CN113801888A - 用于提高枯草芽孢杆菌自发突变频率的质粒 - Google Patents

用于提高枯草芽孢杆菌自发突变频率的质粒 Download PDF

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CN113801888A
CN113801888A CN202111087629.0A CN202111087629A CN113801888A CN 113801888 A CN113801888 A CN 113801888A CN 202111087629 A CN202111087629 A CN 202111087629A CN 113801888 A CN113801888 A CN 113801888A
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闫新
叶斌
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Abstract

本发明公开了用于提高枯草芽孢杆菌自发突变频率的质粒。所述选自pUS20D9M,pUS20XP1以及pUS20XP2中的任意一个；其中，质粒pUS20D9M序列如SEQ ID NO.1所示，质粒pUS20XP1序列如SEQ ID NO.2所示，质粒pUS20XP2序列如SEQ ID NO.3所示。枯草杆菌是一种重要的微生物。很多育种工作需要提高枯草杆菌的基因突变频率。然而，目前尚缺乏理想的控制枯草杆菌自发突变频率的方法。本发明借助CRISPRi技术和过表达易错型DNA聚合酶的方法，提高宿主的自发突变频率(最高达5128倍)。整个操作可通过质粒的转化和消除实现，无需对宿主进行基因敲除。

Description

用于提高枯草芽孢杆菌自发突变频率的质粒
技术领域
本发明属于生物高技术领域，公开了用于提高枯草芽孢杆菌自发突变率的质粒。
背景技术
枯草芽孢杆菌(Bacillus subtilis)在发酵工业、农业和环境保护等领域都发挥重要作用。比如枯草芽孢杆菌被用于重组蛋白表达、化合物生产、植物促生、生物防治和污染物降解等。在实际应用之前，通常要对菌株进行驯化和改造，即育种。物理或化学诱变是常用的育种方法，即通过物理或化学手段提高菌株的基因突变频率，然后筛选目标突变株。该方法的缺点是突变效率低和突变谱窄，且存在危险和污染问题。因此，科研人员尝试提高菌株的自发突变频率。具体策略包括敲除菌株的错配修复系统和表达易错型DNA聚合酶等等。然而，理想的方法应该是不改变宿主的基因型，实现可拔插式操作。迄今，枯草芽孢杆菌中尚无这种理想型的突变方法。鉴于此，本发明提供可以提高枯草芽孢杆菌自发突变频率的质粒，导入质粒即可提高宿主的基因突变频率，丢失质粒后宿主的基因突变频率则恢复正常。该质粒可以应用于枯草芽孢杆菌的育种。
发明内容
技术问题：枯草芽孢杆菌是一种重要的微生物，突变育种在该菌的应用中发挥重要作用。然而，物理和化学诱变育种存在突变效率低、突变谱窄以及危险性高等问题。虽然体内自发突变比体外诱变安全，但目前枯草芽孢杆菌中尚无提高自发突变频率的理想方法。本发明借助 CRISPRi(CRISPR interference)技术和过表达易错型DNA聚合酶的方法，提高宿主的自发突变频率。整个操作可通过质粒的转化和消除实现，无需对宿主进行基因敲除。
本发明的目的可通过以下技术方案实现：
本发明所述的质粒可用于提高枯草芽孢杆菌的自发突变频率，进而对枯草芽孢杆菌进行育种。
用于提高枯草芽孢杆菌自发突变频率的质粒，其特征在于，选自pUS20D9M,pUS20XP1以及pUS20XP2中的任意一个；其中，质粒pUS20D9M序列如SEQ ID NO.1所示，质粒pUS20XP1 序列如SEQ ID NO.2所示，质粒pUS20XP2序列如SEQ ID NO.3所示。
上述质粒均用于提高枯草芽孢杆菌自发突变率。该系列质粒主要功能元件是木糖诱导的 dCas9表达盒，木糖诱导的失去校对功能的polC*表达盒，以及干扰mutSL和polC的两个sgRNA 表达盒。
本发明的原理是抑制错配系统和正常的DNA聚合酶，并表达易错型DNA聚合酶，提高宿主的自发突变频率。根据对突变频率高低的需求，可以通过三种方式实现。第一，将质粒 pUS20D9M转化到枯草芽孢杆菌宿主中，该质粒携带受木糖诱导的dCas9表达盒，可以抑制错配修复基因mutSL(Ginetti et al..1996.Microbiology.226:467-472.doi:10.1099/13500872-142-8-2021)的表达。在添加木糖后，枯草芽孢杆菌中基因mutSL的转录被抑制，导致菌株错配修复系统被干扰，最终菌株的自发突变率增高。第二，将质粒pUS20XP1 转化到染色体整合了木糖诱导的dCas9表达盒的枯草芽孢杆菌宿主中，质粒pUS20XP1包括两个元件，一是抑制正常DNA聚合酶基因polC表达的sgRNA表达盒，另一个是受木糖诱导表达易错型DNA聚合酶PolC*(Sanjanwala et al..1991.Mol Gen Genet.226:467-472.doi: 10.1007/BF00260660)表达盒。添加木糖后polC基因的转录被抑制而polC*获得表达，菌株的自发突变率会明显提高。第三，将质粒pUS20XP2转化到染色体整合了木糖诱导的dCas9表达盒的枯草芽孢杆菌宿主中，质粒pUS20XP2可以同时抑制mutSL和polC的表达，且能表达 PolC*，使菌株的自发突变率进一步提高。以上三种质粒在没有抗性培养的情况下极易丢失，使宿主恢复自然的自发突变频率。
一种提高枯草芽孢杆菌自发突变频率的方法，将本发明所述的任意一种质粒转染枯草芽孢杆菌在含有1％木糖的LB培养基中36℃-38℃培养12h。
质粒pUS20D9M的构建方法，通过同源重组的方式将含有木糖诱导的dCas9表达盒以及干扰mutSL的sgRNA表达盒构建在质粒pUS20上；其中，所述的含有木糖诱导的dCas9表达盒序列如SEQ ID NO.1中第4175bp-8281bp所示；所述的干扰mutSL的sgRNA表达盒序列如SEQ ID NO.1中第第8310bp-8480bp所示。
作为本发明的一种优选，所述的构建方法包括：以质粒pJMP1为模板，用引物P1/P2扩增含有木糖诱导的dCas9表达盒xylR-Pxyl-dcas9部分；合成干扰mutSL的sgRNA表达盒序列，然后经引物P3/P4扩增后得到片段Pveg-sgmutSL；以质粒pUS20为模板，用引物P5/P6扩增质粒pUS20全部序列；然后，将上述3片段通过同源重组的片段融合成环状质粒；最终，将上述融合产物转化到大肠杆菌DH5α后，经100ppm氨苄青霉素筛选后得到质粒pUS20D9M。
质粒pUS20XP1的构建方法，通过同源重组的方式将木糖诱导失去校对功能的DNA聚合酶 PolC*表达盒以及干扰polC的sgRNA表达盒构建在质粒pUS20上，生成质粒pUS20XP1；所述的木糖诱导失去校对功能的DNA聚合酶PolC*表达盒序列如SEQ ID NO.2中的第2574bp-6887bp 所示，所述的干扰polC的sgRNA表达盒序列如SEQ ID NO.2中的第6888bp-7120bp所示。
作为本发明的一种优选，所述的构建方法包括：首先，以枯草芽孢杆菌168基因组为模板，分别用引物P7/P8,P9/P10和P11/P12扩增DNA聚合酶polC*的3个部分，其中碱基突变由 P9和P11引入，将上述3部分融合为失去校对功能的DNA聚合酶PolC*表达盒；合成干扰polC 的sgRNA表达盒序列，然后经引物P13/P14扩增后得到片段Pveg-sgpolC；以权利要求1中所述的质粒pUS20D9M为模板，用引物P15/P16扩增质粒pUS20全部序列和xylR-Pxyl部分；然后，将上述3片段通过同源重组的片段融合成环状质粒；最终，将上述融合产物转化到大肠杆菌DH5α后，经100ppm氨苄青霉素筛选后得到质粒pUS20XP1。
质粒pUS20XP2的构建方法，其特征在于，通过同源重组将片段Pveg-sgmutSL融合到pUS20XP1上，生成质粒pUS20XP2；所述的片段Pveg-sgmutSL序列如SEQ ID NO.3中第6923bp～第7092bp所示。
作为本发明的一种优选，所述的构建方法包括：以质粒pUS20XP1为模板，用引物P17/P18 扩增质粒pUS20XP1全部序列；以质粒pUS20D9M为模板，用引物P19/P20扩增后得到片段 Pveg-sgmutSL；然后，将上述2片段通过同源重组的片段融合成环状质粒；最终，将上述融合产物转化到大肠杆菌DH5α后，经100ppm氨苄青霉素筛选后得到质粒pUS20XP2。
有益效果：
自发突变是生物体遗传变异和进化的最终来源，提高宿主的自发突变率可以更加有效的增加遗传学上的多样性。通过使用CRISPRi技术干扰与DNA错配修复的相关的基因mutSL和 polC的转录，并诱导表达点突变的polC*提高了枯草芽孢杆菌的自发突变率。在枯草芽孢杆菌中开发了一套有效的，可诱导的，广谱的和基于载体的自发诱变系统，该系统在建库后会随着质粒丢失而消失，有利于菌株性状稳定性的提高。
附图说明
图1质粒pUS20D9M图谱及其突变原理
图2质粒pUS20D9M自发突变率统计，其中168(△mutSL)代表Ginetti F,Perego M,Albertini AM.Bacillus subtilis mutS mutL operon:identification,nucleotidesequence and mutagenesis. Microbiology.1996；142:2021-2029.Pedraza-Reyes M,Yasbin RE.Contribution of the Mismatch DNA Repair System to the Generation ofStationary-Phase-Induced Mutants of Bacillus subtilis.J Bacteriol.2004；186:6485-6491.中公开的枯草芽孢杆菌基因工程菌
图3质粒pUS20XP1图谱
图4质粒pUS20XP1自发突变率统计
图5质粒pUS20XP2图谱及其突变原理
图6质粒pUS20XP2自发突变率统计
具体实施方式
实施例1质粒pUS20D9M用于提高枯草芽孢杆菌的自发突变率
大肠杆菌和枯草芽孢杆菌用LB培养基(蛋白胨1％，酵母粉0.5％，氯化钠1％，pH7.0) 在37℃培养。如图1a所示，通过同源重组的方式将含有木糖诱导的dCas9表达盒以及干扰 mutSL的sgRNA表达盒构建在质粒pUS20(Ye et al..2018.J Biotechnol.284:57-62.doi: 10.1016/j.jbiotec.2018.08.001)上。构建流程如下：首先，以质粒pJMP1(Peterset al..2016.Cell. 165:1493-506.doi:10.1016/j.cell.2016.05.003)为模板，用引物P1/P2扩增含有木糖诱导的 dCas9表达盒xylR-Pxyl-dcas9部分；干扰mutSL的sgRNA表达盒序列委托生物公司合成(SEQ ID NO.1中第8310bp-8480bp)，然后经引物P3/P4扩增后得到片段Pveg-sgmutSL；以质粒pUS20 为模板，用引物P5/P6扩增质粒pUS20全部序列。然后，将上述3片段通过同源重组的片段融合成环状质粒。最终，将上述融合产物转化到大肠杆菌DH5α后，经100ppm氨苄青霉素筛选后得到质粒pUS20D9M。
工作原理如图1b所示，添加木糖后会诱使用CRISPRi技术干扰与DNA错配修复的相关的基因mutSL的转录，由于抑制了mutSL的转录，从而提高了自发突变率。将该质粒转化菌株168中枯草芽孢杆菌168(pUS20D9M)，当在含有1％木糖的LB培养基(蛋白胨1％，酵母粉0.5％，氯化钠1％，pH 7.0)37℃培养12h，枯草芽孢杆菌168(pUS20D9M)比原始枯草芽孢杆菌168提高约428.4倍(使用利福平的突变情况来评估菌体的自发突变率)。在没有木糖诱导的情况下，自发突变率仍比原始菌株168高了约78.5倍(图2)。但质粒pUS20D9M 在建库后可丢失(含有质粒pUS20D9M的菌株在没有抗生素的LB培养基中37℃培养12h后，质粒丢失率为80％)，从而使菌体恢复正常状态。
实施例2质粒pUS20XP1用于提高枯草芽孢杆菌的自发突变率
将含有木糖诱导的dCas9表达盒的染色体整合质粒pJMP1(Peters etal..2016.Cell. 165:1493-506.doi:10.1016/j.cell.2016.05.003)转化到枯草芽孢杆菌168中，在基因lacA处通过双交换后将含有木糖诱导的dCas9表达盒的染色体上，由红霉素抗性筛选得到菌株168D。
如图3所示，通过同源重组的方式将木糖诱导失去校对功能的DNA聚合酶PolC*表达盒以及干扰polC的sgRNA表达盒构建在质粒pUS20上，生成质粒pUS20XP1。构建流程如下：首先，以枯草芽孢杆菌168基因组为模板，分别用引物P7/P8,P9/P10和P11/P12扩增DNA 聚合酶polC*的3个部分，其中碱基突变由P9和P11引入，将上述3部分融合为失去校对功能的DNA聚合酶PolC*表达盒；干扰polC的sgRNA表达盒序列委托生物公司合成，然后经引物P13/P14扩增后得到片段Pveg-sgpolC；以质粒pUS20D9M为模板，用引物P15/P16扩增质粒pUS20全部序列和xylR-Pxyl部分。然后，将上述3片段通过同源重组的片段融合成环状质粒。最终，将上述融合产物转化到大肠杆菌DH5α后，经100ppm氨苄青霉素筛选后得到质粒pUS20XP1。
