400
tccgacgcac cagagtacaa gccatgggcg ctcgtgatcc aggattccaa cggcgagaat 8460
aagatcaaga tgctgtctgg cggctccccg aagaagaagc gcaaggtcta gactagtctg 8520
aaatcaccag tctctctcta caaatctatc tctctctata ataatgtgtg agtagttccc 8580
agataaggga attagggttc ttatagggtt tcgctcatgt gttgagcata taagaaaccc 8640
ttagtatgta tttgtatttg taaaatactt ctatcaataa aatttctaat tcctaaaacc 8700
aaaatccagt ggggcgcccg acctgtactc gcgaaggtta acttacagag agtgtccggg 8760
cgcgcctggt ggatcgtccg cctaggctgc agtgcagcgt gacccggtcg tgcccctctc 8820
tagagataat gagcattgca tgtctaagtt ataaaaaatt accacatatt ttttttgtca 8880
cacttgtttg aagtgcagtt tatctatctt tatacatata tttaaacttt actctacgaa 8940
taatataatc tatagtacta caataatatc agtgttttag agaatcatat aaatgaacag 9000
ttagacatgg tctaaaggac aattgagtat tttgacaaca ggactctaca gttttatctt 9060
tttagtgtgc atgtgttctc cttttttttt gcaaatagct tcacctatat aatacttcat 9120
ccattttatt agtacatcca tttagggttt agggttaatg gtttttatag actaattttt 9180
ttagtacatc tattttattc tattttagcc tctaaattaa gaaaactaaa actctatttt 9240
agttttttta tttaataatt tagatataaa atagaataaa ataaagtgac taaaaattaa 9300
acaaataccc tttaagaaat taaaaaaact aaggaaacat ttttcttgtt tcgagtagat 9360
aatgccagcc tgttaaacgc cgtcgacgag tctaacggac accaaccagc gaaccagcag 9420
cgtcgcgtcg ggccaagcga agcagacggc acggcatctc tgtcgctgcc tctggacccc 9480
tctcgagagt tccgctccac cgttggactt gctccgctgt cggcatccag aaattgcgtg 9540
gcggagcggc agacgtgagc cggcacggca ggcggcctcc tcctcctctc acggcaccgg 9600
cagctacggg ggattccttt cccaccgctc cttcgctttc ccttcctcgc ccgccgtaat 9660
aaatagacac cccctccaca ccctctttcc ccaacctcgt gttgttcgga gcgcacacac 9720
acacaaccag atctccccca aatccacccg tcggcacctc cgcttcaagg tacgccgctc 9780
gtcctccccc cccccccctc tctaccttct ctagatcggc gttccggtcc atggttaggg 9840
cccggtagtt ctacttctgt tcatgtttgt gttagatccg tgtttgtgtt agatccgtgc 9900
tgctagcgtt cgtacacgga tgcgacctgt acgtcagaca cgttctgatt gctaacttgc 9960
cagtgtttct ctttggggaa tcctgggatg gctctagccg ttccgcagac gggatcgatt 10020
tcatgatttt ttttgtttcg ttgcataggg tttggtttgc ccttttcctt tatttcaata 10080
tatgccgtgc acttgtttgt cgggtcatct tttcatgctt ttttttgtct tggttgtgat 10140
gatgtggtct ggttgggcgg tcgttctaga tcggagtaga attctgtttc aaactacctg 10200
gtggatttat taattttgga tctgtatgtg tgtgccatac atattcatag ttacgaattg 10260
aagatgatgg atggaaatat cgatctagga taggtataca tgttgatgcg ggttttactg 10320
atgcatatac agagatgctt tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg 10380
tcgttcattc gttctagatc ggagtagaat actgtttcaa actacctggt gtatttatta 10440
attttggaac tgtatgtgtg tgtcatacat cttcatagtt acgagtttaa gatggatgga 10500
aatatcgatc taggataggt atacatgttg atgtgggttt tactgatgca tatacatgat 10560
ggcatatgca gcatctattc atatgctcta accttgagta cctatctatt ataataaaca 10620
agtatgtttt ataattattt tgatcttgat atacttggat gatggcatat gcagcagcta 10680
tatgtggatt tttttagccc tgccttcata cgctatttat ttgcttggta ctgtttcttt 10740
tgtcgatgct caccctgttg tttggtgtta cttctgcagg agctcatgaa aaagcctgaa 10800
ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg 10860
atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga 10920
tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg 10980
cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga gtttagcgag 11040
agcctgacct attgcatctc ccgccgttca cagggtgtca cgttgcaaga cctgcctgaa 11100
accgaactgc ccgctgttct acaaccggtc gcggaggcta tggatgcgat cgctgcggcc 11160
gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact 11220
acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg 11280
atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc 11340
gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg 11400
acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc 11460
caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag 11520
