CN112410366B - 向酿酒酵母中导入线性质粒的方法及制备的酵母菌 - Google Patents

向酿酒酵母中导入线性质粒的方法及制备的酵母菌 Download PDF

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CN112410366B
CN112410366B CN202011451202.XA CN202011451202A CN112410366B CN 112410366 B CN112410366 B CN 112410366B CN 202011451202 A CN202011451202 A CN 202011451202A CN 112410366 B CN112410366 B CN 112410366B
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张生学
吴业春
吴艳斌
王金羽
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Keji Dalong Beijing Biotechnology Co ltd
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Abstract

本申请公开了一种向酿酒酵母中导入线性质粒的方法及制备的酵母菌，该方法包括向源酿酒酵母的线性质粒P1中整合筛选标记基因，并删除酵母交配时负责核融合的KAR1基因的部分序列，由此得到改造后的源酿酒酵母；经改造后的源酿酒酵母与目标酿酒酵母交配，使线性质粒P1和P2导入目标酿酒酵母菌株中。本方法操作简单，且能够高效、高质量的向酿酒酵母中导入线性质粒，经PCR检测，导入成功率可达95％以上。

Description

向酿酒酵母中导入线性质粒的方法及制备的酵母菌
技术领域
本申请涉及分子生物学技术领域，具体而言，涉及一种向酿酒酵母中导入线性质粒的方法及制备的酵母菌。
背景技术
在定向进化过程中，对生物分子进行重新改造以寻找发挥着有益新功能的新型生物分子。这一技术正在彻底改变药物开发、化学工程和其他应用。近年开发的两种定向进化方法PAGE(Esvelt,Kevin,M,et al.A system for the continuous directed evolutionof biomolecules.[J].Nature,2011)和OrthRep(Ravikumar A,Arrieta A,Liu C C.Anorthogonal DNA replication system in yeast.[J].Nature Chemical Biology,2014,10(3):175-7)简化了定向进化流程，减少了实验室工作，提高了定向进化效率。
OrthRep定向进化的原理是乳酸克鲁维酵母(Kluyveromyceslactis)细胞质中存在两个线性质粒pGKl1(P1)和pGKl2(P2)，P1和P2的复制用的是各自线性质粒上编码的一个独立的DNA聚合酶，这两个DNA聚合酶不能复制核基因组DNA和其他质粒DNA，反之，复制核基因组DNA和其他质粒DNA的DNA聚合酶也不能复制P1和P2。这是两套完全不相关的DNA复制系统。将负责复制P1的DNA聚合酶(TP-DNAP1)突变，提高复制P1时错误碱基的掺入率，同时根据酵母核基因密码子的偏好性，将编码TP-DNAP1突变体的基因密码子优化后，在酵母细胞核中进行表达，进而产生突变的TP-DNAP1。该突变的TP-DNAP1在复制P1质粒时，可高频率的掺入错误碱基，从而使P1质粒上的基因高频率的突变。
应用OrthRep定向进化系统已成功进化出抗药性疟疾二氢叶酸还原酶(ArjunRavikumar,et al.Scalable,Continuous Evolution of Genesat Mutation Ratesabove Genomic ErrorThresholds.[J].Cell,2018)。目前，还未见应用OrthRep定向进化系统进化互作蛋白对的报道。
用于研究互作蛋白对最多的酵母菌株为商业化菌株酿酒酵母AH109(MATa,trp1-901,leu2-3,112,ura3-52,his3-200,gal4Δ,gal80Δ,LYS2::GAL1UAS-GAL1TATA-HIS3,MEL1 GAL2UAS-GAL2TATAADE2，URA3::MEL1UAS-MEL1TATA-lacZ)。该菌株遗传背景清楚，已多年用于研究互作蛋白对，积累了丰富的经验，且与该菌株配套的试剂较容易购买。为了建立进化蛋白对的OrthRep定向进化系统，需要向商业化菌株酿酒酵母AH109等目标酿酒酵母中导入P1和P2质粒。向酵母导入P1和P2的方法已有报道，如原生质体融合法(Gunge N,Sakaguchi K.Intergeneric transfer of deoxyribonucleic acid killer plasmids,pGKl1 and pGKl2,from Kluyveromyceslactis into Saccharomyces cerevisiae bycell fusion.[J].Journal of Bacteriology,1981,147(1):155)、纯化的P1和P2线性质粒直接导入原生质体法(Gunge N,Murata K,Sakaguchi K.Transformation ofSaccharomyces cerevisiae with linear DNA killer plasmids fromKluyveromyceslactis.[J].Journal of Bacteriology,1982,151(1):462-4)。这两种方法均用到原生质体，操作繁琐，且效率低下。目前还没有一种简单、高效、高质量的向酿酒酵母导入P1和P2的方法。
发明内容
本发明提供了一种向酿酒酵母中导入线性质粒的方法，该方法包括向源酿酒酵母的线性质粒P1中整合筛选标记基因，并使酵母交配时负责核融合的KAR1基因无法行使核融合功能，由此得到改造后的源酿酒酵母；经改造后的源酿酒酵母与目标酿酒酵母交配，使线性质粒P1和P2导入目标酿酒酵母菌株中。当源酿酒酵母与目标酿酒酵母的交配型相同时，上述方法还包括交配前，先转换改造后的源酿酒酵母的交配型。
具体的，该方法包括下述(1)-(5)中的一个或多个：
(1)所述源酿酒酵母中存在P1和P2两个线性质粒；
(2)所述筛选标记基因为目标酵母菌株的缺陷基因；
(3)所述目标酿酒酵母中不存在线性质粒P1或P2；
(4)所述使酵母交配时负责核融合的KAR1基因无法行使核融合功能是指删除KAR1基因的部分序列，具体的是指敲掉KAR1基因编码区289-573间共285个碱基；
(5)所述转换改造后的源酿酒酵母的交配型是根据实际需要，通过同源重组的方式将MATα或MATa基因序列导入改造后的源酿酒酵母菌株中。
具体的，所述源酿酒酵母可以为GA-Y233菌株，该菌株由酿酒酵母F102-2菌株经URA3基因和Trp1基因失活处理而得，所述酿酒酵母F102-2菌株来源于全球生物资源中心(American Type CultureCollection，PO Box 1549，Manassas,VA 20108USA)，保存目录号为200585；所述目标酿酒酵母为AH109菌株；所述方法具体包括：
(1)在源酿酒酵母GA-Y233菌株的P1质粒上整合Trp1筛选标记基因，便于交配后含有P1和P2质粒的AH109菌株的筛选；
(2)鉴于KAR1基因突变的酵母菌株不能进行细胞核融合，用CRISPR-Cas9介导同源重组的方法敲掉KAR1基因编码区289-573间共285个碱基，便于两酵母菌株交配后细胞质融合而细胞核不融合；
(3)鉴于酿酒酵母GA-Y233和酿酒酵母AH109交配型均为MATa，两菌株不能进行交配，克隆MATα基因序列，并用CRISPR-Cas9介导同源重组的方法将步骤(2)获得的改造后的源酿酒酵母GA-Y233的交配型修改为MATα，便于酿酒酵母GA-Y233和酿酒酵母AH109的交配；
(4)将步骤(3)获得的改造后的酿酒酵母GA-Y233菌株和目标酿酒酵母AH109交配后，在不含尿嘧啶和色氨酸的缺陷型培养基上培养，PCR筛选含有P1和P2质粒的AH109菌株。
进一步的，所述方法包括下述(a)-(g)中的一个或多个：
(a)所述步骤(1)中整合的Trp1筛选标记基因编码序列如SEQ ID No.2所示；
(b)所述步骤(1)中检测所用引物对为F-P1-Trp和R-P1-Trp，其序列分别如SEQ IDNo.4和5所示；
(c)所述步骤(2)中基因重组采用的靶位点为KAR1基因的编辑载体pCAS-KAR1的序列如SEQ ID No.13所示；
(d)所述步骤(2)中检测所用引物对为F-KAR1-JC和R-KAR1-JC，其序列分别如SEQID No.14和15所示；
(e)所述步骤(3)中基因重组采用的靶位点为MATa基因的编辑载体pCAS-MATa的序列如SEQ ID No.22所示；
(f)所述步骤(3)中检测所用引物为F-α-JC、F-a-JC和R-α/a-JC，其序列分别如SEQID No.23-25所示；
(g)所述步骤(4)中检测所用引物对为F-P1-Trp和R-P1-Trp、F-KAR1-JC和R-KAR1-JC、F-α-JC和R-α/a-JC、F-a-JC和R-α/a-JC。
本发明还提供一种酿酒酵母重组工程菌，该酿酒酵母重组工程菌采用前述的方法制备而成。
本发明还提供一种酿酒酵母重组工程菌，该酿酒酵母重组工程菌以GA-Y233菌株为出发菌株，在GA-Y233菌株的P1质粒上整合有Trp1筛选标记基因；且该酿酒酵母重组工程菌的KAR1基因不能行使核融合功能，具体的通过敲掉KAR1基因编码区289-573间共285个碱基使其不能行使核融合功能；其中，所述GA-Y233菌株由酿酒酵母F102-2菌株经URA3基因和Trp1基因失活处理而得，所述酿酒酵母F102-2菌株来源于全球生物资源中心(AmericanType CultureCollection，PO Box 1549，Manassas,VA20108USA)，保存目录号为200585。
进一步的所述酿酒酵母重组工程菌的交配型为MATα型。具体的，通过克隆MATα基因序列，并用CRISPR-Cas9介导同源重组的方法将所述酿酒酵母重组工程菌的交配型修改为MATα。
本发明还提供一种前述的方法和采用前述方法制备的酿酒酵母重组工程菌在定向进化领域的应用。具体的，该应用为互作蛋白对的定向进化领域。
本发明的有益效果包括：本发明提供的向酿酒酵母中导入线性质粒的方法，其操作简单，且能够高效、高质量的向酿酒酵母中导入线性质粒，经PCR检测，导入成功率可达95％以上。
附图说明
图1为酿酒酵母菌株PCR鉴定电泳图，其中第I行检测所用引物为F-P1-Trp和R-P1-Trp；第Ⅱ行检测所用引物为F-KAR1-JC和R-KAR1-JC；第Ⅲ行检测所用引物为F-a-JC和R-α/a-JC；第Ⅳ行检测所用引物为F-α-JC和R-α/a-JC；样品M为Trans2K PlusⅡDNA Marker；样品1为GA-Y233；样品2为GA-Y233-Trp；样品3为GA-Y233-Trp-Kar1；样品4为GA-Y233-Trp-Kar1-MATα；样品5为AH109；样品6为AH109×GA-Y233；样品7为AH109-P1-Trp。
具体实施方式
下面结合实施例对本发明进行进一步说明和描述，但所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明和实施例中，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他发明和实施例，都属于本发明保护的范围。
若未特别指明，实施例均按照常规实验条件，如Sambrook等编写的分子克隆实验手册(New York:Gold Spring Harbor Laboratory Press,1989)、Clontech编写的酵母操作指南(PT3024-1，2009)、或按照制造厂商说明书建议的条件。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、向酿酒酵母AH109中导入线性质粒的方法
包括如下步骤：
(1)酿酒酵母GA-Y233 P1质粒整合Trp1筛选标记基因
全球生物资源中心保藏的酿酒酵母菌株F102-2(MATa,leu2-3 2-112,his4-519,can1,ρ0)(ATCC，catalog no.200585)在缺尿嘧啶的缺陷型培养基(SC-Ura)上可以生长，在缺色氨酸的缺陷型培养基(SC-Trp)上也可以生长。为了方便酵母菌株交配后筛选含有线性质粒P1和P2的酵母菌株AH109，用CRISPR-CAS9介导同源重组的方法，敲除了F102-2菌株的URA3基因和Trp1基因。敲除URA3基因和Trp1基因后的菌株命名为GA-Y233，该菌株不能在缺尿嘧啶的缺陷型培养基(SC-Ura)上生长，也不能在缺色氨酸的缺陷型培养基(SC-Trp)上生长。
酿酒酵母AH109(MATa,trp1-901,leu2-3,112,ura3-52,his3-200,gal4Δ,gal80Δ,LYS2::GAL1UAS-GAL1TATA-HIS3,MEL1 GAL2UAS-GAL2TATAADE2，URA3::MEL1UAS-MEL1TATA-lacZ)在缺尿嘧啶的缺陷型培养基(SC-Ura)上可以生长，在缺色氨酸的缺陷型培养基(SC-Trp)上不能生长。
根据酿酒酵母GA-Y233 P1质粒全序列SEQ ID No.1和酵母中Trp1基因的编码序列SEQ ID No.2，人工合成序列SEQ ID No.3。序列SEQ ID No.3两端分别是BamHⅠ和KpnⅠ酶切位点，便于合成片段的酶切回收。序列SEQ ID No.3Trp1基因编码序列后为增强Trp1基因表达的Poly(A)结构。
SEQ ID No.3序列片段酶切回收后，转化酿酒酵母GA-Y233感受态细胞。转化细胞涂布在缺少色氨酸的SC缺陷型培养基上(SC-Trp)培养。72h后将长出的克隆做PCR检测，所用引物为F-P1-Trp：GAATGGTCATCTGTAAACACTG(SEQ ID No.4)和R-P1-Trp：CGGAATCTAGAGCACATTC(SEQ ID No.5)，扩增片段为1554bp，具体序列见SEQ ID No.6。
整合了Trp1编码基因的P1质粒命名为P1-Trp。P1质粒换为P1-Trp质粒的GA-Y233菌株命名为GA-Y233-Trp。在向P1质粒整合Trp1编码基因时，删掉了P1质粒上原有的第二个和第三个编码基因。P1-Trp具体序列见SEQ ID No.7。
(2)敲掉酿酒酵母GA-Y233-Trp菌株KAR1基因编码区289-573碱基之间的序列
在酵母基因组数据库(简称SGD数据库，https://www.yeastgenome.org/)查询KAR1基因序列。根据查询结果，设计引物F-KAR1:CCAACAATTCAATTTTAACTAAC(SEQ ID No.8)和R-KAR1:CCATCACATGACCAAAAGATATC(SEQ ID No.9)。用上述引物对以酿酒酵母GA-Y233基因组DNA为模板进行PCR扩增，获得2174bp片段。该片段含有KAR1基因起始密码子上游441bp和KAR1基因终止密码子下游431bp，与数据库S288C菌株的序列一致，具体序列见SEQ IDNo.10。
根据SEQ ID No.10序列，人工合成删掉KAR1基因编码区289-573碱基之间序列的900bp片段，具体序列见SEQ ID No.11。该片段两端为EcoRⅠ酶切位点，便于片段回收。
根据SEQ ID No.10序列和KAR1基因的功能结构域，设计CRISPR-Cas9的靶位点序列为ACCCTTTCTACAATGGATCA(SEQ ID No.12)。将该序列插入酵母基因组编辑载体pCAS(Addgene，60847)，获得靶位点为KAR1基因的编辑载体pCAS-KAR1，具体序列见SEQ IDNo.13。