将质粒pUS20XP1转化菌株168D中得到菌株168D(pUS20XP1)，当在LB培养基中添加木糖后，polC基因的转录被抑制并诱导表达失去校对功能的DNA聚合酶polC*后，菌株的自发突变率明显提高。在含有1％木糖培养12h后，菌株168D(pUS20XP1)比原始菌株168 提高约629.5倍(使用利福平的突变情况来评估菌体的自发突变率)。在没有木糖诱导的情况下，自发突变率仍比原始菌株168高了约52.8倍(图4)。但质粒pUS20XP1在建库后可丢失 (丢失方法同质粒pUS20D9M一致)，从而使菌体恢复正常状态。
实施例3质粒pUS20XP2用于提高枯草芽孢杆菌的自发突变率
为了进一步提高质粒型枯草芽孢杆菌自发突变系统中的自发突变率，尝试将干扰mutSL 系统和表达polC*系统结合在一起，以此构建质粒pUS20XP2(图5)。构建流程如下：首先，以质粒pUS20XP1为模板，用引物P17/P18扩增质粒pUS20XP1全部序列；以质粒pUS20D9M为模板，用引物P19/P20扩增后得到片段Pveg-sgmutSL。然后，将上述2片段通过同源重组的片段融合成环状质粒。最终，将上述融合产物转化到大肠杆菌DH5α后，经100ppm氨苄青霉素筛选后得到质粒pUS20XP2。
如图5所示，该质粒含有木糖诱导表达的polC*和两条分别干扰mutSL和polC的sgRNA 表达盒，并将该质粒转化菌株168D中，得到可以通过木糖控制的一株自发突变率更高的菌株。如图6所示，在含有1％木糖培养12h后，菌株168D(pUS20XP2)比WT提高约5128.3倍。在没有木糖诱导的情况下，自发突变率仍比WT高了约437.5倍。综上，该系统有效提高了自发突变率，虽然在不添加诱导剂的情况下也有较高的突变率，但质粒pUS20XP在建库后可丢失(丢失方法同质粒pUS20D9M一致)，从而使菌体恢复正常状态。
表1本发明所用引物
Figure RE-GDA0003318291800000061
序列表
<110> 南京农业大学
<120> 用于提高枯草芽孢杆菌自发突变频率的质粒
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10672
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccgggtaccg agctcgaatt cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 60
tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 120
ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 180
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 240
tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 300
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 360
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 420
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 480
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 540
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 600
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 660
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 720
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 780
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 840
gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 900
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 960
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 1020
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 1080
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 1140
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 1200
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 1260
actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 1320
aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 1380
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 1440
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 1500
cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 1560
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 1620
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 1680
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 1740
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 1800
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 1860
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 1920
gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 1980
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 2040
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 2100
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 2160
atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 2220
taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa 2280
cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag 2340
cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta 2400
tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag 2460
atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg 2520
ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc 2580
tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 2640
ggccagtgcc aagcttgcat gcctgcagtt cagaacgctc ggttgccgcc gggcgttttt 2700
tatgcagcaa tggcaagaac gtcccgggga gctcctaact tataggggta acacttaaaa 2760
aagaatcaat aacgatagaa accgctccta aagcaggtgc attttttcct aacgaagaag 2820
gcaatagttc acatttattg tctaaatgag aatggactct agaagaaact tcgtttttaa 2880
tcgtatttaa aacaatggga tgagattcaa ttatatgatt tctcaagata acagcttcta 2940
tatcaaatgt attaaggata ttggttaatc caattccgat ataaaagcca aagttttgaa 3000
gtgcatttaa catttctaca tcatttttat ttgcgcgttc cacaatctct tttcgagaaa 3060
tattcttttc ttctttagag agcgaagcca gtaacgcttt ttcagaagca tataattccc 3120
aacagcctcg atttccacag ctgcatttgg gtccattaaa atctatcgtc atatgaccca 3180
tttccccaga aaaaccctga acacctttat acaattcgtt gttaataaca agtccagttc 3240
caattccgat attaatactg atgtaaacga tgttttcata gttttttgtc ataccaaata 3300
ctttttcacc gtatgctcct gcattagctt cattttcaac aaaaaccgga acattaaact 3360
cactctcaat taaaaactgc aaatctttga tattccaatt taagttaggc atgaaaataa 3420
tttgctgatg acgatctaca aggcctggaa cacaaattcc tattccgact agaccataag 3480
gggactcagg catatgggtt acaaaaccat gaataagtgc aaataaaatc tcttttactt 3540
cactagcgga agaactagac aagtcagaag tcttctcgag aataatattt ccttctaagt 3600
cggttagaat tccgttaaga tagtcgactc ctatatcaat accaatcgag tagcctgcat 3660
tcttattaaa aacaagcatt acaggtcttc tgccgcctct agattgccct gccccaattt 3720
caaaaataaa atctttttca agcagtgtat ttacttgaga ggagacagta gacttgttta 3780
atcctgtaat ctcagagaga gttgccctgg agacagggga gttcttcaaa atttcatcta 3840
atattaattt ttgattcatt ttttttacta aagcttgatc tgcaatttga ataataacca 3900
ctcctttgtt tatccaccga actaagttgg tgttttttga agcttgaatt agatatttaa 3960
aagtatcata tctaatatta taactaaatt ttctaaaaaa aacattgaaa taaacattta 4020
ttttgtatat gatgagataa agttagttta ttggataaac aaactaactc aattaagata 4080
gttgatggat aaacttgttc acttaaatca aagggggaaa tgacaaatgg tccaaactag 4140
tgatatctaa aaatcaaagg gggaaatggg atccatggat aagaaatact caataggctt 4200
agctatcggc acaaatagcg tcggatgggc ggtgatcact gatgaatata aggttccgtc 4260
taaaaagttc aaggttctgg gaaatacaga ccgccacagt atcaaaaaaa atcttatagg 4320