acgcgctact tcgagcggag gcatccggag cttgcaggat cgccacgact ccgggcgtat 11580
atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat 11640
gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg 11700
cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc 11760
gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaagaaata ggatcgttca 11820
aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 11880
atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 11940
tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 12000
aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 12060
gatctgtagc cctgcaggac gcgtttaatt aagtgcacgc ggccgcctac ttagtcaaga 12120
gcctcgcacg cgactgtcac gcggccagga tcgcctcgtg agcctcgcaa tctgtaccta 12180
gtgtttaaac tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc 12240
gtttattaga ataacggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg 12300
tatgtgcatg ccaaccacag ggttcccctc gggatcaaag tactttgatc caacccctcc 12360
gctgctatag tgcagtcggc ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt 12420
cgcacaagtc ctaagttacg cgacaggctg ccgccctgcc cttttcctgg cgttttcttg 12480
tcgcgtgttt tagtcgcata aagtagaata cttgcgacta gaaccggaga cattacgcca 12540
tgaacaagag cgccgccgct ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg 12600
acttgaccaa ccaacgggcc gaactgcacg cggccggctg caccaagctg ttttccgaga 12660
agatcaccgg caccaggcgc gaccgcccgg agctggccag gatgcttgac cacctacgcc 12720
ctggcgacgt tgtgacagtg accaggctag accgcctggc ccgcagcacc cgcgacctac 12780
tggacattgc cgagcgcatc caggaggccg gcgcgggcct gcgtagcctg gcagagccgt 12840
gggccgacac caccacgccg gccggccgca tggtgttgac cgtgttcgcc ggcattgccg 12900
agttcgagcg ttccctaatc atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc 12960
gaggcgtgaa gtttggcccc cgccctaccc tcaccccggc acagatcgcg cacgcccgcg 13020
agctgatcga ccaggaaggc cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc 13080
gctcgaccct gtaccgcgca cttgagcgca gcgaggaagt gacgcccacc gaggccaggc 13140
ggcgcggtgc cttccgtgag gacgcattga ccgaggccga cgccctggcg gccgccgaga 13200
atgaacgcca agaggaacaa gcatgaaacc gcaccaggac ggccaggacg aaccgttttt 13260
cattaccgaa gagatcgagg cggagatgat cgcggccggg tacgtgttcg agccgcccgc 13320
gcacgtctca accgtgcggc tgcatgaaat cctggccggt ttgtctgatg ccaagctggc 13380
ggcctggccg gccagcttgg ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg 13440
tgtatttgag taaaacagct tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa 13500
taaacaaata cgcaagggga acgcatgaag gttatcgctg tacttaacca gaaaggcggg 13560
tcaggcaaga cgaccatcgc aacccatcta gcccgcgccc tgcaactcgc cggggccgat 13620
gttctgttag tcgattccga tccccagggc agtgcccgcg attgggcggc cgtgcgggaa 13680
gatcaaccgc taaccgttgt cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc 13740
atcggccggc gcgacttcgt agtgatcgac ggagcgcccc aggcggcgga cttggctgtg 13800
tccgcgatca aggcagccga cttcgtgctg attccggtgc agccaagccc ttacgacata 13860
tgggccaccg ccgacctggt ggagctggtt aagcagcgca ttgaggtcac ggatggaagg 13920
ctacaagcgg cctttgtcgt gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt 13980
gccgaggcgc tggccgggta cgagctgccc attcttgagt cccgtatcac gcagcgcgtg 14040
agctacccag gcactgccgc cgccggcaca accgttcttg aatcagaacc cgagggcgac 14100
gctgcccgcg aggtccaggc gctggccgct gaaattaaat caaaactcat ttgagttaat 14160
gaggtaaaga gaaaatgagc aaaagcacaa acacgctaag tgccggccgt ccgagcgcac 14220
gcagcagcaa ggctgcaacg ttggccagcc tggcagacac gccagccatg aagcgggtca 14280
actttcagtt gccggcggag gatcacacca agctgaagat gtacgcggta cgccaaggca 14340
agaccattac cgagctgcta tctgaataca tcgcgcagct accagagtaa atgagcaaat 14400
gaataaatga gtagatgaat tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa 14460
ccaggcaccg acgccgtgga atgccccatg tgtggaggaa cgggcggttg gccaggcgta 14520