将上述900bp酶切回收片段和pCAS-KAR1质粒混合，共转化酿酒酵母GA-Y233-Trp感受态细胞。转化细胞涂布在含有100mg/L G418的YPDA培养基上培养。72h后将长出的克隆在不含G418的YPDA培养基上划线，继续生长36h。将划线的克隆做PCR检测，所用引物为F-KAR1-JC：GGATCTGATTCAGCACATTG(SEQ ID No.14)和R-KAR1-JC：GAAGTCTATGCCATTTAGAC(SEQ ID No.15)。若KAR1基因未发生重组，PCR产物为817bp，具体序列见SEQ ID No.16；若KAR1基因发生了重组，PCR产物为532bp，具体序列见SEQ ID No.17。
将KAR1基因发生了重组的GA-Y233-Trp酵母菌株命名为GA-Y233-Trp-Kar1。该菌株可与交配型为MATα的酵母菌株交配，向后者导入线性质粒P1-Trp和P2，交配后细胞质融合而细胞核不融合。
(3)酿酒酵母GA-Y233-Trp-Kar1菌株的交配型转换为MATα
在SGD数据库(https://www.yeastgenome.org/)查询MATα基因序列。查到S288C菌株的MATα基因的具体序列见SEQ ID No.18。根据该序列设计引物F-α：GAATTCTTGTATTAGACGAGGGACGGAGTG(SEQ ID No.26)和R-α：
GAATTCTTGATTGTTTGCTTGAGTCTGAG(SEQ ID No.27)。用上述引物对以酿酒酵母GA-Y233基因组DNA为模板进行PCR扩增，获得2519bp片段。该片段两端为EcoRⅠ酶切位点，与SEQID No.18比对发现有7个碱基的差异，具体序列见SEQ ID No.19。
根据SGD数据库中查到的S288C菌株MATa基因的具体序列(SEQ ID No.20)和该基因的具体标注信息，设计CRISPR-Cas9的靶位点序列为ACAAAAATATTTCTAACAAT(SEQ IDNo.21)，将该序列插入酵母基因组编辑载体pCAS，获得靶位点为MATa基因的编辑载体pCAS-MATa，具体序列见SEQ ID No.22。
将EcoRⅠ酶切回收的2519bp片段MATα基因片段和pCAS-MATa质粒混合，共转化酿酒酵母GA-Y233-Trp-Kar1感受态细胞。转化细胞涂布在含有100mg/L G418的YPDA培养基上培养。72h后将长出的克隆在不含G418的YPDA培养基上划线，继续生长36h。将划线的克隆做PCR检测，所用引物为F-α-JC:GCACGGAATATGGGACTACTTCG(SEQ ID No.23)、F-a-JC：ACTCCACTTCAAGTAAGAGTTTG(SEQ ID No.24)和R-α/a-JC:AGTCACATCAAGATCGTTTATGG(SEQID No.25)。F-α-JC和R-α/a-JC扩增片为404bp，F-a-JC和R-α/a-JC扩增片段为544bp。若扩增出404bp片段，表明该菌株的交配型为MATα；若扩增出544bp片段，表明该菌株的交配型为MATa。若同时扩增出404bp片段和544bp片段，表明该菌株为二倍体。
将交配型转换为MATα的GA-Y233-Trp-Kar1酵母菌株命名为GA-Y233-Trp-Kar1-MATα。该菌株可与商业化酵母菌株AH109交配，向后者导入线性质粒P1-Trp和P2。
(4)用GA-Y233-Trp-Kar1-MATα酵母菌株向AH109酵母菌株导入P1-Trp和P2
挑取YPDA平板上生长的GA-Y233-Trp-Kar1-MATα酵母菌株克隆和AH109酵母菌株克隆放入盛有5mL YPDA液体培养基的50mL试管，摇菌至吸光值OD600到0.5-0.6。将两种酵母菌液1:1混合，将混合菌液用移液枪一滴滴的滴在YPDA平板上。30℃培养5h，用缺少色氨酸和尿嘧啶的缺陷型液体培养(SC-Trp-Ura)洗脱，将洗脱液涂布在SC-Trp-Ura平板上，30℃培养96h后平板上出现单克隆。
挑取SC-Trp-Ura平板上长出的酵母单克隆，在SC-Trp-Ura平板上划线，生长36h后，PCR检测克隆的基因型。检测的酵母克隆分为两类：一是含有P1-Trp和P2质粒的AH109菌株，命名为AH109-P1-Trp；另一类为AH109菌株和GA-Y233-Trp-Kar1-MATα菌株杂交产生的二倍体，命名为AH109×GA-Y233。检测所用的引物对为：F-P1-Trp和R-P1-Trp、F-KAR1-JC和R-KAR1-JC、F-α-JC和R-α/a-JC、F-a-JC和R-α/a-JC。如附图1所示，AH109-P1-Trp菌株的扩增基因型为：F-P1-Trp和R-P1-Trp PCR扩增产生1554bp片段；F-KAR1-JC和R-KAR1-JC PCR扩增产生817bp片段；F-α-JC和R-α/a-JC PCR扩增不出片段，F-a-JC和R-α/a-JC扩增产生544bp片段。AH109×GA-Y233二倍体菌株的扩增基因型为：F-P1-Trp和R-P1-Trp PCR扩增产生1554bp片段；F-KAR1-JC和R-KAR1-JC PCR扩增产生817bp片段和532bp片段；F-α-JC和R-α/a-JC PCR扩增产生404bp片段，F-a-JC和R-α/a-JC扩增产生544bp片段。
经上述四对引物PCR检测，生长的酵母克隆多为AH109-P1-Trp菌株，占比95％以上。
序列表
<110> 科稷达隆生物技术有限公司
<120> 向酿酒酵母中导入线性质粒的方法及制备的酵母菌
<160> 27
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8874
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
acacataaca taggggagag tactaaaagt gagattattg gaagattagt acgtctccat 60
ttttttctgt ttttttgttt ttatatatta ggttattttt tttcagtttt atatcaactc 120
tgtataacaa gtctattttt ttatatttta agtctatttt acacttttga cctataagtc 180
attttattat acacattttc caactataat atatgaatta cattattaat ttaaaaatgg 240
attacaaaga taaggcttta aatgatctaa gaaatgtata tgccgacttt gattcacttc 300
ctttagattt tagacaaata ttaataaaag atagagccac acttcttcaa aaagaagatg 360
tagaaaagaa aatattggaa agacaagaag atgcaaagaa atatgcagaa tatttaaaac 420
aatcagaaat accagaacga atatctttgc ctaacattaa aagacataaa ggtgtttcta 480
tatcttttga agaaacatca gaagatatgg ttttggaacc aagacctttt atttttgatg 540
gattaaatat tagatgtttt agacgagaga caattttctc tctcaaaaat aaaatattaa 600
acatggtaaa agaaagttct tcttttaaaa atgtttctag acaatcagtt tctttcatgt 660
attttaaaat ttttaataaa gggaaagtta tagcttctac aaaaagtgta aatatttatg 720
aagataaaat agatgagaga ttagaagatt tgtgtaataa ttttgacgat gtattaaaga 780
aaattataga tgtaacttat ggttatgaaa gtttatttgt ttcagaaaca tattcttatg 840
ttatatttta tgctaaatct atatatttcc ctcaacctag atgtgtgaat aattggggta 900
ataatattcc taatattctt actttcgata gttttaagct tttcacagct aataaaaata 960
atgtttcttg tattaaacag tgctctcgtt ttctgtggca aaaagatttt aatacattag 1020
aagaaatgat agaatataaa aatggtaata tttgtatagt tactcctcaa ttacatataa 1080
atgatgtaag agacataaaa tcatttaacg acatacgttt atattcagaa agtcctatta 1140
aaacattcag tgttatagat aatactataa catatttgtt ttattttaaa gaacatttag 1200
gagttatatt taatattact aaatccagac atgatagaag agtcactaaa tttagtcctt 1260
tgtcaaaatt ttctgatgtt aaaaatataa cagtatgttt tgatatagaa tcttattttg 1320
atccagaaaa agaatctaat caagttaata taccctttat atgttgtgca tctataatat 1380
ataataaagt cataggaaat attgtagatt ttgaaggaag agattgtgta gctcaaatga 1440
tagaatatgt tgtagatata tgtggagagc ttaatatatc ttcagtggaa ctaattgcac 1500
ataatggtgg aggttatgat tttcattata ttttaagtag tatgtataat cctgcagcta 1560
ttaaaaatat attaattaga aataactcat ttataagttt taattttgct cacgatggag 1620
tcaaattttc tgtaaaagat tcctatagtt tcttgttatg tagtttagca aatgcttcaa 1680
aagcattttt aaacgaagaa acctttaaga aaacagattt tccccatcat gatttaaaaa 1740
cagcagatga tttatataaa gtatataaag aatggtcatc tgtaaacact gaaataaatc 1800
atgtagtgga aaaagaaaaa cttcttataa catcagaaca tatagttaat ttcactaaaa 1860
atgataaatc taaaactcta atagaatggt ctaaagatta ttgtagaaat gatgttttgg 1920
ttttatctaa ggtatggtta gaatttaaaa atgctgtaga agatattttt aattgtgaat 1980
tagtagatca aactatgaca ttagcaggac taagttataa attatttcaa gcaaatatgc 2040
cttttgatgt tgaattaaga catccaaata aagaagatta ttttaacatg agagaggctt 2100
taataggagg gagatgtatt agtgtcaatg gaatatataa agatgtttta tgtttagatg 2160
taaaatcatt atatccagca tctatggcat tttatgacca gccatatgga tctttcaaaa 2220
gagtatctag tagacctaaa gatgaattag gtatttatta tgtcagagta actcctaata 2280
gaaataataa atccaacttt tttcctataa gaagtcacaa taaaattact tataataatt 2340
ttgaagaaag tacatatata gcatggtata caaatgtaga tatagatata ggtttgtctg 2400
aaggtcataa tatagaatat atcccctttg attcttatgg aaatataggt tattcttggt 2460
ctaaaaaagg taaaatattc gaaaaatata taaaagacgt gctgtacaaa ttaaaaataa 2520
agtatgaaaa acaaaacaat aaagttaaaa gaaatgttat caaaattatt atgaacagtt 2580
tatggggcaa attcgcacaa aaatgggtaa attttgagta ttttataaaa tcagaagatg 2640
atatagattt tgagtcagaa gaggcatata agatatggga cactgatttt atgctgataa 2700
agaaaattaa agaatctact tattcatcta aacctataca aaatggagta tttacattaa 2760
gttgggcaag ataccacatg aaaagtatat gggatgcagg ggctaaagaa ggagcagaat 2820
gtatctattc ggacacagat agtatttttg tacataaaga acattttaaa aagaatgcta 2880
aatttatgtt aaatggttta aaagttccta ttataggatc agaagtagga caattagaat 2940
tagaatgtga gtttgataaa ttgttatgtg caggtaaaaa gcaatacatg ggattttata 3000
cttattttca agatggaaaa ccatgtataa aagaaaagaa aagatttaag ggtattccta 3060
gtaattatat aatacctgaa ttatatgctc atttactttc aggtgcagac aaagaagcta 3120
aaatacaatt tttgaaattt agaagagaat ggggatcagt taaaggatat atagaaaata 3180
agaccgtgaa agctacttaa tatatgaaag tttttataat aattataaaa tgaatatatt 3240
ttacatattt ttgtttttgc tgtcattcgt tcaaggtttg gagcatactc atcgaagagg 3300
ctccttagtc aaaagagcag tatgttatga cactgatcaa gttccactta atattttctt 3360
tggttataat agagcagata agactgattc taataagaat atggctctaa acatctttaa 3420
tgtttttaga ggttttctag ctggagaagg tggagagtct ttttacaatt ctaatggtaa 3480
tgtttatgga tttatgtggg taggtagtat ggttcataat agaggtttta aagataatat 3540
tttacctata atggaaaatg aagttaagaa ttatggtatt cctaaaacct tgtatttaga 3600
atatgacgga ggtggagatc ctatgaaatc ttttggtatt attttagata caacaagtag 3660