ggctctttta tttgacagtg gagagacagc ggaagcgact cgtctcaaac ggacagctcg 4380
tagaaggtat acacgtcgga agaatcgtat ttgttatcta caggagattt tttcaaatga 4440
gatggcgaaa gtagatgata gtttctttca tcgacttgaa gagtcttttt tggtggaaga 4500
agacaagaag catgaacgtc atcctatttt tggaaatata gtagatgaag ttgcttatca 4560
tgagaaatat ccaactatct atcatctgcg aaaaaaattg gtagattcta ctgataaagc 4620
ggatttgcgc ttaatctatt tggccttagc gcatatgatt aagtttcgtg gtcatttttt 4680
gattgaggga gatttaaatc ctgataatag tgatgtggac aaactattta tccagttggt 4740
acaaacctac aatcaattat ttgaagaaaa ccctattaac gcaagtggag tagatgctaa 4800
agcgattctt tctgcacgat tgagtaaatc aagacgatta gaaaatctca ttgctcagct 4860
ccccggtgag aagaaaaatg gcttatttgg gaatctcatt gctttgtcat tgggtttgac 4920
ccctaatttt aaatcaaatt ttgatttggc agaagatgct aaattacagc tttcaaaaga 4980
tacttacgat gatgatttag ataatttatt ggcgcaaatt ggagatcaat atgctgattt 5040
gtttttggca gctaagaatt tatcagatgc tattttactt tcagatatcc taagagtaaa 5100
tactgaaata actaaggctc ccctatcagc ttcaatgatt aaacgctacg atgaacatca 5160
tcaagacttg actcttttaa aagctttagt tcgacaacaa cttccagaaa agtataaaga 5220
aatctttttt gatcaatcaa aaaacggata tgcaggttat attgatgggg gagctagcca 5280
agaagaattt tataaattta tcaaaccaat tttagaaaaa atggatggta ctgaggaatt 5340
attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa cggacctttg acaacggctc 5400
tattccccat caaattcact tgggtgagct gcatgctatt ttgagaagac aagaagactt 5460
ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc 5520
ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc 5580
tgaagaaaca attaccccat ggaattttga agaagttgtc gataaaggtg cttcagctca 5640
atcatttatt gaacgcatga caaactttga taaaaatctt ccaaatgaaa aagtactacc 5700
aaaacatagt ttgctttatg agtattttac ggtttataac gaattgacaa aggtcaaata 5760
tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt gaacagaaga aagccattgt 5820
tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag caattaaaag aagattattt 5880
caaaaaaata gaatgttttg atagtgttga aatttcagga gttgaagata gatttaatgc 5940
ttcattaggt acctaccatg atttgctaaa aattattaaa gataaagatt ttttggataa 6000
tgaagaaaat gaagatatct tagaggatat tgttttaaca ttgaccttat ttgaagatag 6060
ggagatgatt gaggaaagac ttaaaacata tgctcacctc tttgatgata aggtgatgaa 6120
acagcttaaa cgtcgccgtt atactggttg gggacgtttg tctcgaaaat tgattaatgg 6180
tattagggat aagcaatctg gcaaaacaat attagatttt ttgaaatcag atggttttgc 6240
caatcgcaat tttatgcagc tgatccatga tgatagtttg acatttaaag aagacattca 6300
aaaagcacaa gtgtctggac aaggcgatag tttacatgaa catattgcaa atttagctgg 6360
tagccctgct attaaaaaag gtattttaca gactgtaaaa gttgttgatg aattggtcaa 6420
agtaatgggg cggcataagc cagaaaatat cgttattgaa atggcacgtg aaaatcagac 6480
aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa cgaatcgaag aaggtatcaa 6540
agaattagga agtcagattc ttaaagagca tcctgttgaa aatactcaat tgcaaaatga 6600
aaagctctat ctctattatc tccaaaatgg aagagacatg tatgtggacc aagaattaga 6660
tattaatcgt ttaagtgatt atgatgtcga tgccattgtt ccacaaagtt tccttaaaga 6720
cgattcaata gacaataagg tcttaacgcg ttctgataaa aatcgtggta aatcggataa 6780
cgttccaagt gaagaagtag tcaaaaagat gaaaaactat tggagacaac ttctaaacgc 6840
caagttaatc actcaacgta agtttgataa tttaacgaaa gctgaacgtg gaggtttgag 6900
tgaacttgat aaagctggtt ttatcaaacg ccaattggtt gaaactcgcc aaatcactaa 6960
gcatgtggca caaattttgg atagtcgcat gaatactaaa tacgatgaaa atgataaact 7020
tattcgagag gttaaagtga ttaccttaaa atctaaatta gtttctgact tccgaaaaga 7080
tttccaattc tataaagtac gtgagattaa caattaccat catgcccatg atgcgtatct 7140
aaatgccgtc gttggaactg ctttgattaa gaaatatcca aaacttgaat cggagtttgt 7200
ctatggtgat tataaagttt atgatgttcg taaaatgatt gctaagtctg agcaagaaat 7260
aggcaaagca accgcaaaat atttctttta ctctaatatc atgaacttct tcaaaacaga 7320
aattacactt gcaaatggag agattcgcaa acgccctcta atcgaaacta atggggaaac 7380
tggagaaatt gtctgggata aagggcgaga ttttgccaca gtgcgcaaag tattgtccat 7440
gccccaagtc aatattgtca agaaaacaga agtacagaca ggcggattct ccaaggagtc 7500
aattttacca aaaagaaatt cggacaagct tattgctcgt aaaaaagact gggatccaaa 7560
aaaatatggt ggttttgata gtccaacggt agcttattca gtcctagtgg ttgctaaggt 7620
ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag ttactaggga tcacaattat 7680
ggaaagaagt tcctttgaaa aaaatccgat tgacttttta gaagctaaag gatataagga 7740
agttaaaaaa gacttaatca ttaaactacc taaatatagt ctttttgagt tagaaaacgg 7800
tcgtaaacgg atgctggcta gtgccggaga attacaaaaa ggaaatgagc tggctctgcc 7860
aagcaaatat gtgaattttt tatatttagc tagtcattat gaaaagttga agggtagtcc 7920
agaagataac gaacaaaaac aattgtttgt ggagcagcat aagcattatt tagatgagat 7980
tattgagcaa atcagtgaat tttctaagcg tgttatttta gcagatgcca atttagataa 8040
agttcttagt gcatataaca aacatagaga caaaccaata cgtgaacaag cagaaaatat 8100
tattcattta tttacgttga cgaatcttgg agctcccgct gcttttaaat attttgatac 8160
aacaattgat cgtaaacgat atacgtctac aaaagaagtt ttagatgcca ctcttatcca 8220
tcaatccatc actggtcttt atgaaacacg cattgatttg agtcagctag gaggtgacta 8280
actgcagctc gagaagctta tcgaattcct tattaacgtt gatataattt aaattttatt 8340
tgacaaaaat gggctcgtgt tgtacaataa atgtctgtat cataggcgta taacgtttta 8400
gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc 8460
gagtcggtgc tttttttgaa gcttgggccc gaacagtcga cgattggtac caggcccggg 8520
gctttacgtc aaaaaaaaaa tcgtgtaaga taaagtcaag ttaagtcaat gtgaaagttt 8580
tggcattgaa ttctggttct agttattcta aataaacaac aaaaaaccct gttgtttgca 8640
gacaacaagg ttaatagatt tgatgataca tattttgttt gcgagctatt tgcaaaattg 8700
aatacaaata aagaatgaca aataaaaagt gtccgatgtt gcagcatcga acacttggtt 8760
tgtaagccct cacttacaat gtttaagtta tgtcttcatt ttaccgaaaa caagtgtata 8820
cgtcaacact tttcggctat ttttggacga cagaaaacca ctgtttggag ggtatcaaac 8880
cgtggttttc tgtcgttttt tgtttgtcac ccagctgccg ttaaggggtg taaaataaac 8940
tgacgtcagg agttgccccg tccaccgcaa acccggggat ataccacaat aagcgttcct 9000
tgctgcgctt gtggtgaatg gcgaggacgg ccattaacgg tccggagaat gtgcctaaac 9060
cactaggttg cttggcgcac tacggtgcgg gcaactatgg ccaagcttct atgcagcggc 9120
acacaattga gtgtgaccgt gtgagagaac acgagacaaa ccccatagaa gttgcataca 9180