agcggctggg ttgtctgccg gccctgcaat ggcactggaa cccccaagcc cgaggaatcg 14580
gcgtgacggt cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg gtgatgacct 14640
ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg 14700
ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc 14760
ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac cagatttttt 14820
cgttccgatg ctctatgacg tgggcacccg cgatagtcgc agcatcatgg acgtggccgt 14880
tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg atccgctacg agcttccaga 14940
cgggcacgta gaggtttccg cagggccggc cggcatggcc agtgtgtggg attacgacct 15000
ggtactgatg gcggtttccc atctaaccga atccatgaac cgataccggg aagggaaggg 15060
agacaagccc ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt tctgccggcg 15120
agccgatggc ggaaagcaga aagacgacct ggtagaaacc tgcattcggt taaacaccac 15180
gcacgttgcc atgcagcgta cgaagaaggc caagaacggc cgcctggtga cggtatccga 15240
gggtgaagcc ttgattagcc gctacaagat cgtaaagagc gaaaccgggc ggccggagta 15300
catcgagatc gagctagctg attggatgta ccgcgagatc acagaaggca agaacccgga 15360
cgtgctgacg gttcaccccg attacttttt gatcgatccc ggcatcggcc gttttctcta 15420
ccgcctggca cgccgcgccg caggcaaggc agaagccaga tggttgttca agacgatcta 15480
cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat 15540
cgggtcaaat gacctgccgg agtacgattt gaaggaggag gcggggcagg ctggcccgat 15600
cctagtcatg cgctaccgca acctgatcga gggcgaagca tccgccggtt cctaatgtac 15660
ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag gtctctttcc 15720
tgtggatagc acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat 15780
tgggaaccca aagccgtaca ttgggaaccg gtcacacatg taagtgactg atataaaaga 15840
gaaaaaaggc gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg 15900
cctggcctgt gcataactgt ctggccagcg cacagccgaa gagctgcaaa aagcgcctac 15960
ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg 16020
ccgctcaaaa atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc 16080
gtcgccactc gaccgccggc gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg 16140
acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg 16200
atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg 16260
cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc 16320
agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag 16380
gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 16440
cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 16500
atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 16560
taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 16620
aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 16680
tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 16740
gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 16800
cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 16860
cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 16920
atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 16980
tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 17040
ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 17100
acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 17160
aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 17220
aaactcacgt taagggattt tggtcatgca ttctaggtac taaaacaatt catccagtaa 17280
aatataatat tttattttct cccaatcagg cttgatcccc agtaagtcaa aaaatagctc 17340
gacatactgt tcttccccga tatcctccct gatcgaccgg acgcagaagg caatgtcata 17400
ccacttgtcc gccctgccgc ttctcccaag atcaataaag ccacttactt tgccatcttt 17460
cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag acaagttcct cttcgggctt 17520
ttccgtcttt aaaaaatcat acagctcgcg cggatcttta aatggagtgt cttcttccca 17580
gttttcgcaa tccacatcgg ccagatcgtt attcagtaag taatccaatt cggctaagcg 17640
gctgtctaag ctattcgtat agggacaatc cgatatgtcg atggagtgaa agagcctgat 17700
gcactccgca tacagctcga taatcttttc agggctttgt tcatcttcat actcttccga 17760