agatactgta gttaaagctg caaaattatg gagtcaaggt aaaaaattaa atagttatga 3720
aggatctaaa aattatcaag ctactgcatg ctatttatct tatgcatata gaaagcccat 3780
tgttaatgat aattttgtag gaacttgcga ctatttcact ttagaaagtg gtaaaacacc 3840
agcagaccaa tctggtatta atggagagtc tctacaaggt tataatccta atttagattt 3900
ctctaaatta tcagcaggac aacctatttg taaaaccata ggtaatcctc ctaattttaa 3960
accttctaag aattcagacg gttcttgtaa aacatacaag gtatcatctg gagagtcttg 4020
ttcttctata gcagttaaat attatccatt aagtttaaat gatatagaaa attataataa 4080
aggtaattat ggatggaaag gatgttctag tcttcaaaaa gattataact tatgtgtgag 4140
tgatggtagt gctcctagac cagtttcaaa tcctatagca gaatgtggtc cattagctcc 4200
aggagagaaa tataatgcta aatgtccttt aaatgcttgt tgtagtgaat ttggtttctg 4260
tggtttaact aaagattatt gtgacaaaaa gagtagtact actggtgctc ctggtacaga 4320
tggctgtttt tctaattgtg gttatggttc tacttctaat gtaaaatcat ctacttttaa 4380
aaagattgct tattggttag atgctaaaga taaattagct atggatccga agaatattcc 4440
taatggtcct tatgatattt tacattatgc ttttgttaat ataaattcag actttagtat 4500
tgatgattct gcattttcaa aatctgcctt tttaaaagtt acttcttcca aaaagatacc 4560
tagttttggt ggttgggatt ttagtacatc tcctagtact tacactatat ttagaaatgc 4620
tgttaaaaca gatcaaaata gaaatacgtt tgctaacaat ttaatcaatt ttatgaataa 4680
atataatctt gatggtatag atttagattg ggaatatcca ggtgctcctg atattccaga 4740
tattcctgct gatgattcaa gtagtggatc taattatcta actttcctta agttattaaa 4800
gggtaaaatg ccttctggta aaaccttatc tatagccatt ccttcttcct attggtattt 4860
aaaaaatttc cctatttctg atattcaaaa cactgtagat tatatggttt acatgacgta 4920
tgatatacat ggtatatggg aatacggtaa agccaatagt tatataaact gccatactcc 4980
tcgtaaagaa attgaagatg ctataaaaat gttagataaa gctggagtta aatttaataa 5040
agtatttgga ggtgtggcaa attacggtag atcctacaaa atggttaata caaattgtta 5100
taattatgga tgcggttttc aaagagaggg aggaaattct agagatatga ctaatacacc 5160
aggtgttctt tctgattcag aaattattga tattgatagt tcagataaaa agaatgatag 5220
atgggtagat actaacacag attgtatttt tatgaaatat gacggaaatt ctgttgtttc 5280
atggcctaaa agtagatacg atttagaaga tatgtttaaa aattatggat ttgctggtac 5340
ttctttatgg gccgctaatt atttcaaaca tgatgaatgg aagaacgatg aagatgataa 5400
taatgatgat acagaagatc ctttcgatga agagaatgta tatttcgatg tttatgattg 5460
caaaaacaaa gctggttatg atctggacaa tccagtttat gggtgtagat tagaaacagc 5520
tataaatatt attatatgga atggtacaga atctgttaat acagttttaa atatattaaa 5580
tgattacgat aattatatta aatattatga agctctaact agagcacatt atgattcagt 5640
catggaaaaa tacgaaaaat ggctgtttga agaagatgga tattacacat attatactga 5700
tgtagacgga gatgatataa ttataactcc tccagataag aagaaaagag attacataca 5760
agagaaatat tcttttgaaa aagaatttat gatgtctcaa aatatgacag aattaacaga 5820
aattaaagtt aataaaacta ttaattttat gttaaatgga acatctctag ctgtaaaaga 5880
atataacaac gaaaaagttt tatataaaag aggagatata cctcctcctg gttctaataa 5940
tagattaatt agaaacagta ttattttaga taaagataaa gaagcagcta ttgcgtcttt 6000
caaacaatat tctggaatag aattatctaa agattctttt gtacaaagag ataaagataa 6060
aaagtttgat ctaaatggta aacattatac atttatgcat agtactattc tgaatgctat 6120
tgttttattc cctaatgttt taacaaatat tgattctgac tatattcatc atatttcaga 6180
tttaattgaa caagctcata acagtttagg taatgaaagt cctgataata tttatgaggt 6240
cttagaaagt gtggttgttt ttatgtctgt atcagaaata gctgattata catatacaga 6300
aggtaaaaag ataaaagaaa aatacgataa gatgaagaaa actatgattg ttggtattat 6360
attgggtatc ataggtggtt tgtctctatt tttaggacct ataggtatag ctacatctgt 6420
tcttgcagat tttgctctat taggagcaga tgccgctata aacggagagt taaatccatc 6480
agacctagca ttcgctttag caggtttatt cttaccagta tttgcttctt taggaaaaac 6540
atttaaattt gctgaagctt tacaaaaaat taatattaat aaatctaaaa actttgataa 6600
tttaaatgaa tttgagaaaa taagattttt cagatctaaa ttagggaaag ttaagatgtg 6660
tggctcttaa aagtaatgga tgaccattat tcttgtgtaa attgtcaaaa tctacatctt 6720
catatttatg atatttaaat atatattttt cgttttcaaa atctaaatgt tgacacatac 6780
ctccttcttt ttttgcttta ttcatcataa tattataaaa ttcaatacta ccagaagcat 6840
aagctattct tattaaatct atatctggac tataattttc taaatcttca gttatattca 6900
taatagcata atttactaat attgcatatc tttggcgtgg aaaatcgata agtagttttt 6960
gaaccatata tttatttaaa gttttataag tgtaaaaata aaaaggccta taaagagaca 7020
caaagtttga atcataaata tcattcacta ataaatttaa tactgctttt ttacacaaat 7080
catctggata ttctttatga tgtttaagta cataagctga atttaaaaaa ttaaattcaa 7140
ctgtatttat atttatatct aaataaggtt tataagagac catattatag tacacacttt 7200
tatctacaga aacacaatcc ataggaccaa attctgtatt ttgactataa tctatatatg 7260
tatataacat atcatctata atttgttcta tattactttg tttagaagta taattatatt 7320
taaaaaatat ttctaaagtt gtgtctttat tcctgagtat agtttcagga agtaaatatt 7380
tgtctttttc tactttttct aaaatatttt tattttcatg tattttataa ttatatatag 7440
tatcttcttc gcaaaaagat cttctattaa aaattataga taatctaaaa caaacttctg 7500
tacatatttt atcacattta tcacaaacat catcccaacc taataaaaca catattgttt 7560
taataactaa actatattta ggatcttttt ctaataaata tatacaagtt tctttagtag 7620
gaacattagg ataccaaatt cctgaaggca aatatttaaa attaaaatca caaaccttgt 7680
tactcataat atatctagca gatatactgg aaagtattcc agatgttaat tttaaacctg 7740
aattttccat tttaactgca aaattataac tatttcttat tcctatacat aaatgaaagt 7800
ttaaatcatc ttcatcaaaa agctcttcat tatcataata tttaataaaa ttttcataat 7860
ctgaataata agcataagta catgctttaa aataatctga aagattatta tctaattcta 7920
aacacatttt taattaaaat gaagatatat catatattta gtgtttgtta tctaataaca 7980
ttatgtgctg ctgcagctac tactgcgaga gaggagtttt tcttatgtta tgatttaatt 8040
agatatttaa aacaatatga aaaaacagga gagagtaaat tagtagaaca aacatttttt 8100
aatagtatta aaaacttaga cataaactct agagagtata tggaacttgt atataacaaa 8160
atagcaggta tttccaatga aagaaataaa tttgaaaata tatataaaga tggagattct 8220
ataagtcaag ttgtagaaag agctgtaagc gaaaagaaac ttacatttgg attaaacggt 8280
aaaggattat atgttccaga aaacggagaa ccccgactaa aaggttatgc ttctattata 8340
gaaagaataa ctctggattt aatggaaata tattctatta aaggacttaa tgatatacct 8400
agagatataa aatttaatat ggaaaaaata agacaagaaa gatacaacca aatgaaagaa 8460
gctctaaata gtgttgaagg ttataaagga aaaattgtag cctcagactc agattggtgt 8520
ttcaaagatc ctcaaggcaa tagaataaca gattttgata gtattaataa agaattaggt 8580
cttggtagaa gagatgtaaa attagataaa ggtcatgatg atttaattaa attatgtact 8640
gaaaaaatag atagtatgaa taatctacag aatggaaaat gtgtataata aaatgactta 8700
taggtcaaaa gtgtaaaata gacttaaaat ataaaaaaat agacttgtta tacagagttg 8760
atataaaact gaaaaaaaat aacctaatat ataaaaacaa aaaaacagaa aaaaatggag 8820
acgtactaat cttccaataa tctcactttt agtactctcc cctatgttat gtgt 8874
<210> 2
<211> 675
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgtctgtta ttaatttcac aggtagttct ggtccattgg tgaaagtttg cggcttgcag 60
agcacagagg ccgcagaatg tgctctagat tccgatgctg acttgctggg tattatatgt 120
gtgcccaata gaaagagaac aattgacccg gttattgcaa ggaaaatttc aagtcttgta 180
aaagcatata aaaatagttc aggcactccg aaatacttgg ttggcgtgtt tcgtaatcaa 240
cctaaggagg atgttttggc tctggtcaat gattacggca ttgatatcgt ccaactgcat 300
ggagatgagt cgtggcaaga ataccaagag ttcctcggtt tgccagttat taaaagactc 360
gtatttccaa aagactgcaa catactactc agtgcagctt cacagaaacc tcattcgttt 420
attcccttgt ttgattcaga agcaggtggg acaggtgaac ttttggattg gaactcgatt 480
tctgactggg ttggaaggca agagagcccc gaaagcttac attttatgtt agctggtgga 540
