CN113046255B - 大规模基因重排的酵母菌及其构建方法 - Google Patents

大规模基因重排的酵母菌及其构建方法 Download PDF

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CN113046255B
CN113046255B CN202110255090.9A CN202110255090A CN113046255B CN 113046255 B CN113046255 B CN 113046255B CN 202110255090 A CN202110255090 A CN 202110255090A CN 113046255 B CN113046255 B CN 113046255B
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元英进
吴毅
周嗣杰
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Abstract

本发明涉及基因工程技术领域，尤其涉及大规模基因重排的酵母菌及其构建方法。本发明利用Vika/vox技术切除酵母完整染色体。使染色体着丝粒产生同源重组，从而实现整条染色体的敲除，实现六条合成型染色体的整合，并产生大规模的重排事件。本发明对比已有成果具有以下有益效果；简单、高效、快速实现酵母染色体的切割；避免姐妹染色单体的交叉互换，获得六条合成型染色体整合的单倍体酵母；简单、高效、快速产生超大规模基因组重排。

Description

大规模基因重排的酵母菌及其构建方法
技术领域
本发明涉及基因工程技术领域，尤其涉及大规模基因重排的酵母菌及其构建方法。
背景技术
生物基因组携带了决定生物基本性状的遗传信息，人工DNA合成技术和DNA大片段操作技术推动了基因组人工合成研究的进步。合成生物学的发展推动了通过人工设计合成来“写”基因组信息标志着“人造生命”的开始。
合成酵母基因组计划(Sc2.0)旨在构建一个完全设计合成的真核生物基因组，以作为系统研究真核生物染色体的平台。合成型基因组在减少基因组不稳定性的同时提高遗传灵活性，并且保持和野生型细胞一致的性状。2017年3月，Sc2.0项目取得里程碑式进展，Science期刊以五篇研究长文的形式报道了五条染色体的全合成，加上2014年报道的三号染色体的全合成，目前已经完成酿酒酵母二号、三号、五号、六号、十号和十二号染色体的合成，宣布合成型酵母基因组三分之一的工作完成。合成酵母单条染色体的技术日趋成熟，对于Sc2.0项目的下一个公认挑战是如何高效组合多条合成型染色体到单一细胞，直至完整的合成型酵母基因组的构建。
目前，酵母菌的基因发生重排的效率较低，且方法较为复杂。Vika/vox系统是在海洋细菌Vibrio coralliilyticus中发现的一种类似Cre/loxp系统的高效同源重组系统。野生型vox位点全长34bp,包含两个13bp的反向重复序列(Inverted repeats)和一个8bp的非对称间隔区(Spacer)序列。13bp的反向重复序列是Vika重组酶的识别和结合区域,8bp的间隔区是DNA链的断裂与重接等重组交换过程的发生区域,间隔区序列的不对称性显示整个vox位点具有方向性。Vika重组酶与限制酶相似，能识别特异的DNA序列，即vox位点，使vox位点间的基因序列被删除或重组。将Vika/vox系统对酵母染色体进行切除，并利用人工合成的六条染色体使酵母菌基因发生重排，对酵母基因组的研究存在重大意义。
发明内容
有鉴于此，本发明要解决的技术问题在于提供简单、高效、快速产生大规模基因重排的酵母菌及其构建方法。
本发明提供的大规模基因重排的酵母菌，其染色体中野生型II、III、V、VI、IX和X染色体被替换为合成型染色体synII、synIII、synV、synVI、synIXR和synX。
本发明所述酵母菌的构建方法包括：
在酿酒酵母野生型染色体II、III、V、VI、IX或X的着丝粒附近同源重组带vox位点的片段，获得待敲除染色体的酵母菌株；
将两个交配型相反的单倍体酵母菌株融合，构建二倍体酵母菌株；
向二倍体酵母菌株中转化Vika质粒，采用Vika/vox技术删除野生型染色体，然后减数分裂获得单倍体菌株；
所述两个交配型相反的单倍体酵母菌株，其中一个含有合成型染色体synV和synX，另一个含有合成型染色体synII、synIII、synVI和synIXR。
本发明中，所述带vox位点的片段的同源重组包括：合成带筛选抗性基因、vox位点和野生型染色体着丝粒两端同源序列的片段，经Gibson组装获得靶向染色体的片段，连入pUC19质粒，经线性化后转化入酿酒酵母菌株，与待敲除染色体发生同源重组。
在本发明中，所述筛选抗性基因为KanMX基因或hphNT1基因，
所述待敲除染色体着丝粒两端同源序列的长度为1000bp。
一些实施例中，
靶向染色体synII的片段的核酸序列如SEQ ID NO:1所示；
靶向染色体synIII的片段的核酸序列如SEQ ID NO:2所示；
靶向染色体synV的片段的核酸序列如SEQ ID NO:3所示；
靶向染色体synVI的片段的核酸序列如SEQ ID NO:4所示；
靶向染色体synIXR的片段的核酸序列如SEQ ID NO:5所示；
靶向染色体synX的片段的核酸序列如SEQ ID NO:6所示；
本发明中，所述野生型染色体的删除包括，将带有筛选抗性基因和vox位点的片段导入待敲除染色体的菌株，缺陷型筛选获得染色体删除成功的菌株。
本发明中，所述野生型染色体的删除的顺序为II和IX、V、X、VI、III。
本发明还提供了一种酵母菌基因重排的方法，其包括诱导所述的酵母菌，或诱导本发明所述方法构建获得的酵母菌，使其基因发生重排。
所述诱导包括：以His营养缺陷型培养基培养。
本发明利用Vika/vox技术切除酵母完整染色体。使染色体着丝粒产生同源重组，从而实现整条染色体的敲除，实现六条合成型染色体的整合，并产生大规模的重排事件。本发明对比已有成果具有以下有益效果；简单、高效、快速实现酵母染色体的切割；避免姐妹染色单体的交叉互换，获得六条合成型染色体整合的单倍体酵母；简单、高效、快速产生超大规模基因组重排。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍：
图1示本发明的酵母菌株获得的示意图：a片段转化到目标染色体的着丝粒位置，实现染色体的逐条删除,最终获得合成型染色体整合的单倍体酵母；b Vika/vox系统表达，使酵母染色体着丝粒丢失，从而实现酵母整条染色体的敲除系统使染色体产生切口从而达到敲除整条染色体的目的；c所获得的菌株的脉冲场验证长度；
图2示通过翻印初步验证染色体敲除效率，野生型VI敲除效率达100％，野生型III敲除效率98.3％；
图3示本发明实施例1的PCR验证凝胶电泳图；
图4示所获得单倍体菌株的二代测序；
图5产生大规模重排的实验流程；
图6检测到超过260,000的重排事件。
具体实施方式
本发明提供了大规模基因重排的酵母菌及其构建方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试材皆为普通市售品，皆可于市场购得。其中，含合成型染色体synV和synX的酵母菌株来自天津大学(yYW169)，含合成型染色体synII、synIII、synVI和synIXR的酵母菌株来自纽约大学(yZY192)。
本发明产生了可以获得大规模重排的酵母菌株，包括如下步骤：
步骤1：构建带筛选抗性基因、vox位点及待敲除染色体着丝粒两端同源序列的片段；
步骤2：将片段转化入待敲除染色体菌株，与待敲除染色体发生同源重组，在抗性筛选培养平板上筛选成功导入片段的菌株；
步骤3：待敲除染色体菌株与交配型相反的菌株融合交配，挑选二倍体菌株；
步骤4：向二倍体菌株中转化Vika质粒，在SC-Leu培养平板上筛选，并在SC-Leu划线传代。
步骤5：接菌于5mLSC-Leu液体培养基过夜培养，转接SG-Leu(20％棉籽糖，20％D-半乳糖))液体培养基诱导24h,在SC-Leu培养平板表面涂布。
步骤6：Vika/vox系统表达，将SC-Leu培养平板上的菌翻印到抗性筛选培养平板，能初步验证染色体的敲除；
步骤7：挑取SC-Leu培养平板上生长的单菌落在SC-Leu培养平板上划线分纯，选取菌株提取基因组并使用GoTaq GreenMaster Mix利用PCR反应进行验证。利用特异性区分染色体的引物进行PCR反应，PCR结果证明成功切割了整条染色体。
步骤8：将带筛选抗性基因、vox位点的片段导入下一条待删除的染色体着丝粒位置。重复步骤5-7，逐步敲除合成型染色体对应的六条野生型染色体。利用特异性区分染色体的引物进行PCR反应，PCR结果证明六条野生型染色体的敲除。
步骤9：对所获得的的菌株进行生孢处理，减数分裂获得六条合成型染色体整合的单倍体酵母。
步骤10：对所获得的单倍体菌株进行诱导重排，测序在菌株中检测到大规模的重排事件。
下面结合实施例，进一步阐述本发明：
实施例1
利用Vika/vox系统获得可以产生大规模重排的酵母菌株，包括以下步骤：
1.片段构建
a)设计上、下游引物PCR扩增KanMX(或hphNT1)抗性基因，两vox位点间基因合成且左端与KanMX(或hphNT1)抗性基因有20bp同源；
b)提BY4742菌株基因组，设计上游引物左右两端分别带20bp pUC19质粒同源和20bpKanMX(hphNT1)抗性基因、下游引物左右两端分别带20bp pUC19质粒同源和20bp vox序列PCR分别扩增六条野生型号染色体着丝粒两端1000bp同源序列；
c)利用Gibson组装将酶切后的线性化pUC19质粒和双链DNA进行组装，共构建六个质粒；
d)将反应体系转化如DH5α大肠杆菌感受态细胞中，涂布于LB+Carb平板上，37℃培养12h；
e)分别挑取6个单菌落接种于5mLLB+Carb液体培养基中，37℃过夜培养后，提取质粒，部分酶切验证，正确的进行Sanger测序；
f)测序正确的质粒命名为bYW0185(带hphNT1和II号染色体同源段)bYW0186(带hphNT1和V号染色体同源段)，bYW0187(带hphNT1和VI号染色体同源段)，bYW0188(带KanMX和III号染色体同源段)，bYW0189(带KanMX和IX号染色体同源段)，bYW0190(带KanMX和X号染色体同源段)。
2.对步骤1构建的六个质粒酶切，将bYW0185和bYW0189酶切后的片段转化入yYW169(包含synV,X)的野生型II号和IX号的着丝粒位置，PCR验证片段的正确转化，记为yYW169-II,IX。
3.将构建后的yYW169-II,IX和yZY192(包含synII,III,VI,IX)菌株进行mating，成功后PCR验证正确整合的二倍体命名为yYW0268；
4.向二倍体菌株中转化Vika质粒(pLQH135pRS415-Vika-Gal，序列如SEQ ID NO:7)，在SC-Leu培养平板上筛选，并在SC-Leu划线传代。
5.接菌于5mL Sc-Leu液体培养基过夜培养，转接SG-Leu液体培养基(20％棉籽糖，20％D-半乳糖)诱导24h,在SC-Leu培养平板表面涂布。
6.Vika/vox系统表达，将SC-Leu培养平板上的菌翻印到SC-Leu+G418，能初步验证野生型染色体的丢失；
7.挑取SC-Leu培养平板上生长的单菌落在SC-Leu培养平板上划线分纯，选取菌株提取基因组并使用GoTaq GreenMaster Mix利用PCR反应进行验证。利用特异性区分合成型(SYN PCRTags)和野生型(WT PCRTags)的引物进行PCR反应，结果显示存在染色体合成型II和IX染色体，对应的野生型染色体缺失，证明成功切割了整条染色体；
8.往菌株野生型V染色体着丝粒位置转化bYW0186酶切的片段，半乳糖诱导野生型V染色体的删除，并进行PCR验证；
9.之后依次往菌株野生型染色体着丝粒位置转化酶切后的片段，实现野生型X,VI,III染色体的逐条删除，进行PCR验证；
10.对所获得的的菌株进行生孢处理，减数分裂获得六条合成型染色体整合的单倍体酵母。通过脉冲场和二代测序验证包含六条合成型染色体的单倍体；
11.对所获得的正确的菌株，转化Cre质粒(bYW085-pCLB2-Cre，序列如SEQ ID NO:8)，在SC-His培养平板上筛选，并在SC-His划线传代；
12.接菌于5mL SC-His液体培养基过夜培养，转接SC-His液体培养基诱导8h,再离心水洗后在SC-His液体培养基过夜培养，之后进行离心二代测序；
13.通过对测序序列分析，总共检测到超过260,000种重排事件。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 天津大学
<120> 大规模基因重排的酵母菌及其构建方法
<130> MP21003676
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4037
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atatcagtgt gtactctaac atatcctttc tcatgaccgt attcatggat ttcgccaatc 60
aaacccatca catcatccca aggcccttca attgttgttc ctgcactgtg taaagtgctc 120
tttaatgggc tttctctgat ttttttttca atgagtgcaa caaaatcaga aatactagca 180
gagtcggtgc caatagggac catacacacg tccgctaaac aaaagatctt gggcattttc 240
taaacttgta gtttatgtgc tttatagtta atgatcacga cggcaatgac aaaaactgaa 300
tatcttgatc tctccttttg acgatgtcag tgaatcccgg ttaatgatat tctgtttttc 360
acggggtgga aaagagggga gcttgaatca tactaaaaaa ggaatgtcaa agagctatat 420
aattcgaata aaaagtaaaa gaaacgaaag gttttattaa agggtttgga gctgttttcg 480
gactgaaagc cagtaacaag cttgtgtagg agtttgtctg aaaaccatct tcatttcaaa 540
taagagagcg agtaattttt aaataatttc ttttctgaac tacttaaatc gctatgtacc 600
ttgcgaaaat tattctttaa tagtttaact aaaagtaaaa agttagaaat cataaataaa 660