ggacaagcat tgtatgtaaa aaggtcatgg caggcgaaag cctggcgata gcaaggcaga 9240
gctggaaacc tcagacgtct ctatgacgct agtttcaaat cctgatcgat tgcctatgtc 9300
tgtccgtttt tattttttgc gcgacagaca taggcaatcg aaattcgccc tgtttcctag 9360
aacagtcagc tattctgcta gtttcaagtt ctcaaagtca agagaaaaac actaaataaa 9420
atgcgaattt ccttaaaacc cagaggtttt aagttccttt ttaagggttg ttcgtttgat 9480
agaacattag tattttaata tatttttcct gtgggatttc actaacgtat actgttattc 9540
cctttggcct atcttaaaaa cttgtataga ttgtgtttga gccgtttagt atgatatcag 9600
tacatgcttg ctagcaatct ctagaggatc cgattttcgt tcgtgaatac atgttataat 9660
aactataact aataacgtaa cgtgactggc aagagatatt tttaaaacaa tgaataggtt 9720
tacacttact ttagttttat ggaaatgaaa gatcatatca tatataatct agaataaaat 9780
taactaaaat aattattatc tagataaaaa atttagaagc caatgaaatc tataaataaa 9840
ctaaattaag tttatttaat taacaactat ggatataaaa taggtactaa tcaaaatagt 9900
gaggaggata tatttgaata catacgaaca aattaataaa gtgaaaaaaa tacttcggaa 9960
acatttaaaa aataacctta ttggtactta catgtttgga tcaggagttg agagtggact 10020
aaaaccaaat agtgatcttg actttttagt cgtcgtatct gaaccattga cagatcaaag 10080
taaagaaata cttatacaaa aaattagacc tatttcaaaa aaaataggag ataaaagcaa 10140
cttacgatat attgaattaa caattattat tcagcaagaa atggtaccgt ggaatcatcc 10200
tcccaaacaa gaatttattt atggagaatg gttacaagag ctttatgaac aaggatacat 10260
tcctcagaag gaattaaatt cagatttaac cataatgctt taccaagcaa aacgaaaaaa 10320
taaaagaata tacggaaatt atgacttaga ggaattacta cctgatattc cattttctga 10380
tgtgagaaga gccattatgg attcgtcaga ggaattaata gataattatc aggatgatga 10440
aaccaactct atattaactt tatgccgtat gattttaact atggacacgg gtaaaatcat 10500
accaaaagat attgcgggaa atgcagtggc tgaatcttct ccattagaac atagggagag 10560
aattttgtta gcagttcgta gttatcttgg agagaatatt gaatggacta atgaaaatgt 10620
aaatttaact ataaactatt taaataacag attaaaaaaa ttataaggat cc 10672
<210> 2
<211> 10888
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggatccttat aattttttta atctgttatt taaatagttt atagttaaat ttacattttc 60
attagtccat tcaatattct ctccaagata actacgaact gctaacaaaa ttctctccct 120
atgttctaat ggagaagatt cagccactgc atttcccgca atatcttttg gtatgatttt 180
acccgtgtcc atagttaaaa tcatacggca taaagttaat atagagttgg tttcatcatc 240
ctgataatta tctattaatt cctctgacga atccataatg gctcttctca catcagaaaa 300
tggaatatca ggtagtaatt cctctaagtc ataatttccg tatattcttt tattttttcg 360
ttttgcttgg taaagcatta tggttaaatc tgaatttaat tccttctgag gaatgtatcc 420
ttgttcataa agctcttgta accattctcc ataaataaat tcttgtttgg gaggatgatt 480
ccacggtacc atttcttgct gaataataat tgttaattca atatatcgta agttgctttt 540
atctcctatt ttttttgaaa taggtctaat tttttgtata agtatttctt tactttgatc 600
tgtcaatggt tcagatacga cgactaaaaa gtcaagatca ctatttggtt ttagtccact 660
ctcaactcct gatccaaaca tgtaagtacc aataaggtta ttttttaaat gtttccgaag 720
tatttttttc actttattaa tttgttcgta tgtattcaaa tatatcctcc tcactatttt 780
gattagtacc tattttatat ccatagttgt taattaaata aacttaattt agtttattta 840
tagatttcat tggcttctaa attttttatc tagataataa ttattttagt taattttatt 900
ctagattata tatgatatga tctttcattt ccataaaact aaagtaagtg taaacctatt 960
cattgtttta aaaatatctc ttgccagtca cgttacgtta ttagttatag ttattataac 1020
atgtattcac gaacgaaaat cggatcctct agagattgct agcaagcatg tactgatatc 1080
ttcagaacgc tcggttgccg ccgggcgttt tttatgcagc aatggcaaga acgtcccggg 1140
gagctcctaa cttatagggg taacacttaa aaaagaatca ataacgatag aaaccgctcc 1200
taaagcaggt gcattttttc ctaacgaaga aggcaatagt tcacatttat tgtctaaatg 1260
agaatggact ctagaagaaa cttcgttttt aatcgtattt aaaacaatgg gatgagattc 1320
aattatatga tttctcaaga taacagcttc tatatcaaat gtattaagga tattggttaa 1380
tccaattccg atataaaagc caaagttttg aagtgcattt aacatttcta catcattttt 1440
atttgcgcgt tccacaatct cttttcgaga aatattcttt tcttctttag agagcgaagc 1500
cagtaacgct ttttcagaag catataattc ccaacagcct cgatttccac agctgcattt 1560
gggtccatta aaatctatcg tcatatgacc catttcccca gaaaaaccct gaacaccttt 1620
atacaattcg ttgttaataa caagtccagt tccaattccg atattaatac tgatgtaaac 1680
gatgttttca tagttttttg tcataccaaa tactttttca ccgtatgctc ctgcattagc 1740
ttcattttca acaaaaaccg gaacattaaa ctcactctca attaaaaact gcaaatcttt 1800
gatattccaa tttaagttag gcatgaaaat aatttgctga tgacgatcta caaggcctgg 1860
aacacaaatt cctattccga ctagaccata aggggactca ggcatatggg ttacaaaacc 1920
atgaataagt gcaaataaaa tctcttttac ttcactagcg gaagaactag acaagtcaga 1980
agtcttctcg agaataatat ttccttctaa gtcggttaga attccgttaa gatagtcgac 2040
tcctatatca ataccaatcg agtagcctgc attcttatta aaaacaagca ttacaggtct 2100
tctgccgcct ctagattgcc ctgccccaat ttcaaaaata aaatcttttt caagcagtgt 2160
atttacttga gaggagacag tagacttgtt taatcctgta atctcagaga gagttgccct 2220
ggagacaggg gagttcttca aaatttcatc taatattaat ttttgattca ttttttttac 2280
taaagcttga tctgcaattt gaataataac cactcctttg tttatccacc gaactaagtt 2340
ggtgtttttt gaagcttgaa ttagatattt aaaagtatca tatctaatat tataactaaa 2400
ttttctaaaa aaaacattga aataaacatt tattttgtat atgatgagat aaagttagtt 2460
tattggataa acaaactaac tcaattaaga tagttgatgg ataaacttgt tcacttaaat 2520
caaaggggga aatgacaaat ggtccaaact agttagggag ggatactgtc ttaatggaac 2580
agttatcagt aaacagaagg cagtttcaaa ttcttctgca gcagattaat atgacagatg 2640
ataccttcat gacatacttt gaacatggcg agattaaaaa gctgacaatt cacaaagctt 2700
ctaagtcttg gcattttcat tttcaattta aatctttgct gccttttcaa atatatgaca 2760
cattaacaac gaggctgacg caatcgtttg cccacatagc aaaagtgaca tcttcaattg 2820
aagttcagga tgccgaggtc agtgaaagta tcgttcaaga ctactggtca cgctgcattg 2880
aagaactgca gggcatttcg ccgccgatta tcagtctttt aaaccagcaa aaaccgaagc 2940
tgaagggcaa taaactgatt gtcaaaacca aaacagatac agaagcggct gcgctaaaga 3000
acaaatacag ttccatgatt caagcagaat accgtcaatt tggctttccg gatcttcagc 3060
ttgatgctga aatatttgta tccgagcaag aagttcaaaa gtttcgggag caaaagcttg 3120
cggaagacca agagcgggct atgcaggcct tgattgaaat ggagaagaaa gataaagaaa 3180
gtgatgaaga ccaagcacca tctggtcctc ttgttatcgg ttatcaaatt aaagataacg 3240
aggaaatccg aacacttgac agcatcatgg acgaagaacg gagaattacg gtccaaggtt 3300