ctgacgccag aaaatgttgg tgatgcgctt agattaaatg gcgttattgg tgttgatgta 600
agcggaggtg tggagacaaa tggtgtaaaa gactctaaca aaatagcaaa tttcgtcaaa 660
aatgctaaga aatag 675
<210> 3
<211> 2800
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggatccgatc aaactatgac attagcagga ctaagttata aattatttca agcaaatatg 60
ccttttgatg ttgaattaag acatccaaat aaagaagatt attttaacat gagagaggct 120
ttaataggag ggagatgtat tagtgtcaat ggaatatata aagatgtttt atgtttagat 180
gtaaaatcat tatatccagc atctatggca ttttatgacc agccatatgg atctttcaaa 240
agagtatcta gtagacctaa agatgaatta ggtatttatt atgtcagagt aactcctaat 300
agaaataata aatccaactt ttttcctata agaagtcaca ataaaattac ttataataat 360
tttgaagaaa gtacatatat agcatggtat acaaatgtag atatagatat aggtttgtct 420
gaaggtcata atatagaata tatccccttt gattcttatg gaaatatagg ttattcttgg 480
tctaaaaaag gtaaaatatt cgaaaaatat ataaaagacg tgctgtacaa attaaaaata 540
aagtatgaaa aacaaaacaa taaagttaaa agaaatgtta tcaaaattat tatgaacagt 600
ttatggggca aattcgcaca aaaatgggta aattttgagt attttataaa atcagaagat 660
gatatagatt ttgagtcaga agaggcatat aagatatggg acactgattt tatgctgata 720
aagaaaatta aagaatctac ttattcatct aaacctatac aaaatggagt atttacatta 780
agttgggcaa gataccacat gaaaagtata tgggatgcag gggctaaaga aggagcagaa 840
tgtatctatt cggacacaga tagtattttt gtacataaag aacattttaa aaagaatgct 900
aaatttatgt taaatggttt aaaagttcct attataggat cagaagtagg acaattagaa 960
ttagaatgtg agtttgataa attgttatgt gcaggtaaaa agcaatacat gggattttat 1020
acttattttc aagatggaaa accatgtata aaagaaaaga aaagatttaa gggtattcct 1080
agtaattata taatacctga attatatgct catttacttt caggtgcaga caaagaagct 1140
aaaatacaat ttttgaaatt tagaagagaa tggggatcag ttaaaggata tatagaaaat 1200
aagaccgtga aagctactta atatatgaaa gtttttataa taattataaa atgtctgtta 1260
ttaatttcac aggtagttct ggtccattgg tgaaagtttg cggcttgcag agcacagagg 1320
ccgcagaatg tgctctagat tccgatgctg acttgctggg tattatatgt gtgcccaata 1380
gaaagagaac aattgacccg gttattgcaa ggaaaatttc aagtcttgta aaagcatata 1440
aaaatagttc aggcactccg aaatacttgg ttggcgtgtt tcgtaatcaa cctaaggagg 1500
atgttttggc tctggtcaat gattacggca ttgatatcgt ccaactgcat ggagatgagt 1560
cgtggcaaga ataccaagag ttcctcggtt tgccagttat taaaagactc gtatttccaa 1620
aagactgcaa catactactc agtgcagctt cacagaaacc tcattcgttt attcccttgt 1680
ttgattcaga agcaggtggg acaggtgaac ttttggattg gaactcgatt tctgactggg 1740
ttggaaggca agagagcccc gaaagcttac attttatgtt agctggtgga ctgacgccag 1800
aaaatgttgg tgatgcgctt agattaaatg gcgttattgg tgttgatgta agcggaggtg 1860
tggagacaaa tggtgtaaaa gactctaaca aaatagcaaa tttcgtcaaa aatgctaaga 1920
aatagctgca ggaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980
aaaaaaaaaa aaaaaaaaaa aaacctgtca ccggatgtgt tttccggtct gatgagtccg 2040
tgaggacgaa acagggaatt ctgaagatat atcatatatt tagtgtttgt tatctaataa 2100
cattatgtgc tgctgcagct actactgcga gagaggagtt tttcttatgt tatgatttaa 2160
ttagatattt aaaacaatat gaaaaaacag gagagagtaa attagtagaa caaacatttt 2220
ttaatagtat taaaaactta gacataaact ctagagagta tatggaactt gtatataaca 2280
aaatagcagg tatttccaat gaaagaaata aatttgaaaa tatatataaa gatggagatt 2340
ctataagtca agttgtagaa agagctgtaa gcgaaaagaa acttacattt ggattaaacg 2400
gtaaaggatt atatgttcca gaaaacggag aaccccgact aaaaggttat gcttctatta 2460
tagaaagaat aactctggat ttaatggaaa tatattctat taaaggactt aatgatatac 2520
ctagagatat aaaatttaat atggaaaaaa taagacaaga aagatacaac caaatgaaag 2580
aagctctaaa tagtgttgaa ggttataaag gaaaaattgt agcctcagac tcagattggt 2640
gtttcaaaga tcctcaaggc aatagaataa cagattttga tagtattaat aaagaattag 2700
gtcttggtag aagagatgta aaattagata aaggtcatga tgatttaatt aaattatgta 2760
ctgaaaaaat agatagtatg aataatctac agaaggtacc 2800
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gaatggtcat ctgtaaacac tg 22
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cggaatctag agcacattc 19
<210> 6
<211> 1554
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gaatggtcat ctgtaaacac tgaaataaat catgtagtgg aaaaagaaaa acttcttata 60
acatcagaac atatagttaa tttcactaaa aatgataaat ctaaaactct aatagaatgg 120
tctaaagatt attgtagaaa tgatgttttg gttttatcta aggtatggtt agaatttaaa 180
aatgctgtag aagatatttt taattgtgaa ttagtagatc aaactatgac attagcagga 240
ctaagttata aattatttca agcaaatatg ccttttgatg ttgaattaag acatccaaat 300
aaagaagatt attttaacat gagagaggct ttaataggag ggagatgtat tagtgtcaat 360
ggaatatata aagatgtttt atgtttagat gtaaaatcat tatatccagc atctatggca 420
ttttatgacc agccatatgg atctttcaaa agagtatcta gtagacctaa agatgaatta 480
ggtatttatt atgtcagagt aactcctaat agaaataata aatccaactt ttttcctata 540
agaagtcaca ataaaattac ttataataat tttgaagaaa gtacatatat agcatggtat 600
acaaatgtag atatagatat aggtttgtct gaaggtcata atatagaata tatccccttt 660
gattcttatg gaaatatagg ttattcttgg tctaaaaaag gtaaaatatt cgaaaaatat 720
ataaaagacg tgctgtacaa attaaaaata aagtatgaaa aacaaaacaa taaagttaaa 780
agaaatgtta tcaaaattat tatgaacagt ttatggggca aattcgcaca aaaatgggta 840
aattttgagt attttataaa atcagaagat gatatagatt ttgagtcaga agaggcatat 900
aagatatggg acactgattt tatgctgata aagaaaatta aagaatctac ttattcatct 960
aaacctatac aaaatggagt atttacatta agttgggcaa gataccacat gaaaagtata 1020
tgggatgcag gggctaaaga aggagcagaa tgtatctatt cggacacaga tagtattttt 1080
gtacataaag aacattttaa aaagaatgct aaatttatgt taaatggttt aaaagttcct 1140
attataggat cagaagtagg acaattagaa ttagaatgtg agtttgataa attgttatgt 1200
gcaggtaaaa agcaatacat gggattttat acttattttc aagatggaaa accatgtata 1260
aaagaaaaga aaagatttaa gggtattcct agtaattata taatacctga attatatgct 1320
catttacttt caggtgcaga caaagaagct aaaatacaat ttttgaaatt tagaagagaa 1380
tggggatcag ttaaaggata tatagaaaat aagaccgtga aagctactta atatatgaaa 1440
gtttttataa taattataaa atgtctgtta ttaatttcac aggtagttct ggtccattgg 1500
tgaaagtttg cggcttgcag agcacagagg ccgcagaatg tgctctagat tccg 1554
<210> 7
<211> 4975
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
acacataaca taggggagag tactaaaagt gagattattg gaagattagt acgtctccat 60
ttttttctgt ttttttgttt ttatatatta ggttattttt tttcagtttt atatcaactc 120
tgtataacaa gtctattttt ttatatttta agtctatttt acacttttga cctataagtc 180
attttattat acacattttc caactataat atatgaatta cattattaat ttaaaaatgg 240
attacaaaga taaggcttta aatgatctaa gaaatgtata tgccgacttt gattcacttc 300
ctttagattt tagacaaata ttaataaaag atagagccac acttcttcaa aaagaagatg 360
tagaaaagaa aatattggaa agacaagaag atgcaaagaa atatgcagaa tatttaaaac 420
aatcagaaat accagaacga atatctttgc ctaacattaa aagacataaa ggtgtttcta 480
tatcttttga agaaacatca gaagatatgg ttttggaacc aagacctttt atttttgatg 540
gattaaatat tagatgtttt agacgagaga caattttctc tctcaaaaat aaaatattaa 600
acatggtaaa agaaagttct tcttttaaaa atgtttctag acaatcagtt tctttcatgt 660
attttaaaat ttttaataaa gggaaagtta tagcttctac aaaaagtgta aatatttatg 720
aagataaaat agatgagaga ttagaagatt tgtgtaataa ttttgacgat gtattaaaga 780
aaattataga tgtaacttat ggttatgaaa gtttatttgt ttcagaaaca tattcttatg 840
ttatatttta tgctaaatct atatatttcc ctcaacctag atgtgtgaat aattggggta 900
ataatattcc taatattctt actttcgata gttttaagct tttcacagct aataaaaata 960
atgtttcttg tattaaacag tgctctcgtt ttctgtggca aaaagatttt aatacattag 1020
aagaaatgat agaatataaa aatggtaata tttgtatagt tactcctcaa ttacatataa 1080