aaatcaaaga gacaaatctc gttgcaaaag cttgaaaaat agtgctttac caacaggttg 720
gcaaaccaga atgctaacaa aacttaaaat cccatttccc aagaggatca atcaatcata 780
tttaccttga tagtatagga tacaagtgtc tgtgcaggcc tccgataaca tgtaaagtaa 840
taaaagtagg agtacttgct ttcagcagaa aatgaaaaaa agaacgttca aacagactga 900
aaatatagct attcaactgg cacctactaa tatcaataat aataaattaa tcttgagcaa 960
attgatccta cataacagcg acatggaggc ccagaatacc ctccttgaca gtcttgacgt 1020
gcgcagctca ggggcatgat gtgactgtcg cccgtacatt tagcccatac atccccatgt 1080
ataatcattt gcatccatac attttgatgg ccgcacggcg cgaagcaaaa attacggctc 1140
ctcgctgcgg acctgcgagc agggaaacgc tcccctcaca gacgcgttga attgtcccca 1200
cgccgcgccc ctgtagagaa atataaaagg ttaggatttg ccactgaggt tcttctttca 1260
tatacttcct tttaaaatct tgctaggata cagttctcac atcacatccg aacataaaca 1320
accatgggta aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag 1380
ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc 1440
ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac 1500
aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt 1560
gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc 1620
acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc 1680
atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg 1740
caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat 1800
gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc 1860
gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat 1920
ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc 1980
gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg 2040
ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga 2100
tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg 2160
gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga 2220
tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc 2280
gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg 2340
gcaaaggaat aatctcgagt catgtaatta gttatgtcac gcttacattc acgccctccc 2400
cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt 2460
tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt 2520
tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt 2580
ttgggacgaa tgctggtcgc tatactggtt gcaaatgctc cgtcgacggg aataggtctg 2640
agaacgccca ttctcagacg tattgtacag tacctataca tttcataaac atggcatggc 2700
gatcagcgcc aaacaatatg gaaaatccac agaaagctat tcattgaaaa aatagtacaa 2760
ataagtcaca tgatgatatt tgattttatt atatttttaa aaaaagtaaa aaataaaaag 2820
tagtttattt ttaaaaaata aaatttaaaa tattagtgta tttgatttcc gaaagttaaa 2880
aaagaaatag tcttgagaag gttttgggac gaagaaatat atatttcatt gaatggatat 2940
atgaaacgtt tactggtgga agttttgctc atatattatt attcaataga agtaataaag 3000
agttgcaaat gctccgtcga cgggaatagg tctgagaacg cccattctca gacgtattaa 3060
aagttggtaa agcaacttat atttcaacaa gaagtatcaa tatatttctt gcaaaaaatt 3120
gttcaccgag aattgacaaa aaaaccttac gtcggtgacg acgatataca gatcctatat 3180
tcttcatgta tgtaatagat tcccatacct tcttcgcatt taagacacta atagaaatga 3240
aagctttcta ttagtcattc ttcctatgca ctagaccacg caaaggtgaa ccggcttggt 3300
acgagaattc tatcacacgg taatgatagt gttcccgatg tcaagcagtc ttatacagta 3360
ctacccaaga gcttaagaat aagcaagaag atctggcaag ctacaaggta ctcataacga 3420
tcttcaaagc gtaagttatg atcttatgaa aatcagactt ttttgtctca acaaactcct 3480
agtttgaaga tgttactgaa atgcccttca aacactttta cttgaaaatc atcgattact 3540
attctaacct tcacaaatga gcctttacac aacttgatta ataataatat ttgaccttac 3600
tctaaatcgt ctcaaatatt actgaggcac aatctcatgg ttattgatag aaagaagaat 3660
ataacatata tcttatttac atatgttcgt caatattata ggtaatacaa ttttttctca 3720
gagggtttca aactattgaa aaatggcaat ggtggactac aagcagcgag agcccttctt 3780
gcatgattat tcatatattt caatccgagc aaataacttg tgttcaccca accaaagcct 3840
tctgtagcga cgcccttaaa atcagcacct tgattaccat attccgcatc aacgcgatgc 3900
ggatcagtcc ctcttgtcac atcatatttt tctaccacca ttccgttata atcaacaaat 3960
gattttgtaa tcatgtacag ccacctataa gccaatcttg tagctacttg ttgataacca 4020
tatgcagata aaccttt 4037
<210> 2
<211> 3790
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgcttattga aagcgtcttc aattatatta aaactaaaat tgaagtttcc aaaaaagaaa 60
gatagaactg atattagcaa attgtgtgac aagaaagaac ggatgacaca gtggttagaa 120
atttcaattt tgatgaactg agaataataa tattatgctc ccctggattt tatgcgaaga 180
cactgctgaa aaaatttctg atatatgcag agaaggagca aataagccag ttttaaggaa 240
ccgagataag ctgttttcgc ccattgttct agttctacaa aatatctaca gggaatcgat 300
gaggttgtaa gaaatcctga atactctatg atagtgcata acactaaaaa gttgaaagaa 360
tcccgtatca tggacgattt ccttgaacat ttgagcaaag acgataacaa agcatggtat 420
ggcgcggaag aaaccgagag agctgcaaaa ttagatgcaa tagaaacact acttattaca 480
gatagtgtac taaaaaggaa cgacgtgaaa aaacgtgaaa aatacctaga cctaatagag 540
aatagtggaa acaacaatgg caaaatattc gtactcagta cttcaaaaat cacagtgagc 600
aacttgacaa accaacagat ataggctgtg tcttaaaata taccatccaa taccttgatg 660
aacttttcaa agatgactaa aataagtgaa atttcaacat taacttcgaa tttttttctt 720
tttatctaac acttgtcaaa cagaatataa ggattacttg tcttctttgc tacatattgc 780
taccacttct attacacaat agtttcaata gcttgcagcg tagctaaact ctaaaattta 840
tctaaatcac tcatataaac cgaacccttc cccttccgct tatagtacag tacctataca 900
tttcataaac atggcatggc gatcagcgcc aaacaatatg gaaaatccac agaaagctat 960
tcattgaaaa aatagtacaa ataagacatg gaggcccaga ataccctcct tgacagtctt 1020
gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc 1080
catgtataat catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac 1140
ggctcctcgc tgcagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt 1200
ccccacgccg cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc 1260
tttcatatac ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat 1320
aaacaaccat gggtaaggaa aagactcacg tttcgaggcc gcgattaaat tccaacatgg 1380
atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa 1440
tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta 1500
gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc 1560
ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg 1620
cgatccccgg caaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata 1680
ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc 1740
cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt 1800
tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga 1860
aagaaatgca taagcttttg ccattctcac cggattcagt cgtcactcat ggtgatttct 1920
cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag 1980
tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt 2040
ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata 2100
aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agtactgaca ataaaaagat 2160