atgtgtttga tgtggagacg cgcgagctga agagcgggcg cacgctgtgt atcttcaaaa 3360
ttacagacta tacaaatagt attttgatca aaatgtttgc acgtgaaaaa gaagatgcgg 3420
cgctgatgaa gtctctgaaa aaaggaatgt gggtaaaagc acgcggaagc attcaaaatg 3480
atacatttgt cagagacctt gtcatgatcg caaatgacgt aaacgaaata aaagcaaaaa 3540
cccgtgaaga ttcagcacct gaaggagaaa aaagagtgga attgcatctt cattccccaa 3600
tgagccaaat ggatgctgtt acgggtatcg gaaagcttgt cgaacaggcg aaaaaatggg 3660
ggcatgaggc catcgctttg accgaccatg ctgtcgttca atccttccct gatgcgtatt 3720
ctgcggccaa aaagcatgga attaaaatga tttacgggat ggaagcgaat ctcgtggatg 3780
atggcgtgcc aattgcttat aatgccgcac atcgtctgct cgaagaagaa acatatgttg 3840
tttttgacgt tgagacgaca ggattgtctg ctgtatacga taccattatt gagctggctg 3900
cagtaaaagt aaaaggcgga gaaattattg ataaatttga ggcgtttgcg aacccgcatc 3960
gtccgctttc cgccacaatc atagagctga cagggatcac agatgatatg ctacaagacg 4020
ctccggatgt cgtagatgta ataagagatt tcagagaatg gattggcgat gatattcttg 4080
tcgctcataa tgcaagcttt gatatgggat tcttaaatgt agcctataaa aaacttcttg 4140
aagtcgaaaa agctaaaaac ccagtcattg atacgcttga acttggacgt tttctctatc 4200
cggaatttaa gaaccaccgg ttgaacacac tttgtaaaaa gtttgatatc gagctcacac 4260
agcatcaccg tgcgatctat gatactgagg caaccgctta tttgcttctg aaaatgctga 4320
aagacgcagc tgaaaaaggt attcagtacc atgatgagtt gaatgaaaat atgggtcagt 4380
ccaatgctta tcaaagatca agaccgtatc atgcaacatt acttgccgtg aacagcacgg 4440
gacttaaaaa tttatttaag cttgtgtcac tttctcatat tcattatttt tacagagtgc 4500
cgcgtattcc gagatctcag cttgagaaat acagggaagg gcttctgatc ggttctgctt 4560
gtgacagggg agaggttttt gagggaatga tgcaaaaatc gcctgaagag gtggaagata 4620
tcgcgtcatt ctatgattac cttgaggttc agccgcctga agtgtatcgt cacttgctgg 4680
agcttgaact ggtccgtgat gaaaaagcgc tgaaagaaat tattgcgaat atcacgaagc 4740
tgggggaaaa gcttaataaa ccggttgttg ctacgggaaa tgttcattac ttgaatgatg 4800
aggataaaat ctacagaaag attttaatat cctcacaagg cggggcaaat ccgctgaata 4860
ggcatgaact gccgaaagtg catttcagaa cgacagacga aatgcttgaa gctttttctt 4920
tcttaggtga agaaaaagcg aaggagatcg tagtcaccaa tacccaaaag gttgcttctt 4980
tagttgatga catcaagccg attaaagatg atttatatac gccgaaaatc gaaggcgctg 5040
atgaagagat cagagaaatg agctatcagc gtgcaagaag catttacggg gaagagctgc 5100
ctgaaattgt cgaagcgcgg attgaaaaag agttaaagag tattattggc cacggatttg 5160
ctgttattta cttgatctct cacaaacttg taaaacgttc actagatgac gggtatctcg 5220
ttggttcccg tggttccgta ggatcttcat tagttgcgac acttactgag attactgagg 5280
taaacccgct gccgccgcac tatgtttgtc ctgagtgcca gcattctgag ttctttaatg 5340
acggttctgt cggttctggt tttgacctgc ctgacaagac atgccctcat tgcggaacgc 5400
ctttgaaaaa agacggccat gatattccat ttgaaacgtt cttaggattt aaaggggaca 5460
aagtacctga tatcgatttg aacttctcag gggaatatca gccgcaagca cacaattaca 5520
caaaagtatt gttcggagaa gacaatgtat atcgtgcggg aacaataggc acggtggcag 5580
aaaaaacagc ctacggttat gtaaaaggct atgccggaga caacaatctt catatgcgcg 5640
gtgccgaaat agatcggctc gtacagggat gcacaggtgt aaaacgtaca actggacagc 5700
accctggcgg tattatcgta gttccggatt atatggatat ttatgatttt tcaccgatcc 5760
agttcccggc agatgccaca ggttcagagt ggaaaacgac tcattttgat ttccactcca 5820
tccatgacaa cctgttaaaa cttgatattc tcggacacga tgacccgact gttattcgga 5880
tgcttcaaga cttaagcgga atagatccga aaacaattcc gacggatgat cctgaagtga 5940
tgaagatctt ccagggaacc gaatcactcg gtgtgactga agaacagatt ggctgtaaaa 6000
cgggcactct tggaattcct gaattcggaa cccgatttgt ccggcagatg cttgaagata 6060
caaagccgac cactttttct gagctcgttc agatttcagg cttgtctcac ggaactgatg 6120
tatggcttgg caatgcacag gagctcatcc acaataatat ttgtgagctg agtgaggtta 6180
tcggctgccg tgatgacatt atggtttatt taatctatca aggccttgag ccgtcccttg 6240
cctttaaaat catggaattc gtgcgtaaag gaaaaggatt aacgcctgaa tgggaagaag 6300
aaatgaaaaa taacaatgtc ccagactggt atattgattc ctgtaaaaag attaaataca 6360
tgttcccgaa agcccacgcc gcggcatatg tcttaatggc agtccgcatt gcttacttta 6420
aagtgcatca tgctcttttg tattatgcgg cttattttac cgttcgtgca gatgactttg 6480
atattgatac aatgatcaag ggctctacag caatcagagc ggtaatggag gatataaacg 6540
ctaaaggact tgatgcttca ccgaaggaaa agaaccttct gactgtttta gaattagcgc 6600
ttgagatgtg tgagagaggc tattcattcc aaaaagtcga tttatatcgc tccagcgcca 6660
cagagtttat tattgacggc aacagtctta ttccgccgtt taactctatt ccagggttag 6720
ggacgaacgc tgctttgaac attgtaaaag ctcgcgaaga aggcgaattc ctctcaaaag 6780
aagatttgca aaagagaggg aaagtatcaa aaacgatttt agagtactta gatcgccatg 6840
gctgtctgga gtcactgcct gatcaaaacc aattgtcact gttctaaaat cgtcgactgt 6900
tcgggcccaa gcttcaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga 6960
ctagccttat tttaacttgc tatttctagc tctaaaacga ggtaccttca cttatgatac 7020
atttattgta caacacgagc ccatttttgt caaataaaat ttaaattata tcaacgttaa 7080
taaggaattc gataagcttc tcgactgttc gggcccaagc gatatcatac taaacggctc 7140
aaacacaatc tatacaagtt tttaagatag gccaaaggga ataacagtat acgttagtga 7200
aatcccacag gaaaaatata ttaaaatact aatgttctat caaacgaaca acccttaaaa 7260
aggaacttaa aacctctggg ttttaaggaa attcgcattt tatttagtgt ttttctcttg 7320
actttgagaa cttgaaacta gcagaatagc tgactgttct aggaaacagg gcgaatttcg 7380
attgcctatg tctgtcgcgc aaaaaataaa aacggacaga cataggcaat cgatcaggat 7440
ttgaaactag cgtcatagag acgtctgagg tttccagctc tgccttgcta tcgccaggct 7500
ttcgcctgcc atgacctttt tacatacaat gcttgtcctg tatgcaactt ctatggggtt 7560
tgtctcgtgt tctctcacac ggtcacactc aattgtgtgc cgctgcatag aagcttggcc 7620
atagttgccc gcaccgtagt gcgccaagca acctagtggt ttaggcacat tctccggacc 7680
gttaatggcc gtcctcgcca ttcaccacaa gcgcagcaag gaacgcttat tgtggtatat 7740
ccccgggttt gcggtggacg gggcaactcc tgacgtcagt ttattttaca ccccttaacg 7800
gcagctgggt gacaaacaaa aaacgacaga aaaccacggt ttgataccct ccaaacagtg 7860
gttttctgtc gtccaaaaat agccgaaaag tgttgacgta tacacttgtt ttcggtaaaa 7920
tgaagacata acttaaacat tgtaagtgag ggcttacaaa ccaagtgttc gatgctgcaa 7980
catcggacac tttttatttg tcattcttta tttgtattca attttgcaaa tagctcgcaa 8040
acaaaatatg tatcatcaaa tctattaacc ttgttgtctg caaacaacag ggttttttgt 8100
tgtttattta gaataactag aaccagaatt caatgccaaa actttcacat tgacttaact 8160