gcaaaggacg ccatcggcct cactcatgag cagattgctc cagccatcat gccgttcaaa 17820
gtgcaggacc tttggaacag gcagctttcc ttccagccat agcatcatgt ccttttcccg 17880
ttccacatca taggtggtcc ctttataccg gctgtccgtc atttttaaat ataggttttc 17940
attttctccc accagcttat ataccttagc aggagacatt ccttccgtat cttttacgca 18000
gcggtatttt tcgatcagtt ttttcaattc cggtgatatt ctcattttag ccatttatta 18060
tttccttcct cttttctaca gtatttaaag ataccccaag aagctaatta taacaagacg 18120
aactccaatt cactgttcct tgcattctaa aaccttaaat accagaaaac agctttttca 18180
aagttgtttt caaagttggc gtataacata gtatcgacgg agccgatttt gaaaccgcgg 18240
tgatcacagg cagcaacgct ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc 18300
atccgtgttt caaacccggc agcttagttg ccgttcttcc gaatagcatc ggtaacatga 18360
gcaaagtctg ccgccttaca acggctctcc cgctgacgcc gtcccggact gatgggctgc 18420
ctgtatcgag tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggct 18476
<210> 2
<211> 4269
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggactaca aggaccacga cggggattac aaagaccacg acatagacta caaggatgac 60
gatgacaaaa tggcaccgaa gaaaaaaagg aaggtcggaa tccatggcgt tccagctgcc 120
gataagaaat attccatcgg actcgccatt ggcacgaata gcgtcggatg ggctgttatt 180
actgatgagt acaaagttcc gtctaagaag ttcaaggtgc tgggcaacac agaccgccac 240
agcataaaga aaaatctcat cggtgcactc cttttcgata gtggggagac tgcagaagcg 300
acaagattga aaaggactgc gagaaggcgc tatacacggc gtaagaatag aatctgctac 360
cttcaggaga ttttctctaa cgaaatggct aaggtcgatg acagtttctt tcatagactt 420
gaggaatcgt tcttggttga ggaggataag aaacatgaga ggcacccgat atttggaaac 480
atcgtggatg aggtcgcata tcatgaaaag taccccacaa tctaccacct gagaaagaaa 540
ctcgttgatt ccaccgacaa agcggatttg agactcatct acctcgctct tgcccatatg 600
ataaagttcc gcggacactt tctgatcgag ggcgacctca accctgataa tagcgacgtc 660
gataagctct tcatccagtt ggttcaaacc tacaatcagc tctttgagga aaacccaatt 720
aatgctagtg gagtggatgc aaaagcgata ctgtcggcca gactctccaa gagcagaagg 780
ttggagaacc tgatcgctca acttcctgga gaaaagaaaa acggtctttt tgggaatttg 840
attgccttgt ctctgggcct cacaccaaac ttcaagtcaa attttgacct cgctgaggat 900
gccaaacttc agttgtctaa ggatacctat gatgacgatc ttgacaattt gctggcacaa 960
attggcgacc agtacgcgga tctgttcctc gcagcgaaga atctgagtga tgctattctc 1020
ctttcggaca tactcagggt taacactgag atcacaaaag cacctttgag tgcgtcgatg 1080
attaagcgct atgatgaaca tcaccaagac ctcactttgc tgaaggccct tgtgcggcag 1140
caattgccag agaagtacaa agaaatcttc tttgaccaat ctaagaacgg atacgctggc 1200
tatattgatg gaggagcttc tcaggaggaa ttctataagt ttatcaaacc tatacttgag 1260
aagatggatg gtacagagga actccttgtt aaattgaaca gagaagattt gctgcgcaag 1320
caacggacct ttgacaacgg atcaattccg catcagatac acctcggcga gcttcatgcc 1380
atccttcgcc ggcaggaaga tttctacccc tttttgaagg acaaccgcga gaagatagaa 1440
aaaatcctta cgttccggat tccttactat gtgggtccat tggcaagggg gaattcccgc 1500
tttgcgtgga tgactcggaa aagcgaggaa actatcacac cgtggaactt cgaggaagtt 1560
gtggacaagg gagcttctgc ccaatcattc attgagagga tgactaactt cgataagaac 1620
ctgccgaacg agaaagttct ccccaagcac tccctccttt acgagtattt caccgtgtat 1680
aacgaactta cgaaggttaa atacgtgact gagggtatga ggaagccagc attcttgagc 1740
ggggaacaaa agaaagcgat tgttgatttg ctgtttaaaa ctaatcgcaa ggtgacagtc 1800
aagcagctca aagaggatta tttcaagaaa attgaatgtt tcgactctgt ggagatatca 1860
ggagtcgaag ataggtttaa cgcttccctt ggcacatacc atgacctcct taagatcatt 1920
aaggacaaag atttcctgga taacgaggaa aatgaggaca tcctcgaaga tattgttctt 1980
accttgacgc tgtttgagga tcgcgaaatg atcgaggaac ggcttaagac gtatgctcac 2040
ttgttcgacg ataaggttat gaagcagctc aagcgtagaa ggtacactgg atggggccgt 2100
ctgtctagaa agctcatcaa cggaatacgt gataaacaaa gtggcaagac aattttggat 2160
tttctgaagt cggacggatt cgccaacaga aattttatgc agctgattca tgacgatagt 2220
ctcaccttca aagaggacat acagaaggct caagtgagtg gtcaagggga ttcgctgcat 2280