tcttgttttc aagaacttgt catttgtata gtttttttat attgtagttg ttctatttta 2220
atcaaatgtt agcgtgattt atattttttt tcgcctcgac atcatctgcc cagatgcgaa 2280
gttaagtgcg cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg tcgctatact 2340
ggttgcaaat gctccgtcga cgggaatagg tctgagaacg cccattctca gacgtattgt 2400
acagtaccta tacatttcat aaacatggca tggcgatcag cgccaaacaa tatggaaaat 2460
ccacagaaag ctattcattg aaaaaatagt acaaataagt cacatgatga tatttgattt 2520
tattatattt ttaaaaaaag taaaaaataa aaagtagttt atttttaaaa aataaaattt 2580
aaaatattag tgtatttgat ttccgaaagt taaaaaagaa atagtaagaa atatatattt 2640
cattgaatgg atatatgaaa cgtttactgg tggaagtttt gctcatatat tattattcaa 2700
tagaagtaat aaagagttgc aaatgctccg tcgacgggaa taggtctgag aacgcccatt 2760
ctcagacgta ttaaaagttg gtaaagcaac ttaagaaata gtaagaaata tatatttcat 2820
tgaatggata tatgaaacgt ttactggtgg aagttttgct catatattat tattcaatag 2880
aagtaataaa gaaaaagttg gtaaagcaac ttaacagtaa aaaggtaatg attgaaaaag 2940
tttttgaaca tctaagctat atgttgatgg gtttacaatt ttaccattag tactcatgcc 3000
tatacttttc tgttcgtcct taatgtccgc gatttagagc aatcattgaa agtactagat 3060
acattttagc cagagaggac tcgttgacgt agaattaaaa ttcaaatgaa tttccgcccc 3120
attcatatac cccaaataac aaacatatta aaacttcata attattcaaa atgtggagta 3180
gtatagaaga gcagtacctt caaaattgat ttcttcagtt tcccacccgg gatccacttg 3240
tcatgcggtg agaatcgtat attgcgtata atccgtgttt catcacccac attatagtac 3300
aaacctactg gtgtaaccat tatcatattc atgacttgta gttaaaaatc tttcacgata 3360
aattgaggaa aaaatcacgt taaatagttt catatcattc tttagttaaa gtctgtgcat 3420
aaaaagagga tcgggtgttg tactcgttct atcaaaatat tgttattgtt gcattatcac 3480
acgaattaca atcaattttt ttatctacag gccagagtac ctgaaataat ggaattagat 3540
tcaaaaagga tagcttaatc ggtctttata ctaattattt tacgcccaga acgaaacata 3600
acgtgcatta aatctcactg tcacaacatt gtatataatg gtagcatcgt actaactatt 3660
ggcattcatt caatattata caattatctt attaatactt tctcaagtag tcttgaagac 3720
cagcatgtag gaaggtgatg atatgctccg taaataattg gaaatattga gatataataa 3780
cgatgacaat 3790
<210> 3
<211> 4102
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ttgccagcta taaagaagtg acagtgaact tttacttgtt tcttttgttt aaccactagt 60
atgtaattgt ttttgcagaa tttgaaactt caccaaaatc ttcttaacgt tcaatgacaa 120
aattacttac ccgaactacc gaactacgaa acatgaataa ataaactgga ggcataaact 180
catttccaaa ttcccggaat acatcgtgta gtcaagcagc atacatcact atcaaaaatc 240
atgcgcttgt gattatcgta tagtgtattt atataatata tatagatagc gataaatggc 300
aagatcctct atgataaaag tcatcataat gaaaacagat ggcgcttgtc tactgtttgc 360
aaggcttgaa atcacaggtt tttattatat cgtggatttt tgccattgcc taaatcaaaa 420
ataatgatcg attttttgtg aattagggaa cggaaggaaa aaatcagcaa acagtcagcc 480
ctagcaccat aggccgacag caacaaaaac tgttttttta ttctcaaata ttcatgatat 540
tgattacacc gtacttcttt tcaatgcgta aacaactaga gttgacaaat attataaact 600
aaaaactatc atattaataa tatacctcct agcacttcgt aatgtttctg tcattgaatg 660
gcgagaacat aattttaaag aatcaacacc ttaaattgtt aaccttaaca aatgaagtaa 720
attcaaatat ttactcttga attctttttg ctttaactaa cacattgaaa tatcaaatta 780
gtccactttt cttccttgtt gaatgggttt cttcattaac aggggaacgc ttgcctacca 840
tcaagcccat tcaatgcaga tgtgattaat tggtaaacaa agggccaagc aaaaatacat 900
ctctcctaca tgctacataa gtccgagata tgttttattt aagaactatg aatctgtaaa 960
tgactgattc aatattaaac taaaatttct atgaaacatc aaattacagc gacatggagg 1020
cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc 1080
gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata cattttgatg 1140
gccgcacggc gcgaagcaaa aattacggct cctcgctgcg gacctgcgag cagggaaacg 1200
ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag 1260
gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat 1320
acagttctca catcacatcc gaacataaac aaccatgggt aaaaagcctg aactcaccgc 1380
gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct 1440
ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct 1500
gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc 1560
atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac 1620
ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact 1680
gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag 1740
ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg 1800
tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga 1860
caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg 1920
ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa 1980
tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga 2040
ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta 2100
cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg 2160
cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg 2220
ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca 2280
aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag 2340
tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa taatctcgag tcatgtaatt 2400
agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg 2460
agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag 2520
aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa 2580
cattatactg aaaaccttgc ttgagaaggt tttgggacga atgctggtcg ctatactggt 2640
tgcaaatgct ccgtcgacgg gaataggtct gagaacgccc attctcagac gtattgtaca 2700
gtacctatac atttcataaa catggcatgg cgatcagcgc caaacaatat ggaaaatcca 2760
cagaaagcta ttcattgaaa aaatagtaca aataagtcac atgatgatat ttgattttat 2820
tatattttta aaaaaagtaa aaaataaaaa gtagtttatt tttaaaaaat aaaatttaaa 2880
atattagtgt atttgatttc cgaaagttaa aaaagaaata gtcttgagaa ggttttggga 2940
cgaagaaata tatatttcat tgaatggata tatgaaacgt ttactggtgg aagttttgct 3000
catatattat tattcaatag aagtaataaa gagttgcaaa tgctccgtcg acgggaatag 3060
gtctgagaac gcccattctc agacgtatta aaagttggta aagcaactta aggtttcaag 3120
aaaaggaaaa gaaaaactaa acgtagtata aagtcttgtt tagtgcaagc cactgttggc 3180
gtttcaacta aactattcct taaaaataaa gtacaatgtt actgctaaac aatgttgaag 3240
gtgagcttaa gacagtaaat caaggaaatg cggtataatt cctgctgaca aagccgcaca 3300
taaatagcag ttgttagcta taaggagatt tttagctacg aacatggttg taattctctt 3360
tatcctgatt accttcaaga ttaagaaaaa aacaaccatg gtaactactt tggctcaaag 3420
tcaaacaaaa cttttaccat ttcacggtac acgtttccga taaaatatca ctagctattc 3480
aatgtctgta ttcatcgata agccagagta cgacgtacaa aaaataacgt aaatgcatat 3540
cctgcatcaa gaaagaaccc ttatggacga acaacgaaag agatctaaat ttagaagaca 3600
gcaagactaa ccatttgata gcgtagtaag gcttatctag aaatgttgga cagataggaa 3660
gagaaagtat aataatggtg tatggtagat gactctataa tcgtggtcgg aacgtgcaat 3720
atatgagaac gatgaatgca tatacatatg tcgcgactcc tttttttcca ttatagcggc 3780
atcgctttat ttcgcgtttt aattcatttt cttttttctt catttctttc tgcatatgca 3840
aacgcggcaa gaagtgccca cggatccgaa gaaaacctaa tacattgaag tttctctccc 3900