tgactttatc ttacacgatt ttttttttga cgtaaagccc cgggcctggt accagtcgac 8220
ctgcaggcat gcaagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 8280
tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 8340
cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg 8400
cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 8460
tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc 8520
cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 8580
cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg 8640
aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta 8700
gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 8760
aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 8820
ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 8880
ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 8940
agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 9000
tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 9060
tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 9120
ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 9180
gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 9240
acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 9300
tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 9360
gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 9420
actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 9480
aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 9540
cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 9600
tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 9660
cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 9720
tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 9780
ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 9840
ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 9900
cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 9960
aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 10020
agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 10080
tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 10140
ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 10200
cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 10260
atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 10320
ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 10380
tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 10440
gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 10500
gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 10560
cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 10620
gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 10680
tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 10740
aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 10800
tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 10860
tgattacgaa ttcgagctcg gtacccgg 10888
<210> 3
<211> 11093
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggatccttat aattttttta atctgttatt taaatagttt atagttaaat ttacattttc 60
attagtccat tcaatattct ctccaagata actacgaact gctaacaaaa ttctctccct 120
atgttctaat ggagaagatt cagccactgc atttcccgca atatcttttg gtatgatttt 180
acccgtgtcc atagttaaaa tcatacggca taaagttaat atagagttgg tttcatcatc 240
ctgataatta tctattaatt cctctgacga atccataatg gctcttctca catcagaaaa 300
tggaatatca ggtagtaatt cctctaagtc ataatttccg tatattcttt tattttttcg 360
ttttgcttgg taaagcatta tggttaaatc tgaatttaat tccttctgag gaatgtatcc 420
ttgttcataa agctcttgta accattctcc ataaataaat tcttgtttgg gaggatgatt 480
ccacggtacc atttcttgct gaataataat tgttaattca atatatcgta agttgctttt 540
atctcctatt ttttttgaaa taggtctaat tttttgtata agtatttctt tactttgatc 600
tgtcaatggt tcagatacga cgactaaaaa gtcaagatca ctatttggtt ttagtccact 660
ctcaactcct gatccaaaca tgtaagtacc aataaggtta ttttttaaat gtttccgaag 720
tatttttttc actttattaa tttgttcgta tgtattcaaa tatatcctcc tcactatttt 780
gattagtacc tattttatat ccatagttgt taattaaata aacttaattt agtttattta 840
tagatttcat tggcttctaa attttttatc tagataataa ttattttagt taattttatt 900
ctagattata tatgatatga tctttcattt ccataaaact aaagtaagtg taaacctatt 960
cattgtttta aaaatatctc ttgccagtca cgttacgtta ttagttatag ttattataac 1020
atgtattcac gaacgaaaat cggatcctct agagattgct agcaagcatg tactgatatc 1080
ttcagaacgc tcggttgccg ccgggcgttt tttatgcagc aatggcaaga acgtcccggg 1140
gagctcctaa cttatagggg taacacttaa aaaagaatca ataacgatag aaaccgctcc 1200
taaagcaggt gcattttttc ctaacgaaga aggcaatagt tcacatttat tgtctaaatg 1260
agaatggact ctagaagaaa cttcgttttt aatcgtattt aaaacaatgg gatgagattc 1320
aattatatga tttctcaaga taacagcttc tatatcaaat gtattaagga tattggttaa 1380
tccaattccg atataaaagc caaagttttg aagtgcattt aacatttcta catcattttt 1440
atttgcgcgt tccacaatct cttttcgaga aatattcttt tcttctttag agagcgaagc 1500
cagtaacgct ttttcagaag catataattc ccaacagcct cgatttccac agctgcattt 1560
gggtccatta aaatctatcg tcatatgacc catttcccca gaaaaaccct gaacaccttt 1620
atacaattcg ttgttaataa caagtccagt tccaattccg atattaatac tgatgtaaac 1680
gatgttttca tagttttttg tcataccaaa tactttttca ccgtatgctc ctgcattagc 1740
ttcattttca acaaaaaccg gaacattaaa ctcactctca attaaaaact gcaaatcttt 1800
gatattccaa tttaagttag gcatgaaaat aatttgctga tgacgatcta caaggcctgg 1860
aacacaaatt cctattccga ctagaccata aggggactca ggcatatggg ttacaaaacc 1920
atgaataagt gcaaataaaa tctcttttac ttcactagcg gaagaactag acaagtcaga 1980
agtcttctcg agaataatat ttccttctaa gtcggttaga attccgttaa gatagtcgac 2040
tcctatatca ataccaatcg agtagcctgc attcttatta aaaacaagca ttacaggtct 2100
tctgccgcct ctagattgcc ctgccccaat ttcaaaaata aaatcttttt caagcagtgt 2160
atttacttga gaggagacag tagacttgtt taatcctgta atctcagaga gagttgccct 2220
ggagacaggg gagttcttca aaatttcatc taatattaat ttttgattca ttttttttac 2280
taaagcttga tctgcaattt gaataataac cactcctttg tttatccacc gaactaagtt 2340
ggtgtttttt gaagcttgaa ttagatattt aaaagtatca tatctaatat tataactaaa 2400
ttttctaaaa aaaacattga aataaacatt tattttgtat atgatgagat aaagttagtt 2460