gaacacatcg caaacctcgc gggttcaccg gccataaaga aaggaatcct tcaaactgtt 2340
aaggtcgttg atgagttggt taaagtgatg ggtaggcaca agcccgaaaa catagtgatc 2400
gagatggctc gcgaaaatca gactacacaa aaagggcaga agaactctcg cgagcggatg 2460
aaaaggattg aggaaggaat caaggaactg ggctcacaga ttctcaaaga gcatccagtc 2520
gaaaacacac agctgcaaaa tgagaagctc tatctttact atctccaaaa tggccgggac 2580
atgtatgttg atcaggagct tgacatcaac cgtttgtccg actatgatgt ggaccacatt 2640
gtcccgcaat ctttccttaa ggacgattca atcgataata aggtgttgac ccggagcgat 2700
aaaaaccgtg gaaagtctga caatgtccct tcagaggaag tggttaagaa gatgaagaac 2760
tactggagac aattgctgaa tgcaaaactg atcacacaga gaaagttcga caacctcacc 2820
aaagcagaga gaggtgggct cagtgaactt gataaagcgg gcttcattaa gcgtcagctc 2880
gttgagacta gacagatcac gaagcatgtc gcgcagattt tggattcgcg gatgaacacg 2940
aagtacgacg agaatgataa actgatacgt gaagtcaagg ttatcactct taagtccaaa 3000
ttggtgagcg atttcagaaa ggacttccaa ttctataagg tcagggagat caacaattat 3060
catcacgctc acgatgccta ccttaatgct gttgtgggga ccgcccttat taagaaatac 3120
cctaaattgg agtctgaatt cgtttacggg gattataagg tctacgacgt taggaaaatg 3180
atagctaaga gtgagcagga gatcggtaaa gcaactgcga agtatttctt ttactcgaac 3240
atcatgaatt tctttaagac cgagataacg ctggcaaatg gcgaaattag aaagaggcct 3300
ctcatagaga ctaacggtga gacaggggaa atcgtctggg ataagggtag ggactttgcg 3360
acagtgcgca aggtcctctc tatgccgcaa gttaatattg tgaagaaaac cgaggtgcag 3420
acgggaggct tctccaagga aagcatactt cccaaacgga actctgataa gttgatcgct 3480
cgtaagaaag attgggaccc taagaaatat ggtgggttcg attccccaac tgttgcttac 3540
agcgtgctgg tcgttgccaa ggtcgagaag ggtaaatcca agaaactcaa aagcgttaag 3600
gaactccttg ggattactat catggagaga tcttcattcg aaaagaatcc tatcgacttt 3660
cttgaggcca aaggatataa ggaagttaag aaagatctga taatcaaact cccaaagtac 3720
tcattgtttg agctggaaaa cggcaggaag cgcatgcttg cttccgccgg agagttgcag 3780
aaagggaacg agttggctct gccttctaag tatgttaact tcctctatct tgcctctcat 3840
tacgagaagc tcaaaggctc accagaggac aacgaacaga aacaactttt tgtcgagcaa 3900
cataagcact atttggatga gattatagaa cagatcagtg aattctcgaa aagggttatc 3960
cttgcagatg cgaatcttga caaggtgttg tctgcataca acaaacatag agataagccg 4020
atcagggagc aagcggaaaa tatcattcac ctcttcactc ttacaaactt gggtgctccc 4080
gctgccttca agtattttga taccacgatt gaccggaaac gttacacctc aacgaaggag 4140
gtgctggatg ccaccctcat ccaccaatct attaccggac tctacgagac tagaatcgat 4200
ctctcacagc tcggcgggga taaaagacca gcagcgacga aaaaggcagg acaggctaag 4260
aagaagaaa 4269
<210> 3
<211> 1183
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gaagcaactt aaagttatca ggcatgcatg gatcttggag gaatcagatg tgcagtcagg 60
gaccatagca caagacaggc gtcttctact ggtgctacca gcaaatgctg gaagccggga 120
acactgggta cgttggaaac cacgtgatgt gaagaagtaa gataaactgt aggagaaaag 180
catttcgtag tgggccatga agcctttcag gacatgtatt gcagtatggg ccggcccatt 240
acgcaattgg acgacaacaa agactagtat tagtaccacc tcggctatcc acatagatca 300
aagctgattt aaaagagttg tgcagatgat ccgtggcgga tccaacaaag caccagtggt 360
ctagtggtag aatagtaccc tgccacggta cagacccggg ttcgattccc ggctggtgca 420
cgcgtccatg gagatccacc gtttcagagc tatgctggaa acagcatagc aagttgaaat 480
aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcaaca aagcaccagt 540
ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt cccggctggt 600
gcagaccagc cagcgtctgg cgcgtttcag agctatgctg gaaacagcat agcaagttga 660
aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca acaaagcacc 720
agtggtctag tggtagaata gtaccctgcc acggtacaga cccgggttcg attcccggct 780
ggtgcacggc gacggcgagc aagtgggttt cagagctatg ctggaaacag catagcaagt 840
tgaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttt 900
ttcgttttgc attgagtttt ctccgtcgca tgtttgcagt tttattttcc gttttgcatt 960
gaaatttctc cgtctcatgt ttgcagcgtg ttcaaaaagt acgcagctgt atttcactta 1020
tttacggcgc cacattttca tgccgtttgt gccaactatc ccgagctagt gaatacagct 1080