tgagatagga actctctctt ctgcgtcaga aaaaaaaaaa agtactcttt ctgcatatat 3960
tgctcactac tgactcactg atccttttac gaatagttgg gcaacaccta ttaagttgta 4020
aaacaatcgc gaaaaaaaaa agaaaacaac caaggcatcc cacttttcga accctggcgc 4080
tcggaagact taatcattgc ga 4102
<210> 4
<211> 4115
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ttggaggcca tgaataagtg taaacttgtt ccctcgatgg ttttacaaga ctataaattt 60
agcagatgtg gcagaacaga caagggagtt agcgccatga accaagttat atcgttagaa 120
gtccgttcca atttaacaga tgaggaacag cgggatccga ccaacgacag cagggaaata 180
ccctatgttc acgttttaaa tcaattacta cccgacgata ttcgtatatc agctgtctgc 240
ctcagaccgc cacctaactt tgacgcaaga ttcagctgtg ttcaccggca ctataagtac 300
atctttaatg gaaaaaacct taatattgaa aaaatgtcta aagccgcatc atattttgtt 360
ggagagagag acttcaggaa tttttgtaag cttgatggct caaaacaaat taccaatttt 420
aaacgaacaa taataagctc aaaaattctt cccctttctg aaactttcta ttgttttgat 480
ctcgttggtt cagcattcct atggcaccaa gttcgttgca tgatggccat tcttttctta 540
gttggtcaat cacttgaagt gccggaaatt gtcttgcgtc tgactgatat tgagaaaacc 600
cctcaaaggc ctgtttacga aatggcaaat gatataccat tattgttata tgattgtaaa 660
tttcctgaaa tggattggca agaacctact gtagatgact ataaggcaat aaaattcacg 720
acagctactg aagcattgac gttacactac gaacttaagg ccgcagtatg caatattttt 780
aaagatgttt tgcccacagc aaatacaaac aacttctcaa agacaattat taatttgggc 840
gatggaagag gtaaagtagt cggtacctat gtgaaactag aggatagaag cgttatggaa 900
cctgtcgaag ttgttaatgc taaatactcc aagaaaaaga acaacaaaaa taagtaaaat 960
aatatataaa cctgtataat ataaccttga agactatatt tcttttccag cgacatggag 1020
gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt 1080
cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat acattttgat 1140
ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc ggacctgcga gcagggaaac 1200
gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa 1260
ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga 1320
tacagttctc acatcacatc cgaacataaa caaccatggg taaaaagcct gaactcaccg 1380
cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc 1440
tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc 1500
tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg 1560
catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga 1620
cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac 1680
tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta 1740
gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc 1800
gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg 1860
acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact 1920
gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca 1980
atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg 2040
aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct 2100
acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc 2160
gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt 2220
gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac 2280
aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata 2340
gtggaaaccg acgccccagc actcgtccga gggcaaagga ataatctcga gtcatgtaat 2400
tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc gaaaaggaag 2460
gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa 2520
gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta 2580
acattatact gaaaaccttg cttgagaagg ttttgggacg aatgctggtc gctatactgg 2640
ttgcaaatgc tccgtcgacg ggaataggtc tgagaacgcc cattctcaga cgtattgtac 2700
agtacctata catttcataa acatggcatg gcgatcagcg ccaaacaata tggaaaatcc 2760
acagaaagct attcattgaa aaaatagtac aaataagtca catgatgata tttgatttta 2820
ttatattttt aaaaaaagta aaaaataaaa agtagtttat ttttaaaaaa taaaatttaa 2880
aatattagtg tatttgattt ccgaaagtta aaaaagaaat agtcttgaga aggttttggg 2940
acgaagaaat atatatttca ttgaatggat atatgaaacg tttactggtg gaagttttgc 3000
tcatatatta ttattcaata gaagtaataa agagttgcaa atgctccgtc gacgggaata 3060
ggtctgagaa cgcccattct cagacgtatt aaaagttggt aaagcaactt ataggttgaa 3120
agttagaaat tagtattata atagcaaaaa aaatttaaag ttagaaatta gaatttaagg 3180
ctctacacac gtttacgatg atattggacg accgacacga aaagacagtt ttaggctttc 3240
ttttgtcttg ctcttgcgtt gaaagtacgt aatttccatt cgattactta ctcaacactt 3300
gatatagaga ccaactataa aataaactgt ttgggaagga aagcagaact cttgtaatta 3360
cctcatctca ttatcaagaa gcaaaatttt aactatgtat tttgacctta tttttgtact 3420
ccactttttt tttttagttt tttttagtta ttttttaatt ttgaagtaac ggcgatgagg 3480
tgaagttttt tttaaactga aaaagaagat cgaatgcaaa ccataaatta gagatctaat 3540
tgaggtaaaa ttataaggtt agatatctgt aaatctatac aattaaggaa agatggcacc 3600
aaagaaacct tctaagagac aaaatctgag aagagaagtc gcaccagagg tgtttcaaga 3660
ttcacaagct aggaatcaac tagcgaatgt tcctcatctt accgaaaaat ctgcccagcg 3720
taagccttct aaaaccaagg ttaaaaaaga acagtctttg gctagacttt atggtgcgaa 3780
gaaggacaag aaggggaaat attctgagaa agacttgaat attccaacac tcaatagagc 3840
tatcgttccg ggtgttaaaa taaggagggg aaagaaaggt aagaaattca ttgctgataa 3900
cgacactctg actttaaacc gtttaataac aactattggt gacaagtacg acgatatagc 3960
tgagagtaag cttgaaaagg ctagaagatt agaagagata cgagaattga aaagaaagga 4020
aattgaaaga aaggaagcgc ttaaacaaga taaactagaa gaaaaaaaag acgagattaa 4080
aaagaagtct tctgtcgcaa ggactatacg tagaa 4115
<210> 5
<211> 3793
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tataagttta ccgaggttgc aagaagtcaa cttttaatat ttgtgaacaa ttgtatcttt 60
agatctactg ttgatcttgc taatagtgaa cgacgagttt tttccttgat gaactgtcca 120
gcttatccgg atgttcaact tatggtaaaa aaccgaaatg gagccattcg aatataccct 180
tgtcacctgg ctgttttgag ccgcgcagaa tatttcaagg tgatgttcac aaacaacttc 240
aaggagaaag ttacatatat caaagccaag cacgttacag gaaagtataa tagtatcatt 300
ccacagctga cattacccaa ttgtgagttt gaggttgccg aaattattct ccgctactta 360
tatgcagata atacagacat tccttggatg tatgccgtcg atgttttgtt acttgccgat 420
atacttttag aagaccgctt aaaaactatc gcttctacaa ttataacaca atcaaaagag 480
tttattcagc aatacaatgt ttttgacgta ctctatttat catgggaaat aggagtggaa 540
cgcttagaac aatttgcagc taaatttata gctatacatt tgcaagagtt atataaagat 600
ccagagatta aaagggcaat tatgttgagt tcacaacgaa tttctctccg ccaggaaact 660
gatactattg aactagtgga tgacattagg tactatttgc tccgaaaata ttcttttgaa 720
ccggatgatg tggagctgtt cgaaaaccaa gacgatttag aatacctaaa acaagtaggg 780
tatttggagt acagaaaaga tatgggtatg ttagataata tcctagctga tctggaactt 840
gatgtataat tccgcattca ataaccttat gtatttttac gcgtcactgc gaacggatga 900
atattgccaa tgactgctgc gcactcttaa aaaatataat gacaattata ataagaagta 960
tgatactttt tctgtaaaaa atatatatac atatgagaca tggaggccca gaataccctc 1020
cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc gtacatttag 1080
cccatacatc cccatgtata atcatttgca tccatacatt ttgatggccg cacggcgcga 1140