atgatgtaag agacataaaa tcatttaacg acatacgttt atattcagaa agtcctatta 1140
aaacattcag tgttatagat aatactataa catatttgtt ttattttaaa gaacatttag 1200
gagttatatt taatattact aaatccagac atgatagaag agtcactaaa tttagtcctt 1260
tgtcaaaatt ttctgatgtt aaaaatataa cagtatgttt tgatatagaa tcttattttg 1320
atccagaaaa agaatctaat caagttaata taccctttat atgttgtgca tctataatat 1380
ataataaagt cataggaaat attgtagatt ttgaaggaag agattgtgta gctcaaatga 1440
tagaatatgt tgtagatata tgtggagagc ttaatatatc ttcagtggaa ctaattgcac 1500
ataatggtgg aggttatgat tttcattata ttttaagtag tatgtataat cctgcagcta 1560
ttaaaaatat attaattaga aataactcat ttataagttt taattttgct cacgatggag 1620
tcaaattttc tgtaaaagat tcctatagtt tcttgttatg tagtttagca aatgcttcaa 1680
aagcattttt aaacgaagaa acctttaaga aaacagattt tccccatcat gatttaaaaa 1740
cagcagatga tttatataaa gtatataaag aatggtcatc tgtaaacact gaaataaatc 1800
atgtagtgga aaaagaaaaa cttcttataa catcagaaca tatagttaat ttcactaaaa 1860
atgataaatc taaaactcta atagaatggt ctaaagatta ttgtagaaat gatgttttgg 1920
ttttatctaa ggtatggtta gaatttaaaa atgctgtaga agatattttt aattgtgaat 1980
tagtagatca aactatgaca ttagcaggac taagttataa attatttcaa gcaaatatgc 2040
cttttgatgt tgaattaaga catccaaata aagaagatta ttttaacatg agagaggctt 2100
taataggagg gagatgtatt agtgtcaatg gaatatataa agatgtttta tgtttagatg 2160
taaaatcatt atatccagca tctatggcat tttatgacca gccatatgga tctttcaaaa 2220
gagtatctag tagacctaaa gatgaattag gtatttatta tgtcagagta actcctaata 2280
gaaataataa atccaacttt tttcctataa gaagtcacaa taaaattact tataataatt 2340
ttgaagaaag tacatatata gcatggtata caaatgtaga tatagatata ggtttgtctg 2400
aaggtcataa tatagaatat atcccctttg attcttatgg aaatataggt tattcttggt 2460
ctaaaaaagg taaaatattc gaaaaatata taaaagacgt gctgtacaaa ttaaaaataa 2520
agtatgaaaa acaaaacaat aaagttaaaa gaaatgttat caaaattatt atgaacagtt 2580
tatggggcaa attcgcacaa aaatgggtaa attttgagta ttttataaaa tcagaagatg 2640
atatagattt tgagtcagaa gaggcatata agatatggga cactgatttt atgctgataa 2700
agaaaattaa agaatctact tattcatcta aacctataca aaatggagta tttacattaa 2760
gttgggcaag ataccacatg aaaagtatat gggatgcagg ggctaaagaa ggagcagaat 2820
gtatctattc ggacacagat agtatttttg tacataaaga acattttaaa aagaatgcta 2880
aatttatgtt aaatggttta aaagttccta ttataggatc agaagtagga caattagaat 2940
tagaatgtga gtttgataaa ttgttatgtg caggtaaaaa gcaatacatg ggattttata 3000
cttattttca agatggaaaa ccatgtataa aagaaaagaa aagatttaag ggtattccta 3060
gtaattatat aatacctgaa ttatatgctc atttactttc aggtgcagac aaagaagcta 3120
aaatacaatt tttgaaattt agaagagaat ggggatcagt taaaggatat atagaaaata 3180
agaccgtgaa agctacttaa tatatgaaag tttttataat aattataaaa tgtctgttat 3240
taatttcaca ggtagttctg gtccattggt gaaagtttgc ggcttgcaga gcacagaggc 3300
cgcagaatgt gctctagatt ccgatgctga cttgctgggt attatatgtg tgcccaatag 3360
aaagagaaca attgacccgg ttattgcaag gaaaatttca agtcttgtaa aagcatataa 3420
aaatagttca ggcactccga aatacttggt tggcgtgttt cgtaatcaac ctaaggagga 3480
tgttttggct ctggtcaatg attacggcat tgatatcgtc caactgcatg gagatgagtc 3540
gtggcaagaa taccaagagt tcctcggttt gccagttatt aaaagactcg tatttccaaa 3600
agactgcaac atactactca gtgcagcttc acagaaacct cattcgttta ttcccttgtt 3660
tgattcagaa gcaggtggga caggtgaact tttggattgg aactcgattt ctgactgggt 3720
tggaaggcaa gagagccccg aaagcttaca ttttatgtta gctggtggac tgacgccaga 3780
aaatgttggt gatgcgctta gattaaatgg cgttattggt gttgatgtaa gcggaggtgt 3840
ggagacaaat ggtgtaaaag actctaacaa aatagcaaat ttcgtcaaaa atgctaagaa 3900
atagctgcag gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3960
aaaaaaaaaa aaaaaaaaaa aacctgtcac cggatgtgtt ttccggtctg atgagtccgt 4020
gaggacgaaa cagggaattc tgaagatata tcatatattt agtgtttgtt atctaataac 4080
attatgtgct gctgcagcta ctactgcgag agaggagttt ttcttatgtt atgatttaat 4140
tagatattta aaacaatatg aaaaaacagg agagagtaaa ttagtagaac aaacattttt 4200
taatagtatt aaaaacttag acataaactc tagagagtat atggaacttg tatataacaa 4260
aatagcaggt atttccaatg aaagaaataa atttgaaaat atatataaag atggagattc 4320
tataagtcaa gttgtagaaa gagctgtaag cgaaaagaaa cttacatttg gattaaacgg 4380
taaaggatta tatgttccag aaaacggaga accccgacta aaaggttatg cttctattat 4440
agaaagaata actctggatt taatggaaat atattctatt aaaggactta atgatatacc 4500
tagagatata aaatttaata tggaaaaaat aagacaagaa agatacaacc aaatgaaaga 4560
agctctaaat agtgttgaag gttataaagg aaaaattgta gcctcagact cagattggtg 4620
tttcaaagat cctcaaggca atagaataac agattttgat agtattaata aagaattagg 4680
tcttggtaga agagatgtaa aattagataa aggtcatgat gatttaatta aattatgtac 4740
tgaaaaaata gatagtatga ataatctaca gaatggaaaa tgtgtataat aaaatgactt 4800
ataggtcaaa agtgtaaaat agacttaaaa tataaaaaaa tagacttgtt atacagagtt 4860
gatataaaac tgaaaaaaaa taacctaata tataaaaaca aaaaaacaga aaaaaatgga 4920
gacgtactaa tcttccaata atctcacttt tagtactctc ccctatgtta tgtgt 4975
<210> 8
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ccaacaattc aattttaact aac 23
<210> 9
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccatcacatg accaaaagat atc 23
<210> 10
<211> 2174
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccaacaattc aattttaact aactgaatgt gaagaaaaag atcaactaaa taagtttatt 60
cagataaatc ttcattatct gatccattat ttttaagttt ttcttttaat gtatataaga 120
aattctaatc ttttaattcg cacaatagta ttttcatgta ctagttcgta cagtattctc 180
tttctctggg gtttttccct tttatagaag ttgacttgtt ctatagttgt tatatatgta 240
ttgctgggtt ttttaatcta ttttgcggaa gacgccaaaa ataagaaaaa aattggaaag 300
acacaagaaa aaagtaagaa tatccacagc tgaaagatct aacaggcgta tttttatttt 360
cgaagaaacg tcctaaaagg atctgattca gcacattgat aatagcatag gcatttttat 420
ctttcttttt tgagttcatg aatgaatgta acttctccaa aagatgggaa tcacagtttc 480
tcgaagaaaa atagatttaa tacaaataaa ccgcgattcc acaaactaaa tgagcaggcg 540
cagagtataa atttaccaga agaccgtgat tcaattgttt caagtaatac aacgtcaatt 600
atgacagatg atgcatttga ttacaatgag ggcattgcat cgcgtaccaa aaatattaat 660
tctgatagtg atagaagcaa tgataccata aaacaaaaca actacaataa aagggagacc 720
ggatataacc ctttctacaa tggatcaggg atcaatcagc gatatacaca gtttcgaaaa 780
agagagtttg aaccaacact tgcagaaaac aaagccgaag agtacatatc ggacgaagat 840
aatgtaaaaa ttgatgaaga taatatagag aacgaactcc agtttacgcc gaaaattaaa 900
gaggctagta tactccgatc tagtttacta ggacaaagaa atgttttaaa tactcggaat 960
ccaaaatcaa aagaatcaca cattaaagta aaacccatca tcaataacaa gagctcctca 1020
caaagaaaat ctagtgcagc acttcggaaa caattaggaa aacccctacc gctgccgtat 1080
ttgaatagcc ctaatagtga tagtacaccc acattacaga gaaaagaaga agtattcaca 1140
gacgaagtgc ttcaaaaaaa gagagaattg attgagtcta aatggcatag acttctcttt 1200
catgacaaaa aaatggtgga aaaaaagcta gaaagtttaa gagaatacga aaggaaaaga 1260
atgcctccac gaggaactga tgtttctagc tctgagcagg acaattcttt caaaatatcg 1320
acgccaacaa aatcgtatgt ttctttggag caaaaaccct taccaaatct ctctgctatg 1380
aataacttta atgatgttac cgacaataag gagaaagaag aaacgaacaa caatatatta 1440
aagttccaag cgcaacgaga tccattacaa atactacagt ctgagatcga aatgcatact 1500
aagaaacttg acacgataat agagttacta aaagacgata ccgattcaaa ggaaaaaagg 1560
aaagtagtga ctaatgacaa cgcagcgcct gaacaaatgg tcaacaaagg atggcggaaa 1620
aacgtgatga tgatctacaa aaaatcagga aatattatga aaaagtatag ggaatatttc 1680