tattggataa acaaactaac tcaattaaga tagttgatgg ataaacttgt tcacttaaat 2520
caaaggggga aatgacaaat ggtccaaact agttagggag ggatactgtc ttaatggaac 2580
agttatcagt aaacagaagg cagtttcaaa ttcttctgca gcagattaat atgacagatg 2640
ataccttcat gacatacttt gaacatggcg agattaaaaa gctgacaatt cacaaagctt 2700
ctaagtcttg gcattttcat tttcaattta aatctttgct gccttttcaa atatatgaca 2760
cattaacaac gaggctgacg caatcgtttg cccacatagc aaaagtgaca tcttcaattg 2820
aagttcagga tgccgaggtc agtgaaagta tcgttcaaga ctactggtca cgctgcattg 2880
aagaactgca gggcatttcg ccgccgatta tcagtctttt aaaccagcaa aaaccgaagc 2940
tgaagggcaa taaactgatt gtcaaaacca aaacagatac agaagcggct gcgctaaaga 3000
acaaatacag ttccatgatt caagcagaat accgtcaatt tggctttccg gatcttcagc 3060
ttgatgctga aatatttgta tccgagcaag aagttcaaaa gtttcgggag caaaagcttg 3120
cggaagacca agagcgggct atgcaggcct tgattgaaat ggagaagaaa gataaagaaa 3180
gtgatgaaga ccaagcacca tctggtcctc ttgttatcgg ttatcaaatt aaagataacg 3240
aggaaatccg aacacttgac agcatcatgg acgaagaacg gagaattacg gtccaaggtt 3300
atgtgtttga tgtggagacg cgcgagctga agagcgggcg cacgctgtgt atcttcaaaa 3360
ttacagacta tacaaatagt attttgatca aaatgtttgc acgtgaaaaa gaagatgcgg 3420
cgctgatgaa gtctctgaaa aaaggaatgt gggtaaaagc acgcggaagc attcaaaatg 3480
atacatttgt cagagacctt gtcatgatcg caaatgacgt aaacgaaata aaagcaaaaa 3540
cccgtgaaga ttcagcacct gaaggagaaa aaagagtgga attgcatctt cattccccaa 3600
tgagccaaat ggatgctgtt acgggtatcg gaaagcttgt cgaacaggcg aaaaaatggg 3660
ggcatgaggc catcgctttg accgaccatg ctgtcgttca atccttccct gatgcgtatt 3720
ctgcggccaa aaagcatgga attaaaatga tttacgggat ggaagcgaat ctcgtggatg 3780
atggcgtgcc aattgcttat aatgccgcac atcgtctgct cgaagaagaa acatatgttg 3840
tttttgacgt tgagacgaca ggattgtctg ctgtatacga taccattatt gagctggctg 3900
cagtaaaagt aaaaggcgga gaaattattg ataaatttga ggcgtttgcg aacccgcatc 3960
gtccgctttc cgccacaatc atagagctga cagggatcac agatgatatg ctacaagacg 4020
ctccggatgt cgtagatgta ataagagatt tcagagaatg gattggcgat gatattcttg 4080
tcgctcataa tgcaagcttt gatatgggat tcttaaatgt agcctataaa aaacttcttg 4140
aagtcgaaaa agctaaaaac ccagtcattg atacgcttga acttggacgt tttctctatc 4200
cggaatttaa gaaccaccgg ttgaacacac tttgtaaaaa gtttgatatc gagctcacac 4260
agcatcaccg tgcgatctat gatactgagg caaccgctta tttgcttctg aaaatgctga 4320
aagacgcagc tgaaaaaggt attcagtacc atgatgagtt gaatgaaaat atgggtcagt 4380
ccaatgctta tcaaagatca agaccgtatc atgcaacatt acttgccgtg aacagcacgg 4440
gacttaaaaa tttatttaag cttgtgtcac tttctcatat tcattatttt tacagagtgc 4500
cgcgtattcc gagatctcag cttgagaaat acagggaagg gcttctgatc ggttctgctt 4560
gtgacagggg agaggttttt gagggaatga tgcaaaaatc gcctgaagag gtggaagata 4620
tcgcgtcatt ctatgattac cttgaggttc agccgcctga agtgtatcgt cacttgctgg 4680
agcttgaact ggtccgtgat gaaaaagcgc tgaaagaaat tattgcgaat atcacgaagc 4740
tgggggaaaa gcttaataaa ccggttgttg ctacgggaaa tgttcattac ttgaatgatg 4800
aggataaaat ctacagaaag attttaatat cctcacaagg cggggcaaat ccgctgaata 4860
ggcatgaact gccgaaagtg catttcagaa cgacagacga aatgcttgaa gctttttctt 4920
tcttaggtga agaaaaagcg aaggagatcg tagtcaccaa tacccaaaag gttgcttctt 4980
tagttgatga catcaagccg attaaagatg atttatatac gccgaaaatc gaaggcgctg 5040
atgaagagat cagagaaatg agctatcagc gtgcaagaag catttacggg gaagagctgc 5100
ctgaaattgt cgaagcgcgg attgaaaaag agttaaagag tattattggc cacggatttg 5160
ctgttattta cttgatctct cacaaacttg taaaacgttc actagatgac gggtatctcg 5220
ttggttcccg tggttccgta ggatcttcat tagttgcgac acttactgag attactgagg 5280
taaacccgct gccgccgcac tatgtttgtc ctgagtgcca gcattctgag ttctttaatg 5340
acggttctgt cggttctggt tttgacctgc ctgacaagac atgccctcat tgcggaacgc 5400
ctttgaaaaa agacggccat gatattccat ttgaaacgtt cttaggattt aaaggggaca 5460
aagtacctga tatcgatttg aacttctcag gggaatatca gccgcaagca cacaattaca 5520
caaaagtatt gttcggagaa gacaatgtat atcgtgcggg aacaataggc acggtggcag 5580
aaaaaacagc ctacggttat gtaaaaggct atgccggaga caacaatctt catatgcgcg 5640
gtgccgaaat agatcggctc gtacagggat gcacaggtgt aaaacgtaca actggacagc 5700
accctggcgg tattatcgta gttccggatt atatggatat ttatgatttt tcaccgatcc 5760
agttcccggc agatgccaca ggttcagagt ggaaaacgac tcattttgat ttccactcca 5820
tccatgacaa cctgttaaaa cttgatattc tcggacacga tgacccgact gttattcgga 5880
tgcttcaaga cttaagcgga atagatccga aaacaattcc gacggatgat cctgaagtga 5940
tgaagatctt ccagggaacc gaatcactcg gtgtgactga agaacagatt ggctgtaaaa 6000
cgggcactct tggaattcct gaattcggaa cccgatttgt ccggcagatg cttgaagata 6060
caaagccgac cactttttct gagctcgttc agatttcagg cttgtctcac ggaactgatg 6120
tatggcttgg caatgcacag gagctcatcc acaataatat ttgtgagctg agtgaggtta 6180
tcggctgccg tgatgacatt atggtttatt taatctatca aggccttgag ccgtcccttg 6240
cctttaaaat catggaattc gtgcgtaaag gaaaaggatt aacgcctgaa tgggaagaag 6300
aaatgaaaaa taacaatgtc ccagactggt atattgattc ctgtaaaaag attaaataca 6360
tgttcccgaa agcccacgcc gcggcatatg tcttaatggc agtccgcatt gcttacttta 6420
aagtgcatca tgctcttttg tattatgcgg cttattttac cgttcgtgca gatgactttg 6480
atattgatac aatgatcaag ggctctacag caatcagagc ggtaatggag gatataaacg 6540
ctaaaggact tgatgcttca ccgaaggaaa agaaccttct gactgtttta gaattagcgc 6600
ttgagatgtg tgagagaggc tattcattcc aaaaagtcga tttatatcgc tccagcgcca 6660
cagagtttat tattgacggc aacagtctta ttccgccgtt taactctatt ccagggttag 6720
ggacgaacgc tgctttgaac attgtaaaag ctcgcgaaga aggcgaattc ctctcaaaag 6780
aagatttgca aaagagaggg aaagtatcaa aaacgatttt agagtactta gatcgccatg 6840
gctgtctgga gtcactgcct gatcaaaacc aattgtcact gttctaagca tgcctgcagc 6900
tcgagaagct tatcgaattc cttattaacg ttgatataat ttaaatttta tttgacaaaa 6960
atgggctcgt gttgtacaat aaatgtctgt atcataggcg tataacgttt tagagctaga 7020
aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 7080
gctttttttg aagcttgggc ccgaacagtc gagaagctta tcgaattcct tattaacgtt 7140
gatataattt aaattttatt tgacaaaaat gggctcgtgt tgtacaataa atgtatcata 7200
agtgaaggta cctcgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat 7260
caacttgaaa aagtggcacc gagtcggtgc tttttttgaa gcttgggccc gaacagtcga 7320