agcaaaaatt acggctcctc gctgcagacc tgcgagcagg gaaacgctcc cctcacagac 1200
gcgttgaatt gtccccacgc cgcgcccctg tagagaaata taaaaggtta ggatttgcca 1260
ctgaggttct tctttcatat acttcctttt aaaatcttgc taggatacag ttctcacatc 1320
acatccgaac ataaacaacc atgggtaagg aaaagactca cgtttcgagg ccgcgattaa 1380
attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat 1440
caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac 1500
atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga 1560
cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt 1620
tactcaccac tgcgatcccc ggcaaaacag cattccaggt attagaagaa tatcctgatt 1680
caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg 1740
tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa 1800
tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg 1860
aacaagtctg gaaagaaatg cataagcttt tgccattctc accggattca gtcgtcactc 1920
atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg 1980
atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc 2040
tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc 2100
ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagtactga 2160
caataaaaag attcttgttt tcaagaactt gtcatttgta tagttttttt atattgtagt 2220
tgttctattt taatcaaatg ttagcgtgat ttatattttt tttcgcctcg acatcatctg 2280
cccagatgcg aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct 2340
ggtcgctata ctggttgcaa atgctccgtc gacgggaata ggtctgagaa cgcccattct 2400
cagacgtatt gtacagtacc tatacatttc ataaacatgg catggcgatc agcgccaaac 2460
aatatggaaa atccacagaa agctattcat tgaaaaaata gtacaaataa gtcacatgat 2520
gatatttgat tttattatat ttttaaaaaa agtaaaaaat aaaaagtagt ttatttttaa 2580
aaaataaaat ttaaaatatt agtgtatttg atttccgaaa gttaaaaaag aaatagtaag 2640
aaatatatat ttcattgaat ggatatatga aacgtttact ggtggaagtt ttgctcatat 2700
attattattc aatagaagta ataaagagtt gcaaatgctc cgtcgacggg aataggtctg 2760
agaacgccca ttctcagacg tattaaaagt tggtaaagca actttataat tattacttca 2820
gaatagttat tttcacgaat acgagataca gggtaatgaa aacaggtgtt atccaaaaaa 2880
aaaaattgat atattttaga tactttacat acagaggcaa gcttcagctg tagccaggta 2940
cataatattc cctataaagt gatcaatatt ttttaacgaa aaattaaaaa aaaaaatcct 3000
tgaataaagg aatgaaaata ttgtaacaag cgatgacatt tctttttttg attgtagttg 3060
tggcatttta tatttgcttt tgcatttacc ctattcttga aggtttggat agacaagcga 3120
gataacacca gttgtccttc aacttattca aagcagcagc tcacttaatt attattaagt 3180
atacacatga aagccttgtg tttatttcga atctttttct tgggtagaac ttttaacgtt 3240
gtcagacggc atgacaacag gtttttggga agtagatcta atattttttg tagagtcttt 3300
ggcactgttg aagtgttttt tttggaaggt tctattcttg ttattgtaag gactcctata 3360
ataattgttc ttattatatc cgaaattttg atttggagac tgatttggag ggtacggtgg 3420
gtaataaggg aaggtatcgg gattggggta ggccattaag ggataattcc attgccattg 3480
ttggaaatac tggttttgcg agttgtaatg cctattaaat cctggaatgt tagttctttt 3540
tctggagaca gcaatttttt tgcctttaag ttctcctcca tttaattgca gggctttttc 3600
tctgtatgcc ggactttcaa attcaatata gccgtagcct tttggtgttc ccgtattcct 3660
atcataaaga agtgttattc tttttatctg gccacagtct ttgaaatgat cttctatttg 3720
ctctggggtg acatccggag tgatgttacc gacaaagata gaacgcgagt cagcttcaag 3780
ttgatgagcg tgt 3793
<210> 6
<211> 3862
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aagtaaagtc tcaattggcc ttattactaa ctaataggta tcttataatc acctaataaa 60
atagtatggc cgtgacattt aaggatggtg tgatactagg tgctgattca cgtaccacca 120
ctggtgcgta catagctaac cgtgtgacag ataaattaac gagagtacat gacaaaattt 180
ggtgttgtag gtccggttct gcagcagaca cgcaggcgat tgccgacatt gttcagtacc 240
atttggaatt atatacttct caatatggta ccccctccac agagactgct gcctcggtgt 300
tcaaagaatt atgttacgaa aataaagata accttactgc tggtataatt gtggctggtt 360
atgatgacaa aaacaaaggg gaagtatata ctattccatt gggtggctcc gtccataagc 420
tgccttatgc gatagcagga tctggctcta ctttcatata tgggtattgt gataaaaact 480
ttagagaaaa tatgtcaaag gaagaaaccg tagatttcat aaagcattcg ctatcgcaag 540
ccattaaatg ggacggatct tccggtggtg ttataagaat ggttgttttg acagctgctg 600
gtgtggaacg tttgatattc taccctgatg aatatgaaca actataatag accccaaaag 660
aacaaattct gagtgattac ttctctttat atttgttaac aaatgcctct ggatctacac 720
tgatataaca tataatagtc tataacctct ttctcttttt ctcttcgctt tacgcttttt 780
tctctaactg ttagaacgac aaagtatctc agaagggaat ttcgtaagca agatatttta 840
cgactaatca tttttttgtt gtataaaaac gtatgtgaga ttgattcact aattcttcgg 900
agtcctggaa ggcttacctt atctatgcat tcgtacagtt gtgtgcaatg ttaaaaatgg 960
tgactgtatc tacgtatcta taaaaaaagg ttaactaccg gaaaatcatt tcttctcgta 1020
aagtgtagac atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg 1080
ggcatgatgt gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc 1140
atccatacat tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac 1200
ctgcgagcag ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct 1260
gtagagaaat ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt 1320
taaaatcttg ctaggataca gttctcacat cacatccgaa cataaacaac catgggtaag 1380
gaaaagactc acgtttcgag gccgcgatta aattccaaca tggatgctga tttatatggg 1440
tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg 1500
aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt 1560
acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag 1620
cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggcaaaaca 1680
gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca 1740
gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc 1800
gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat 1860
tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt 1920
ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt 1980
tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga 2040
taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa 2100
cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg 2160
atgctcgatg agtttttcta atcagtactg acaataaaaa gattcttgtt ttcaagaact 2220
tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga 2280
tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag 2340
taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actggttgca aatgctccgt 2400
cgacgggaat aggtctgaga acgcccattc tcagacgtat tgtacagtac ctatacattt 2460
cataaacatg gcatggcgat cagcgccaaa caatatggaa aatccacaga aagctattca 2520
ttgaaaaaat agtacaaata agtcacatga tgatatttga ttttattata tttttaaaaa 2580
aagtaaaaaa taaaaagtag tttattttta aaaaataaaa tttaaaatat tagtgtattt 2640
gatttccgaa agttaaaaaa gaaatagtaa gaaatatata tttcattgaa tggatatatg 2700
aaacgtttac tggtggaagt tttgctcata tattattatt caatagaagt aataaagagt 2760
tgcaaatgct ccgtcgacgg gaataggtct gagaacgccc attctcagac gtattaaaag 2820