ttatggacaa tttgtatttt aatattgtta tattgcaata tatatgtgta ttataggttt 1740
taaaccatat aaaggttagt acatgaagaa gtgtgtacag ttatggcaat aactgttctc 1800
ttccgtttta aagagagtat tatcacggtc tgaaaattaa gcaaaaaaag aaaagcgtgc 1860
acttaaaaat cggtggtgga cagaccgcat attccataaa catagatggt acaagggcat 1920
tatgagtcct tgaatagaaa aaatgatgaa agaatgaatg caattccgta ttgcgacact 1980
ggcgaagtgt tcgaggctga cacgatagcg aatgtatgga aaagagaaga taaggaatgg 2040
ttaaaaagga ctcaaagtga tcgaagtgga tacgagtatc cacgactagg agaatcacca 2100
tatatcaata tgacagacga cttcagaatg gaaaagaagg taatatgtca agatatcttt 2160
tggtcatgtg atgg 2174
<210> 11
<211> 900
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gaattcatga atgtaacttc tccaaaagat gggaatcaca gtttctcgaa gaaaaataga 60
tttaatacaa ataaaccgcg attccacaaa ctaaatgagc aggcgcagag tataaattta 120
ccagaagacc gtgattcaat tgtttcaagt aatacaacgt caattatgac agatgatgca 180
tttgattaca atgagggcat tgcatcgcgt accaaaaata ttaattctga tagtgataga 240
agcaatgata ccataaaaca aaacaactac aataaaaggg agaccggata taactcctca 300
caaagaaaat ctagtgcagc acttcggaaa caattaggaa aacccctacc gctgccgtat 360
ttgaatagcc ctaatagtga tagtacaccc acattacaga gaaaagaaga agtattcaca 420
gacgaagtgc ttcaaaaaaa gagagaattg attgagtcta aatggcatag acttctcttt 480
catgacaaaa aaatggtgga aaaaaagcta gaaagtttaa gagaatacga aaggaaaaga 540
atgcctccac gaggaactga tgtttctagc tctgagcagg acaattcttt caaaatatcg 600
acgccaacaa aatcgtatgt ttctttggag caaaaaccct taccaaatct ctctgctatg 660
aataacttta atgatgttac cgacaataag gagaaagaag aaacgaacaa caatatatta 720
aagttccaag cgcaacgaga tccattacaa atactacagt ctgagatcga aatgcatact 780
aagaaacttg acacgataat agagttacta aaagacgata ccgattcaaa ggaaaaaagg 840
aaagtagtga ctaatgacaa cgcagcgcct gaacaaatgg tcaacaaagg atgggaattc 900
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
accctttcta caatggatca 20
<210> 13
<211> 8743
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg 60
ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt 120
tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt 180
taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc 240
cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc 300
atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata 360
gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat 420
atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt 480
tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag 540
gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat 600
agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc 660
aatattttag tagcccgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca 720
gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagag aataggaact 780
tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc 840
tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata 900
tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtatat gtgttatgta 960
gtatactctt tcttcaacaa ttaaatactc tcggtagcca agttggttta aggcgcaaga 1020
ctgtaattta tcactacgaa atcttgagat cgggcgttcg actcgccccc gggagagatg 1080
gccggcatgg tcccagcctc ctcgctggcg ccggctgggc aacaccttcg ggtggcgaat 1140
gggactttac cctttctaca atggatcagt tttagagcta gaaatagcaa gttaaaataa 1200
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tattttttgt 1260
cactattgtt atgtaaaatg ccacctctga cagtatggaa cgcaaacttc tgtctagtgg 1320
atataggtct agagatctgt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac 1380
atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt 1440
gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat 1500
tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac ctgcgagcag 1560
ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat 1620
ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg 1680
ctaggataca gttctcacat cacatccgaa cataaacaac catgggtaag gaaaagactc 1740
acgtttcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg 1800
ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg 1860
cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga 1920
tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc 1980
gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggcaaaaca gcattccagg 2040
tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc 2100
gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc 2160
tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg 2220
agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt ttgccattct 2280
caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg 2340
ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc 2400
ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc 2460
aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg 2520
agtttttcta atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt 2580
atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt 2640
ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg 2700
cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca 2760
tccagtgtcg aaaacgagct ctcgagaacc cttaatagtc gaacaagaag caggcaaagt 2820
ttagagcact gcccctccgc actcaaaaaa gaaaaaacta ggaggaaaat aaaattctca 2880
accacacaaa cacataaaca catacaaata caaatacaag cttatttact tgacatcgcg 2940
cgatcttcca ctattcagcg ccgtccgccc tctctcgtgt tttttgttta cgcgacaact 3000
atgcgaaatc cggagcaacg ggcaaccgtt tggggaaaga ccacacccac gcgcgatcgc 3060
catggcaacg aggtcgcaca cgccccacac ccagacctcc ctgcgagcgg gcatgggtac 3120
aatgtccccg ttgccacaga caccacttcg tagcacagcg cagagcgtag cgtgttgttg 3180
ctgctgacaa aagaaaattt ttcttagcaa agcaaaggag gggaagcacg ggcagatagc 3240
accgtaccat acccttggaa actcgaaatg aacgaagcag gaaatgagag aatgagagtt 3300
ttgtaggtat atatagcggt agtgtttgcg cgttaccatc atcttctgga tctatctatt 3360
gttcttttcc tcatcacttt cccctttttc gctcttcttc ttgtctttta tttctttctt 3420
ttttttaatt gttccctcga ttggctatct accaaagaat ccaaacttaa tacacgtatt 3480
tatttgtcca attaccatgg ataagaaata ctcaataggc ttagatatcg gcacaaatag 3540
cgtcggatgg gcggtgatca ctgatgaata taaggttccg tctaaaaagt tcaaggttct 3600
gggaaataca gaccgccaca gtatcaaaaa aaatcttata ggggctcttt tatttgacag 3660
tggagagaca gcggaagcga ctcgtctcaa acggacagct cgtagaaggt atacacgtcg 3720
gaagaatcgt atttgttatc tacaggagat tttttcaaat gagatggcga aagtagatga 3780
tagtttcttt catcgacttg aagagtcttt tttggtggaa gaagacaaga agcatgaacg 3840
tcatcctatt tttggaaata tagtagatga agttgcttat catgagaaat atccaactat 3900
ctatcatctg cgaaaaaaat tggtagattc tactgataaa gcggatttgc gcttaatcta 3960
tttggcctta gcgcatatga ttaagtttcg tggtcatttt ttgattgagg gagatttaaa 4020
tcctgataat agtgatgtgg acaaactatt tatccagttg gtacaaacct acaatcaatt 4080
atttgaagaa aaccctatta acgcaagtgg agtagatgct aaagcgattc tttctgcacg 4140
attgagtaaa tcaagacgat tagaaaatct cattgctcag ctccccggtg agaagaaaaa 4200
tggcttattt gggaatctca ttgctttgtc attgggtttg acccctaatt ttaaatcaaa 4260
ttttgatttg gcagaagatg ctaaattaca gctttcaaaa gatacttacg atgatgattt 4320
agataattta ttggcgcaaa ttggagatca atatgctgat ttgtttttgg cagctaagaa 4380
tttatcagat gctattttac tttcagatat cctaagagta aatactgaaa taactaaggc 4440
tcccctatca gcttcaatga ttaaacgcta cgatgaacat catcaagact tgactctttt 4500
aaaagcttta gttcgacaac aacttccaga aaagtataaa gaaatctttt ttgatcaatc 4560