tggcttcaca caacactggt gacccgctga cctgctcgta cctcgtaccg tcgtacggca 1140
cagcatttgg aattaaaggg tgtgatcgat actgcttgct gct 1183
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tagcgacggc gagcaagtgg 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgatgacggc gagcaagtgg 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cggcagcggc gagcaagtgg 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cggcgatagc gagcaagtgg 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cggcgacgat gagcaagtgg 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cggcgacggc aggcaagtgg 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cggcgacggc gaataagtgg 20
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cggcgacggc gagcgggtgg 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cggcgacggc gagcaaacgg 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cggcgacggc gagcaagtaa 20
<210> 14
<211> 817
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gaagcaactt aaagttatca ggcatgcatg gatcttggag gaatcagatg tgcagtcagg 60
gaccatagca caagacaggc gtcttctact ggtgctacca gcaaatgctg gaagccggga 120
acactgggta cgttggaaac cacgtgatgt gaagaagtaa gataaactgt aggagaaaag 180
catttcgtag tgggccatga agcctttcag gacatgtatt gcagtatggg ccggcccatt 240
acgcaattgg acgacaacaa agactagtat tagtaccacc tcggctatcc acatagatca 300
aagctgattt aaaagagttg tgcagatgat ccgtggcgga tccaacaaag caccagtggt 360
ctagtggtag aatagtaccc tgccacggta cagacccggg ttcgattccc ggctggtgca 420
tagcgacggc gagcaagtgg gtttcagagc tatgctggaa acagcatagc aagttgaaat 480
aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttttttcgtt 540
ttgcattgag ttttctccgt cgcatgtttg cagttttatt ttccgttttg cattgaaatt 600
tctccgtctc atgtttgcag cgtgttcaaa aagtacgcag ctgtatttca cttatttacg 660
gcgccacatt ttcatgccgt ttgtgccaac tatcccgagc tagtgaatac agcttggctt 720
cacacaacac tggtgacccg ctgacctgct cgtacctcgt accgtcgtac ggcacagcat 780
ttggaattaa agggtgtgat cgatactgct tgctgct 817
<210> 15
<211> 1833
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp
1 5 10 15
Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys Val
20 25 30
Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu
35 40 45
Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr
50 55 60
Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His
65 70 75 80
Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu
85 90 95
Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr
100 105 110
Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu
115 120 125
Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe
130 135 140
Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn
145 150 155 160
Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His
165 170 175
Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu
180 185 190
Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu
195 200 205
Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe
210 215 220
Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile
225 230 235 240
Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser
245 250 255
Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys
260 265 270
Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr
275 280 285
Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln
290 295 300
Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln
305 310 315 320
Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser
325 330 335
Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr
340 345 350
Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His
355 360 365
Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu
370 375 380
Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly
385 390 395 400
Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys
405 410 415
Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu
420 425 430
Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser
435 440 445
Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg
450 455 460
Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu
465 470 475 480
Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg
485 490 495
Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile
500 505 510
Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln
515 520 525
Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu
530 535 540
Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr
545 550 555 560
Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro
565 570 575
Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe
580 585 590
Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe
595 600 605
Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp
610 615 620
Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile
625 630 635 640
Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu
645 650 655
Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu
660 665 670
Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys
675 680 685
Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys
690 695 700
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp
705 710 715 720
Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
725 730 735
His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val
740 745 750
Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly
755 760 765
Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp
770 775 780
Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile
785 790 795 800
Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
805 810 815
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser
820 825 830
Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
835 840 845
Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp
850 855 860
Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
865 870 875 880
Val Pro Gln Ser Phe Leu Ala Asp Asp Ser Ile Asp Asn Lys Val Leu
885 890 895
Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu
900 905 910
Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala
915 920 925
Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
930 935 940
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu
945 950 955 960
Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
965 970 975
Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val
980 985 990
Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp
995 1000 1005
Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His
1010 1015 1020
Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr
1025 1030 1035 1040
Pro Ala Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1045 1050 1055
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr
1060 1065 1070
Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu
1075 1080 1085
Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Ala Pro Leu Ile Glu Thr
1090 1095 1100
Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1105 1110 1115 1120
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys
1125 1130 1135
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1140 1145 1150
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1155 1160 1165
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val
1170 1175 1180
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys
1185 1190 1195 1200
Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn
1205 1210 1215
Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp
1220 1225 1230
Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly
1235 1240 1245
Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu
1250 1255 1260
Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His
1265 1270 1275 1280
Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1285 1290 1295
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile
1300 1305 1310
Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
1315 1320 1325
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln
1330 1335 1340
Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1345 1350 1355 1360
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr
1365 1370 1375
Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1380 1385 1390
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys
1395 1400 1405
Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Glu
1410 1415 1420
Leu Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser Ala Glu Tyr Val Arg
1425 1430 1435 1440
Ala Leu Phe Asp Phe Asn Gly Asn Asp Glu Glu Asp Leu Pro Phe Lys
1445 1450 1455
Lys Gly Asp Ile Leu Arg Ile Arg Asp Lys Pro Glu Glu Gln Trp Trp
1460 1465 1470
Asn Ala Glu Asp Ser Glu Gly Lys Arg Gly Met Ile Leu Val Pro Tyr
1475 1480 1485
Val Glu Lys Tyr Ser Gly Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys
1490 1495 1500
Asp His Asp Ile Asp Tyr Lys Asp Asp Asp Asp Lys Ser Gly Val Asp
1505 1510 1515 1520
Met Thr Asp Ala Glu Tyr Val Arg Ile His Glu Lys Leu Asp Ile Tyr
1525 1530 1535
Thr Phe Lys Lys Gln Phe Phe Asn Asn Lys Lys Ser Val Ser His Arg
1540 1545 1550
Cys Tyr Val Leu Phe Glu Leu Lys Arg Arg Gly Glu Arg Arg Ala Cys
1555 1560 1565
Phe Trp Gly Tyr Ala Val Asn Lys Pro Gln Ser Gly Thr Glu Arg Gly
1570 1575 1580
Ile His Ala Glu Ile Phe Ser Ile Arg Lys Val Glu Glu Tyr Leu Arg
1585 1590 1595 1600
Asp Asn Pro Gly Gln Phe Thr Ile Asn Trp Tyr Ser Ser Trp Ser Pro
1605 1610 1615
Cys Ala Asp Cys Ala Glu Lys Ile Leu Glu Trp Tyr Asn Gln Glu Leu
1620 1625 1630
Arg Gly Asn Gly His Thr Leu Lys Ile Trp Ala Cys Lys Leu Tyr Tyr
1635 1640 1645
Glu Lys Asn Ala Arg Asn Gln Ile Gly Leu Trp Asn Leu Arg Asp Asn
1650 1655 1660
Gly Val Gly Leu Asn Val Met Val Ser Glu His Tyr Gln Cys Cys Arg
1665 1670 1675 1680
Lys Ile Phe Ile Gln Ser Ser His Asn Gln Leu Asn Glu Asn Arg Trp
1685 1690 1695
Leu Glu Lys Thr Leu Lys Arg Ala Glu Lys Trp Arg Ser Glu Leu Ser
1700 1705 1710
Ile Met Ile Gln Val Lys Ile Leu His Thr Thr Lys Ser Pro Ala Val
1715 1720 1725
Gly Gly Gly Gly Ser Glu Phe Ser Gly Gly Ser Thr Asn Leu Ser Asp
1730 1735 1740
Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile
1745 1750 1755 1760
Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu
1765 1770 1775
Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn
1780 1785 1790
Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu
1795 1800 1805
Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly
1810 1815 1820
Gly Ser Pro Lys Lys Lys Arg Lys Val
1825 1830

Claims (10)

1.一种碱基编辑系统，包括融合蛋白和sgRNA；
所述融合蛋白由切刻酶、胞嘧啶核苷脱氨酶PmCDA1和尿嘧啶DNA糖化酶抑制剂UGI组成；所述切刻酶如序列表的序列15自N端第1-1423位氨基酸所示；
所述sgRNA的核苷酸序列如序列表的序列3自5’端第441-526位所示。
2.表达权利要求1所述的碱基编辑系统的重组表达载体、表达盒、重组细胞或重组菌。
3.一种用于基因组碱基替换的重组表达载体，包括表达盒甲和表达盒乙；所述表达盒甲表达权利要求1中所述的融合蛋白；所述表达盒乙包括n个元件乙；所述元件乙包括权利要求1中所述的sgRNA和靶序列；所述重组表达载体可靶向n个不同的靶序列进行碱基替换。
4.如权利要求3所述的重组表达载体，其特征在于：所述元件乙还包括pre-tRNA的核苷酸序列；所述pre-tRNA的核苷酸序列如序列表的序列3自5’端第344-420所示。
5.如权利要求4所述的重组载体，其特征在于：所述元件乙自5’端依次为pre-tRNA的核苷酸序列、靶点序列和sgRNA的核苷酸序列。
6.权利要求1中所述的融合蛋白。
7.权利要求6所述融合蛋白的编码基因。
8.一种植物基因组碱基替换的方法，包括如下步骤：利用权利要求1所述的碱基编辑系统完成植物基因组碱基替换。
9.一种植物基因组碱基替换的方法，包括如下步骤：将权利要求3至5任一所述的重组表达载体导入目的植物，实现植物基因组碱基替换。
10.权利要求1所述的碱基编辑系统，或，权利要求2所述的重组表达载体、表达盒、重组细胞或重组菌，或，权利要求3至5任一所述的重组表达载体，或，权利要求6所述的融合蛋白，在植物基因组碱基替换中的应用。
CN201811123556.4A 2018-09-26 2018-09-26 一种基因组核苷酸定点替换的方法 Pending CN109266686A (zh)

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