ttggtaaagc aactttaagt tactctaata taatcctatt taataattag tagagttgtt 2880
gagaacgagt ataatattat ttttttttta tttttttgta ggttctttgg ccatcctaaa 2940
tagaataagc atccacaaca actaaactgg tattttgtta tacacaacgc gtcttggtta 3000
ggcatctgtt ttgttttagc tattgttcat cgcgtttttt tttttcttat ctcttttacc 3060
gaggggcgaa aatagcgata gatcgagaat ataaataaat aaaatattgt tgtgttgtgt 3120
agttctaaag aaaaatttta cagtgaaaag gtaacaccgg ggagtcatta cgtagactta 3180
cgtattctgt ataactgatt ccgagacgca aatgcctgag caagaaccat tgagtcccaa 3240
tggccgtaag cgctctgagg ttcactatat ctcaatccca ttgaacagag gttccgcatt 3300
ctcaccagat gattctgtat cacagtttca gtctgacggg ttcatgacac gtaggcaatc 3360
catattggac catcctgtgg gctcatttaa gggagttaat tctttaagtc gatttgcgac 3420
ttcgttgagg agagccaatt cgtttcgtaa tatcgagcta aatgcggata atgaaagatc 3480
ctttttcaaa gagagtaacg atgaaaccta cgatccggat actttagcac cagctttgga 3540
cggtagaaga ttatcagtaa ctttaaataa tgctggtcgc ccgcgcatta ctaatttggc 3600
taacaacgat agagttagta cagccagcat ggctattcac gatgatgatt atgggtccat 3660
ccaaaattca acaattgggg attctgggtc aatattacgc cctactgcct ccttaacaga 3720
aatgatgagt ggtggagcgg ggagaaggtt tacaaataat gacatggatt caattgtagt 3780
gaagagagtg gagggtgtag atggtaaggt agtaactctt cttgctggtc agtcgaccgc 3840
tccacagacc atattcaact ca 3862
<210> 7
<211> 7880
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc 240
accattatgg gaaatggttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca 300
ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat 360
taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc 420
ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc 480
aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt 540
agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg 600
tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct 660
ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg 720
ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct 780
tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac 840
aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat 900
ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc 960
aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg 1020
ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat catggcggca 1080
gaatcaatca attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc 1140
acagtttttc tccataatct tgaagaggcc aaaacattag ctttatccaa ggaccaaata 1200
ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact 1260
tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc 1320
ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca 1380
aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt 1440
aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca 1500
ctaccggtac cccatttagg accacccaca gcacctaaca aaacggcatc aaccttcttg 1560
gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca 1620
attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga 1680
accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc 1740
ttcttagggg cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata 1800
tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat 1860
tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat 1920
ttagtcatga acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct 1980
ttttctccca atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca 2040
aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat 2100
gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga 2160
gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg 2220
gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt 2280
ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg 2340
atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta 2400
gaagttctcc tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa 2460
ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt 2520
taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa 2580
gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag 2640
aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt 2700
gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 2760
cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 2820
gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg 2880
cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg 2940
ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc 3000
cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc 3060
cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc 3120
gaattgggta ccgggccccc cctcgaggtc gacggtatcg ataagcttga tatcgaattc 3180
cggattagaa gccgccgagc gggtgacagc cctccgaagg aagactctcc tccgtgcgtc 3240
ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca ctgctccgaa 3300
caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt ggcagtaacc 3360
tggccccaca aaccttcaaa tgaacgaatc aaattaacaa ccataggatg ataatgcgat 3420
tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt ttgatctatt 3480
aacagatata taaatgcaaa aactgcataa ccactttaac taatactttc aacattttcg 3540
gtttgtatta cttcttattc aaatgtaata aaagtatcaa caaaaaattg ttaatatacc 3600
tctatacttt aacgtcaagg agaaaaaacc ccggatcgga ctactagcag ctgtaatacg 3660
actcactata gggaatatta agcttatgac tgatttgact cctttcccac cattagagca 3720
cttggagcca gacgagtttg cagacttggt caggaaggct attaagaggg acccacaagc 3780
aggtgctcac ccagcaattc aatctgctat ttctcacttt caggatgagt tcgtcaggag 3840
gcaaggtgaa tggcaaccag ctactttgca aagattgaga aatgcttgga acgttttcgt 3900
cagatggtgc acacaccagg gtattccagc tttgccagca aggcaccaag atgtcgaaag 3960
gtacttgatt gagagaagaa atgaattgca tagaaatact ttgaaagttc atttatgggc 4020
tattggtaaa acacatgtca tttctggttt gccaaaccct tgcgctcata ggtacgttaa 4080
ggctcaaatg gcacaaatta ctcaccagaa ggtcagggaa agggaaagga ttgagcaggc 4140
tcctgctttt agggagtctg acttggacag gttgacagag ttgtggtctg ctactaggtc 4200
tgtcactcag caaagagatt taatgattgt ttctttggct tatgagacat tattgagaaa 4260
gaacaatttg gaacaaatga aagttggtga tattgaattt tgtcaagatg gttcagcttt 4320
gattactatt ccattctcta aaacaaacca ctctggtaga gatgacgtca ggtggatttc 4380
tccacaggtc gctaaccagg ttcatgctta cttgcaatta ccaaatattg atgctgatcc 4440
tcaatgtttt ttattgcaaa gagttaaaag atctggtaag gcgctgaatc cagaatcaca 4500
caatactttg aatggtcatc atccagtttc tgaaaaattg attagcaggg tcttcgaaag 4560
ggcatggaga gctttgaacc acgagactgg tccaaggtat actggtcact ctgctagagt 4620
cggtgctgct caagacttgt tgcaggaggg ttactcaact ttgcaggtca tgcaggctgg 4680
tggatggtct tcagagaaga tggtcttgag gtacggtagg cacttgcatg cacacacatc 4740
agctatggct cagaagagga gacaaagata atctagaggg ccgcatcatg taattagtta 4800
tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta 4860
gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt 4920
tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta 4980
tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcgaattcc 5040
tgcagcccgg gggatccact agttctagag cggccgccac cgcggtggag ctccagcttt 5100
tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct 5160
gtgtgaaatt gttatccgct cacaattcca cacaacatag gagccggaag cataaagtgt 5220
aaagcctggg gtgcctaatg agtgaggtaa ctcacattaa ttgcgttgcg ctcactgccc 5280
gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 5340
agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 5400
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 5460
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 5520
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 5580
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 5640
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 5700
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 5760
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 5820
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 5880
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 5940
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 6000
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 6060
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 6120
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 6180
gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 6240
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 6300
gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 6360
tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 6420
ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 6480
ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 6540
atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 6600
cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 6660
tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 6720
aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 6780
tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 6840
ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 6900
agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 6960
gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 7020
agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 7080
accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 7140
gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 7200
cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 7260
ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt ccttttcatc acgtgctata 7320
aaaataatta taatttaaat tttttaatat aaatatataa attaaaaata gaaagtaaaa 7380
aaagaaatta aagaaaaaat agtttttgtt ttccgaagat gtaaaagact ctagggggat 7440
cgccaacaaa tactaccttt tatcttgctc ttcctgctct caggtattaa tgccgaattg 7500
tttcatcttg tctgtgtaga agaccacaca cgaaaatcct gtgattttac attttactta 7560
tcgttaatcg aatgtatatc tatttaatct gcttttcttg tctaataaat atatatgtaa 7620
agtacgcttt ttgttgaaat tttttaaacc tttgtttatt tttttttctt cattccgtaa 7680
ctcttctacc ttctttattt actttctaaa atccaaatac aaaacataaa aataaataaa 7740
cacagagtaa attcccaaat tattccatca ttaaaagata cgaggcgcgt gtaagttaca 7800
ggcaagcgat ccgtcctaag aaaccattat tatcatgaca ttaacctata aaaataggcg 7860
tatcacgagg ccctttcgtc 7880
<210> 8
<211> 7798
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc 60
gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt 120
gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg cgtgttggcg 180
ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata 240
aattcccgtt ttaagagctt ggtgagcgct aggagtcact gccaggtatc gtttgaacac 300
ggcattagtc agggaagtca taacacagtc ctttcccgca attttctttt tctattactc 360
ttggcctcct ctagtacact ctatattttt ttatgcctcg gtaatgattt tcattttttt 420
ttttccccta gcggatgact cttttttttt cttagcgatt ggcattatca cataatgaat 480
tatacattat ataaagtaat gtgatttctt cgaagaatat actaaaaaat gagcaggcaa 540
gataaacgaa ggcaaagatg acagagcaga aagccctagt aaagcgtatt acaaatgaaa 600
ccaagattca gattgcgatc tctttaaagg gtggtcccct agcgatagag cactcgatct 660
tcccagaaaa agaggcagaa gcagtagcag aacaggccac acaatcgcaa gtgattaacg 720
tccacacagg tatagggttt ctggaccata tgatacatgc tctggccaag cattccggct 780
ggtcgctaat cgttgagtgc attggtgact tacacataga cgaccatcac accactgaag 840
actgcgggat tgctctcggt caagctttta aagaggccct actggcgcgt ggagtaaaaa 900
ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg gtagatcttt 960
cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta ggagatctct 1020
cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga attaccctcc 1080
acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg ttcaaggctc 1140
ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt ccctccacca 1200
aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat atatatacat 1260
gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta tgatactgaa 1320
gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg ctttcctttt 1380
ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg gtgtgaaata 1440
ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaaacgtt aatattttgt 1500
taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg 1560
gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt 1620
ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct 1680
atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt 1740
gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa 1800
agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc 1860
tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc 1920
tacagggcgc gtcgcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 1980
tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 2040
gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg 2100
cgtaatacga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag gtcgacggta 2160
tcgataagct tgatatcgaa ttcctgcagc ccgccaattt agctagactt ttgaaaagat 2220
ccaaaaattt actaaaattg aatctttctg gtattaattt tgtcctatac ctcaagaagt 2280
aaagtattct cgaattgata cttctgaaaa gcgaaagaaa tatagctaaa ggatactggt 2340
tatttattct ttcttttttt ctttcttttt gattgagcca gtgtataata aaaacctact 2400
gcgagctatt caataaattg acatagaata tctgttgaag cggttctttg attgagcatc 2460
tgttttcctt ttgtattttt gtccttttag attcttttaa cgacattgga gaaattttaa 2520
tcattgtata aaaacttcat tcgctcgttt gtcagaagtt tttttataac ttgctatttc 2580
cttccaagaa gccttttatt gattaccccc tctctctctt cattgatctt atagatgtcc 2640
aatttactga ccgtacacca aaatttgcct gcattaccgg tcgatgcaac gagtgatgag 2700
gttcgcaaga acctgatgga catgttcagg gatcgccagg cgttttctga gcatacctgg 2760