aaaaaacgga tatgcaggtt atattgatgg gggagctagc caagaagaat tttataaatt 4620
tatcaaacca attttagaaa aaatggatgg tactgaggaa ttattggtga aactaaatcg 4680
tgaagatttg ctgcgcaagc aacggacctt tgacaacggc tctattcccc atcaaattca 4740
cttgggtgag ctgcatgcta ttttgagaag acaagaagac ttttatccat ttttaaaaga 4800
caatcgtgag aagattgaaa aaatcttgac ttttcgaatt ccttattatg ttggtccatt 4860
ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag tctgaagaaa caattacccc 4920
atggaatttt gaagaagttg tcgataaagg tgcttcagct caatcattta ttgaacgcat 4980
gacaaacttt gataaaaatc ttccaaatga aaaagtacta ccaaaacata gtttgcttta 5040
tgagtatttt acggtttata acgaattgac aaaggtcaaa tatgttactg aaggaatgcg 5100
aaaaccagca tttctttcag gtgaacagaa gaaagccatt gttgatttac tcttcaaaac 5160
aaatcgaaaa gtaaccgtta agcaattaaa agaagattat ttcaaaaaaa tagaatgttt 5220
tgatagtgtt gaaatttcag gagttgaaga tagatttaat gcttcattag gtacctacca 5280
tgatttgcta aaaattatta aagataaaga ttttttggat aatgaagaaa atgaagatat 5340
cttagaggat attgttttaa cattgacctt atttgaagat agggagatga ttgaggaaag 5400
acttaaaaca tatgctcacc tctttgatga taaggtgatg aaacagctta aacgtcgccg 5460
ttatactggt tggggacgtt tgtctcgaaa attgattaat ggtattaggg ataagcaatc 5520
tggcaaaaca atattagatt ttttgaaatc agatggtttt gccaatcgca attttatgca 5580
gctgatccat gatgatagtt tgacatttaa agaagacatt caaaaagcac aagtgtctgg 5640
acaaggcgat agtttacatg aacatattgc aaatttagct ggtagccctg ctattaaaaa 5700
aggtatttta cagactgtaa aagttgttga tgaattggtc aaagtaatgg ggcggcataa 5760
gccagaaaat atcgttattg aaatggcacg tgaaaatcag acaactcaaa agggccagaa 5820
aaattcgcga gagcgtatga aacgaatcga agaaggtatc aaagaattag gaagtcagat 5880
tcttaaagag catcctgttg aaaatactca attgcaaaat gaaaagctct atctctatta 5940
tctccaaaat ggaagagaca tgtatgtgga ccaagaatta gatattaatc gtttaagtga 6000
ttatgatgtc gatcacattg ttccacaaag tttccttaaa gacgattcaa tagacaataa 6060
ggtcttaacg cgttctgata aaaatcgtgg taaatcggat aacgttccaa gtgaagaagt 6120
agtcaaaaag atgaaaaact attggagaca acttctaaac gccaagttaa tcactcaacg 6180
taagtttgat aatttaacga aagctgaacg tggaggtttg agtgaacttg ataaagctgg 6240
ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt 6300
ggatagtcgc atgaatacta aatacgatga aaatgataaa cttattcgag aggttaaagt 6360
gattacctta aaatctaaat tagtttctga cttccgaaaa gatttccaat tctataaagt 6420
acgtgagatt aacaattacc atcatgccca tgatgcgtat ctaaatgccg tcgttggaac 6480
tgctttgatt aagaaatatc caaaacttga atcggagttt gtctatggtg attataaagt 6540
ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa 6600
atatttcttt tactctaata tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg 6660
agagattcgc aaacgccctc taatcgaaac taatggggaa actggagaaa ttgtctggga 6720
taaagggcga gattttgcca cagtgcgcaa agtattgtcc atgccccaag tcaatattgt 6780
caagaaaaca gaagtacaga caggcggatt ctccaaggag tcaattttac caaaaagaaa 6840
ttcggacaag cttattgctc gtaaaaaaga ctgggatcca aaaaaatatg gtggttttga 6900
tagtccaacg gtagcttatt cagtcctagt ggttgctaag gtggaaaaag ggaaatcgaa 6960
gaagttaaaa tccgttaaag agttactagg gatcacaatt atggaaagaa gttcctttga 7020
aaaaaatccg attgactttt tagaagctaa aggatataag gaagttaaaa aagacttaat 7080
cattaaacta cctaaatata gtctttttga gttagaaaac ggtcgtaaac ggatgctggc 7140
tagtgccgga gaattacaaa aaggaaatga gctggctctg ccaagcaaat atgtgaattt 7200
tttatattta gctagtcatt atgaaaagtt gaagggtagt ccagaagata acgaacaaaa 7260
acaattgttt gtggagcagc ataagcatta tttagatgag attattgagc aaatcagtga 7320
attttctaag cgtgttattt tagcagatgc caatttagat aaagttctta gtgcatataa 7380
caaacataga gacaaaccaa tacgtgaaca agcagaaaat attattcatt tatttacgtt 7440
gacgaatctt ggagctcccg ctgcttttaa atattttgat acaacaattg atcgtaaacg 7500
atatacgtct acaaaagaag ttttagatgc cactcttatc catcaatcca tcactggtct 7560
ttatgaaaca cgcattgatt tgagtcagct aggaggtgac ggtggagggc caaaaaagaa 7620
aagaaaagtt gaagatgctt ctgctcatca tcaccatcac catcaccatt aaatccgctc 7680
taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag 7740
ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac 7800
gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga 7860
agtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 7920
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 7980
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 8040
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 8100
tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 8160
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 8220
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 8280
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 8340
actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 8400
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 8460
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 8520
tcttttctac cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc 8580
taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag 8640
cgctatttta ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag 8700
agcgctaatt tttcaaacaa agaatctgag ctgcattttt aca 8743
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ggatctgatt cagcacattg 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gaagtctatg ccatttagac 20
<210> 16
<211> 817
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ggatctgatt cagcacattg ataatagcat aggcattttt atctttcttt tttgagttca 60
tgaatgaatg taacttctcc aaaagatggg aatcacagtt tctcgaagaa aaatagattt 120
aatacaaata aaccgcgatt ccacaaacta aatgagcagg cgcagagtat aaatttacca 180
gaagaccgtg attcaattgt ttcaagtaat acaacgtcaa ttatgacaga tgatgcattt 240
gattacaatg agggcattgc atcgcgtacc aaaaatatta attctgatag tgatagaagc 300
aatgatacca taaaacaaaa caactacaat aaaagggaga ccggatataa ccctttctac 360
aatggatcag ggatcaatca gcgatataca cagtttcgaa aaagagagtt tgaaccaaca 420
cttgcagaaa acaaagccga agagtacata tcggacgaag ataatgtaaa aattgatgaa 480
gataatatag agaacgaact ccagtttacg ccgaaaatta aagaggctag tatactccga 540
tctagtttac taggacaaag aaatgtttta aatactcgga atccaaaatc aaaagaatca 600
cacattaaag taaaacccat catcaataac aagagctcct cacaaagaaa atctagtgca 660
gcacttcgga aacaattagg aaaaccccta ccgctgccgt atttgaatag ccctaatagt 720
gatagtacac ccacattaca gagaaaagaa gaagtattca cagacgaagt gcttcaaaaa 780
aagagagaat tgattgagtc taaatggcat agacttc 817
<210> 17
<211> 532
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ggatctgatt cagcacattg ataatagcat aggcattttt atctttcttt tttgagttca 60
tgaatgaatg taacttctcc aaaagatggg aatcacagtt tctcgaagaa aaatagattt 120
aatacaaata aaccgcgatt ccacaaacta aatgagcagg cgcagagtat aaatttacca 180
gaagaccgtg attcaattgt ttcaagtaat acaacgtcaa ttatgacaga tgatgcattt 240
gattacaatg agggcattgc atcgcgtacc aaaaatatta attctgatag tgatagaagc 300
aatgatacca taaaacaaaa caactacaat aaaagggaga ccggatataa ctcctcacaa 360
agaaaatcta gtgcagcact tcggaaacaa ttaggaaaac ccctaccgct gccgtatttg 420
aatagcccta atagtgatag tacacccaca ttacagagaa aagaagaagt attcacagac 480
gaagtgcttc aaaaaaagag agaattgatt gagtctaaat ggcatagact tc 532
<210> 18
<211> 2507
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ttgtattaga cgagggacgg agtgattttt gtgtttgttt ttattaattg tgggatagga 60
tagtagcaac tcttggagga gagcattgtc agttgtccag tctctgaagt taagtagtaa 120