cgattgatat catactaaac ggctcaaaca caatctatac aagtttttaa gataggccaa 7380
agggaataac agtatacgtt agtgaaatcc cacaggaaaa atatattaaa atactaatgt 7440
tctatcaaac gaacaaccct taaaaaggaa cttaaaacct ctgggtttta aggaaattcg 7500
cattttattt agtgtttttc tcttgacttt gagaacttga aactagcaga atagctgact 7560
gttctaggaa acagggcgaa tttcgattgc ctatgtctgt cgcgcaaaaa ataaaaacgg 7620
acagacatag gcaatcgatc aggatttgaa actagcgtca tagagacgtc tgaggtttcc 7680
agctctgcct tgctatcgcc aggctttcgc ctgccatgac ctttttacat acaatgcttg 7740
tcctgtatgc aacttctatg gggtttgtct cgtgttctct cacacggtca cactcaattg 7800
tgtgccgctg catagaagct tggccatagt tgcccgcacc gtagtgcgcc aagcaaccta 7860
gtggtttagg cacattctcc ggaccgttaa tggccgtcct cgccattcac cacaagcgca 7920
gcaaggaacg cttattgtgg tatatccccg ggtttgcggt ggacggggca actcctgacg 7980
tcagtttatt ttacacccct taacggcagc tgggtgacaa acaaaaaacg acagaaaacc 8040
acggtttgat accctccaaa cagtggtttt ctgtcgtcca aaaatagccg aaaagtgttg 8100
acgtatacac ttgttttcgg taaaatgaag acataactta aacattgtaa gtgagggctt 8160
acaaaccaag tgttcgatgc tgcaacatcg gacacttttt atttgtcatt ctttatttgt 8220
attcaatttt gcaaatagct cgcaaacaaa atatgtatca tcaaatctat taaccttgtt 8280
gtctgcaaac aacagggttt tttgttgttt atttagaata actagaacca gaattcaatg 8340
ccaaaacttt cacattgact taacttgact ttatcttaca cgattttttt tttgacgtaa 8400
agccccgggc ctggtaccag tcgacctgca ggcatgcaag cttggcactg gccgtcgttt 8460
tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 8520
cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 8580
tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg 8640
gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 8700
gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 8760
catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 8820
cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta 8880
atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg 8940
gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 9000
aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 9060
gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 9120
cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 9180
tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 9240
tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 9300
agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 9360
cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 9420
tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 9480
ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 9540
tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 9600
cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 9660
actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 9720
ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 9780
tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 9840
ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 9900
aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 9960
ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 10020
agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 10080
ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 10140
tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 10200
cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 10260
ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 10320
gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 10380
ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 10440
aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 10500
cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 10560
ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 10620
gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 10680
ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 10740
ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 10800
gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 10860
cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 10920
ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 10980
aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 11040
ttcacacagg aaacagctat gaccatgatt acgaattcga gctcggtacc cgg 11093

Claims (9)

1.用于提高枯草芽孢杆菌自发突变频率的质粒，其特征在于，选自pUS20D9M,pUS20XP1以及pUS20XP2中的任意一个；其中，质粒pUS20D9M序列如SEQ ID NO.1所示，质粒pUS20XP1序列如SEQ ID NO.2所示，质粒pUS20XP2序列如SEQ ID NO.3所示。
2.权利要求1所述的质粒在提高枯草芽孢杆菌自发突变频率中的应用。
3.一种提高枯草芽孢杆菌自发突变频率的方法，其特征在于将权利要求1中所述的任意一种质粒转染枯草芽孢杆菌在含有1％木糖的LB培养基中36℃-38℃培养10-14h,优选培养12h。
4.质粒pUS20D9M的构建方法，其特征在于通过同源重组的方式将含有木糖诱导的dCas9表达盒以及干扰mutSL的sgRNA表达盒构建在质粒pUS20上；其中，所述的含有木糖诱导的dCas9表达盒序列如SEQ ID NO.1中第4175bp-8281bp所示；所述的干扰mutSL的sgRNA表达盒序列如SEQ ID NO.1中第第8310bp-8480bp所示。
5.根据权利要求4所述的构建方法，其特征在于以质粒pJMP1为模板，用引物P1/P2扩增含有木糖诱导的dCas9表达盒xylR-Pxyl-dcas9部分；合成干扰mutSL的sgRNA表达盒序列，然后经引物P3/P4扩增后得到片段Pveg-sgmutSL；以质粒pUS20为模板，用引物P5/P6扩增质粒pUS20全部序列；然后，将上述3片段通过同源重组的片段融合成环状质粒；最终，将上述融合产物转化到大肠杆菌DH5α后，经100ppm氨苄青霉素筛选后得到质粒pUS20D9M；其中各引物序列如下所示：
Figure FDA0003266118330000011
6.质粒pUS20XP1的构建方法，其特征在于，通过同源重组的方式将木糖诱导失去校对功能的DNA聚合酶PolC*表达盒以及干扰polC的sgRNA表达盒构建在质粒pUS20上，生成质粒pUS20XP1；所述的木糖诱导失去校对功能的DNA聚合酶PolC*表达盒序列如SEQ ID NO.2中的第2574bp-6887bp所示，所述的干扰polC的sgRNA表达盒序列如SEQ ID NO.2中的第6888bp-7120bp所示。
7.根据权利要求6所述的构建方法，其特征在于，首先，以枯草芽孢杆菌168基因组为模板，分别用引物P7/P8,P9/P10和P11/P12扩增DNA聚合酶polC*的3个部分，其中碱基突变由P9和P11引入，将上述3部分融合为失去校对功能的DNA聚合酶PolC*表达盒；合成干扰polC的sgRNA表达盒序列，然后经引物P13/P14扩增后得到片段Pveg-sgpolC；以权利要求1中所述的质粒pUS20D9M为模板，用引物P15/P16扩增质粒pUS20全部序列和xylR-Pxyl部分；然后，将上述3片段通过同源重组的片段融合成环状质粒；最终，将上述融合产物转化到大肠杆菌DH5α后，经100ppm氨苄青霉素筛选后得到质粒pUS20XP1；其中各引物序列如下所示：
Figure FDA0003266118330000021
8.质粒pUS20XP2的构建方法，其特征在于，通过同源重组将片段Pveg-sgmutSL融合到pUS20XP1上，生成质粒pUS20XP2；所述的片段Pveg-sgmutSL序列如SEQ ID NO.3中第6923bp～第7092bp所示。
9.根据权利要求8所述的构建方法，其特征在于，以质粒pUS20XP1为模板，用引物P17/P18扩增质粒pUS20XP1全部序列；以质粒pUS20D9M为模板，用引物P19/P20扩增后得到片段Pveg-sgmutSL；然后，将上述2片段通过同源重组的片段融合成环状质粒；最终，将上述融合产物转化到大肠杆菌DH5α后，经100ppm氨苄青霉素筛选后得到质粒pUS20XP2；其中各引物序列如下所示：
Figure FDA0003266118330000022
Figure FDA0003266118330000031
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