aaaatgcttc tgtccgtttg ccggtcgtgg gcggcatggt gcaagttgaa taaccggaaa 2820
tggtttcccg cagaacctga agatgttcgc gattatcttc tatatcttca ggcgcgcggt 2880
ctggcagtaa aaactatcca gcaacatttg ggccagctaa acatgcttca tcgtcggtcc 2940
gggctgccac gaccaagtga cagcaatgct gtttcactgg ttatgcggcg gatccgaaaa 3000
gaaaacgttg atgccggtga acgtgcaaaa caggctctag cgttcgaacg cactgatttc 3060
gaccaggttc gttcactcat ggaaaatagc gatcgctgcc aggatatacg taatctggca 3120
tttctgggga ttgcttataa caccctgtta cgtatagccg aaattgccag gatcagggtt 3180
aaagatatct cacgtactga cggtgggaga atgttaatcc atattggcag aacgaaaacg 3240
ctggttagca ccgcaggtgt agagaaggca cttagcctgg gggtaactaa actggtcgag 3300
cgatggattt ccgtctctgg tgtagctgat gatccgaata actacctgtt ttgccgggtc 3360
agaaaaaatg gtgttgccgc gccatctgcc accagccagc tatcaactcg cgccctggaa 3420
gggatttttg aagcagctca tcgactgatt tacggcgctg aggatgactc tggtcagagg 3480
tacctggcct ggtctggaca cagtgcccgt gtcggagccg cgcgagatat ggcccgcgct 3540
ggagtttcaa taccggagat catgcaagct ggtggctgga ccaatgtaaa tattgtcatg 3600
aactatatcc gtaacctgga tagtgaaaca ggggcaatgg tggtcctgct ggaagatggc 3660
gatctcgagc catccgctgg agacatgaga gctgccaacc tttggccaag cccgctcatg 3720
atcaaacgct ctaaggagaa cagcctggcc ttgtccctga cggccgacca gatggtcagt 3780
gccttgttgg atgctgagcc ccccatactc tattccgagt atgatcctac cagacccttc 3840
agtgaagctt cgatgatggg cttactgacc aacctggcag acagggagct ggttcacatg 3900
atcaactggg cgaagagggt gccaggcttt gtggatttga ccctccatga tcaggtccac 3960
cttctagaat gtgcctggct agagatcctg atgattggtc tcgtctggcg ctccatggaa 4020
cacccgggga agctcctgtt tgctcctaac ttgctcctgg acaggaatca aggtaaatgt 4080
gtggaaggca tggtggagat cttcgacatg ctgctggtta catcatctcg gttccgcatg 4140
atgaatctgc agggagagga gtttgtgtgc ctcaaatcta ttattttgct taattctgga 4200
gtatacacat ttctgtccag caccctgaag tctctcgagg agaaggacca tatccaccga 4260
gtcctggaca agatcacaga cactttgatc cacctgatgg ccaaggcagg cctgaccctg 4320
cagcagcagc accagcggct agcccagctc ctcctcctcc tctcccacat caggcacatg 4380
agtaacgaag gcatggagca tctgtacagc atgaagtgca agaacgtggt acccctctat 4440
gacctgctgc tggagatgct agacgcccac cgcctacatg cgcccactag tcgtggaggg 4500
gcatccgtgg aggagacgga ccaaagccac ttggccactg cgggctctac ttcatcgcat 4560
tccttgcaaa agtattacat cacgggggag gcagagggtt tccctgccac agtctgagta 4620
gaatgcctga tattgactca atatccgttg cgtttcctgt caaaagtatg cgtagtgctg 4680
aacatttcgt gatgaatgcc accgaggaag aagcacggcg cggttttgct aaagtgatgt 4740
ctgagtttgg cgaactcttg ggtaaggttg gaattgtcga cctcgagtca tgtaattagt 4800
tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt 4860
tagacaacct gaagtctagg tccctattta tttttttata gttatgttag tattaagaac 4920
gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat 4980
tatactgaaa accttgcttg agaaggtttt gggacgctcg gggggatcca ctagttctag 5040
agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc 5100
gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 5160
cacacaacat aggagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgaggt 5220
aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 5280
agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 5340
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 5400
ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 5460
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 5520
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 5580
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 5640
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 5700
tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5760
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5820
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5880
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5940
actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 6000
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 6060
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 6120
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 6180
tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 6240
caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 6300
cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 6360
agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 6420
acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 6480
gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 6540
ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 6600
tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 6660
ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 6720
tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 6780
attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 6840
agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 6900
ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 6960
ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 7020
cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 7080
gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 7140
tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 7200
tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 7260
tgccacctgg gtccttttca tcacgtgcta taaaaataat tataatttaa attttttaat 7320
ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg 7380
ttttccgaag atgtaaaaga ctctaggggg atcgccaaca aatactacct tttatcttgc 7440
tcttcctgct ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta gaagaccaca 7500
cacgaaaatc ctgtgatttt acattttact tatcgttaat cgaatgtata tctatttaat 7560
ctgcttttct tgtctaataa atatatatgt aaagtacgct ttttgttgaa attttttaaa 7620
cctttgttta tttttttttc ttcattccgt aactcttcta ccttctttat ttactttcta 7680
aaatccaaat acaaaacata aaaataaata aacacagagt aaattcccaa attattccat 7740
cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg atccgtccta agaaacca 7798

Claims (6)

1.大规模基因重排的酵母菌的构建方法，其特征在于，包括：
在酿酒酵母野生型染色体II、III、V、VI、IX或X的着丝粒附近同源重组带vox位点的片段，获得待敲除染色体的酵母菌株；
将两个交配型相反的单倍体酵母菌株融合，构建二倍体酵母菌株；
向二倍体酵母菌株中转化Vika质粒，采用Vika/vox技术删除野生型染色体，然后减数分裂获得单倍体菌株；所述野生型染色体的删除的顺序为II和IX、V、X、VI、III；
所述两个交配型相反的单倍体酵母菌株，其中一个含有合成型染色体synV和synX，另一个含有合成型染色体synII、synIII、synVI和synIXR；所述带vox位点的片段的同源重组包括：合成带筛选抗性基因、两个vox位点和野生型染色体着丝粒两端同源序列的片段，经Gibson组装获得靶向染色体的片段，连入pUC19 质粒，经线性化后转化入酿酒酵母菌株，与待敲除染色体发生同源重组。
2.根据权利要求1所述的构建方法，其特征在于，
所述筛选抗性基因为KanMX基因或hphNT1基因，
所述待敲除染色体着丝粒两端同源序列的长度为1000bp。
3.根据权利要求1所述的构建方法，其特征在于，所述野生型染色体的删除包括，将带有筛选抗性基因和vox位点的片段导入待敲除染色体的菌株，缺陷型筛选获得染色体删除成功的菌株。
4.大规模基因重排的酵母菌，其由权利要求1~3任一项所述的方法构建获得，其染色体中野生型II、III、V、VI、IX和X染色体被替换为合成型染色体synII、synIII、synV、synVI、synIXR和synX。
5.一种酵母菌基因重排的方法，其特征在于，诱导权利要求4所述的酵母菌，或诱导权利要求1~3任一项所述方法构建获得的酵母菌，使其基因发生重排。
6.根据权利要求5所述的方法，其特征在于，所述诱导包括：以His营养缺陷型培养基培养。
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