gtttgcggag tcaaaggggg atggcttttg ccatttgtga gagttgtgcg gcagcatctt 180
attcaaatag agctgtattc tgaagacctc ttgtagaaca tcatccatac taaaaagtaa 240
atcgtcctgt cccattacga gctgtattag tgctgtgacc ctctgtatat ttacgttgcc 300
atgaagaagg taatgggcga tattttgata caattcctga gttgcatgtt ggattgagtt 360
tacgaagggt cgccagacgg ccagaaacct ccaggcggag ttaacaacta gtaatacggc 420
atccatgttt gcatcagcgc cgagcctata ccagtcactg agtagacgtt ttcttgctct 480
ttttatgtcc tgacttcttt tgacgagggg gcattctcta gagacacagg cagttgcttc 540
cagcaactgc cgtacggccg ttctcatgct gtcgaggatt ttttttggga cgatattgtc 600
attatagggc agtgtgtgac ttatgaattg ttgtagaagg acgtctgtga tgttggagat 660
atgtattttg ttaactcttc ttgagacgat ttggccctgg atagcgaagc gtgcggttac 720
aaataggtcg tcttgttcaa gaaggtaggc gaggacatta tctatcagta caaacatctt 780
agtagtgtct gaggagaggg ttgattgttt atgtattttt gcgaaatata tatatatata 840
ttctacacag atatatacat atttgttttt cgggctcatt ctttcttctt tgccagaggc 900
tcaccgctca agaggtccgc taattctgga gcgattgtta ttgttttttc ttttcttctt 960
ctattcgaaa cccagttttt gatttgaatg cgagataaac tggtattctt cattagattc 1020
tctaggccct tggtatctag atatgggttc tcgatgttct ttgcaaacca actttctagt 1080
attcggacat tttcttttgt aaaccggtgt cctctgtaag gtttagtact tttgtttatc 1140
atatcttgag ttaccacatt aaataccaac ccatccgccg atttattttt ctgtgtaagt 1200
tgataattac ttctatcgtt ttctatgctg cgcatttctt tgagtaatac agtaatggta 1260
gtagtgagtt gagatgttgt ttgcaacaac ttcttctcct catcactaat cttacggttt 1320
ttgttggccc tagataagaa tcctaatata tcccttaatt caacttcttc ttctgttgtt 1380
acactctctg gtaacttagg taaattacag caaatagaaa agagcttttt atttatgtct 1440
agtatgctgg atttaaactc atctgtgatt tgtggattta aaaggtcttt aatgggtatt 1500
ttattcattt tttcttgctt atcttccttt ttttcttgcc cacttctaag ctgatttcaa 1560
tctctccttt atatatattt ttaagttcca acattttatg tttcaaaaca ttaatgatgt 1620
ctgggttttg tttgggatgc aatttattgc ttcccaatgt agaaaagtac atcatatgaa 1680
acaacttaaa ctcttaacta cttcttttaa ccttcacttt ttatgaaatg tatcaaccat 1740
atataataac ttaatagacg acattcacaa tatgtttact tcgaagcctg ctttcaaaat 1800
taagaacaaa gcatccaaat catacagaaa cacagcggtt tcaaaaaagc tgaaagaaaa 1860
acgtctagct gagcatgtga ggccaagctg cttcaatatt attcgaccac tcaagaaaga 1920
tatccagatt cctgttcctt cctctcgatt tttaaataaa atccaaattc acaggatagc 1980
gtctggaagt caaaatactc agtttcgaca gttcaataag acatctataa aatcttcaaa 2040
gaaatattta aactcattta tggcttttag agcatattac tcacagtttg gctccggtgt 2100
aaaacaaaat gtcttgtctt ctctgctcgc tgaagaatgg cacgcggaca aaatgcagca 2160
cggaatatgg gactacttcg cgcaacagta taattttata aaccctggtt ttggttttgt 2220
agagtggttg acgaataatt atgctgaagt acgtggtgac ggatattggg aagatgtgtt 2280
tgtacatttg gccttataga gtgtggtcgt ggcggaggtt gtttatcttt cgagtactga 2340
atgttgtcag tatagctatc ctatttgaaa ctccccatcg tcttgctctt gttcccaatg 2400
tttgtttata cactcatatg gctataccct tatctacttg cctcttttgt ttatgtctat 2460
gtatttgtat aaaatatgat attactcaga ctcaagcaaa caatcaa 2507
<210> 19
<211> 2519
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gaattcttgt attagacgag ggacggagtg atttttgtgt ttgtttttat taattgtggg 60
ataggatagt agcaactctt ggaggagagc attgtcagtt gtccagtctc tgaagttaag 120
tagtaagttt gcggagtcaa agggggatgg cttttgccat ttgtgagagt tgtgcggcag 180
catcttattc aaatagagct gtattctgaa gacctcttgt agaacatcat ccatactaaa 240
aagtaaatcg tcctgtccca ttacgagctg tattagtgca gtgaccctct gtatatttac 300
gttgccatga agaaggtaat gggcgatatt ttgatacaat tcctgagttg catgttggat 360
tgagtttacg aagggtcgcc agacggccag aaacctccag gcggagttaa caactagtaa 420
tacggcatcc atgtttgcat cagcgccgag cctataccag tcactgagta gacgttttct 480
tgctcttttt atgtcctgac ttcttttgac gagggggcat tctctagaga cacaggcagt 540
tgcttccagc aactgccgta cggccgttct catgctgtcg aggatttttt ttgggacgat 600
attgtcatta tagggcagtg tgtgacttat gaattgttgt aggaggacgt ctgtgatgtt 660
ggagatatgt attttgttaa ctcttcttga gacgatttgg ccctggatag cgaagcgtgc 720
ggttacaaat aggtcgtctt gttcaagaag gtaggcgagg acattatcta tcagtacaaa 780
catcttagta gtgtctgagg agagggttga ttgtttatgt atttttgcga aatatatata 840
tatatattct acacagatat atacatattt gtttttcggg ctcattcttt cttctttgcc 900
agaggctcac cgctcaagag gtccgctaat tctggagcga ttgttattgt tttttctttt 960
cttcttctat tcgaaaccca gtttttgatt tgaatgcgag ataaactggt attcttcatt 1020
agattctcta ggcccttggt atctagatat gggttctcga tgttctttgc aaaccaactt 1080
tctagtattc ggacattttc ttttgtaaac cggtgtcctc tgtaaggttt agtacttttg 1140
tttatcatat cttgagttac cacattaaat accaacccat ccgccgattt atttttctgt 1200
gtaagttgat aattacttct atcgttttct atgctgcgca tttctttgag taatacagta 1260
atggtagtag tgagttgaga tgttgtttgc aacaacttct tctcctcatc actaatctta 1320
cggtttttgt tggccctaga taagaatcct aatatatccc ttaattcaac ttcttcttct 1380
gttgttacac tctctggtaa cttaggtaaa ttacagcaaa tagaaaagag ctttttattt 1440
atgtctagta tgctggattt aaactcatct gtgatttgtg gatttaaaag gtctttaatg 1500
ggtattttat tcattttttc ttgcttatct tccttttttt cttgcccact tctaagctga 1560
tttcaatctc tcctttatat atatttttaa gttccaacat tttatgtttc aaaacattaa 1620
tgatgtctgg gttttgtttg ggatgcaatt tattgcttcc caatgtagaa aagtacatca 1680
tatgaaacaa cttaaactct taactacttc ttctaacctt cactttttat gaaatgtatc 1740
aaccatatat aataacttaa tagaccacat tcacaatatg tttacttcga agcctgcttt 1800
caaaattaag aacaaagcat ccaaatcata cagaaacaca gcggtttcaa aaaagctgaa 1860
agaaaaacgt ctagctgagc atgtgaggcc aagctgcttc aatattattc gaccactcaa 1920
gaaagatatc cagattcctg taccttcctc tcgattttta aataagatcc aaattcacag 1980
gatagcgtct ggaactcaaa atactcagtt tcgacagttc aataagacat ctataaaatc 2040
ttcaaagaaa tatttaaact catttatggc ttttagagca tattactcac agtttggctc 2100
cggtgtaaaa caaaatgtct tgtcttctct gctcgctgaa gaatggcacg cggacaaaat 2160
gcagcacgga atatgggact acttcgcgca acagtataat tttataaacc ctggttttgg 2220
ttttgtagag tggttgacga ataattatgc tgaagtacgt ggtgacggat attgggaaga 2280
tgtgtttgta catttggcct tatagagtgt ggtcgtggcg gaggttgttt atctttcgag 2340
tactgaatgt tgtcagtata gctatcctat ttgaaactcc ccatcgtctt gctcttgttc 2400
ccaatgtttg tttatacact catatggcta tacccttatc tacttgcctc ttttgtttat 2460
gtctatgtat ttgtataaaa tatgatatta ctcagactca agcaaacaat caagaattc 2519
<210> 20
<211> 2640
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ctagtactta aaaaaactgt agtttcagtg caaaaaagtt ttaacattac gtatcttgta 60
ccctttttat tgcatataga aaggtcaaat aatccttcac atcatgaaat ataagctaaa 120
tcgcatttct tttcgtccac atttgcaaac aaaacttttc aataataatt ttataaatag 180
tatcaatata tatatatata tatatattta tttgtttact ttttctatca gtgttttcaa 240
ttttttatta aacaatgttt gattttttaa atcgcaattt aatacctaaa tataaaaaat 300
gttattatat tgcaaaaacc catcaacctt gaaaaaaagt agaaacgttt tatttaattc 360
tatcaataca tcataaaata cgaacgatcc ccgtccaagt tatgagctta atcttccata 420
aaaatatttg aaagcaatag atcatgtact aaactaaaat cagggaaatt aagactcctt 480
ttgaagtaat acctattact tactaatacg tttgagaata agcgcaggta ctcctggttt 540
ttgttaaaac tacaaattta tacttagcat tacgaagatt ctcgattccg aaaaacaaaa 600
attttatcgt catatacaaa tctagaaatt accagagcta tccatcttgt tcaagaaggt 660
aggcgaggac attatctatc agtacaaaca tcttagtagt gtctgaggag agggttgatt 720
gtttatgtat ttttgcgaaa tatatatata tatatattct acacagatat atacatattt 780
gtttttcggg ctcattcttt cttctttgcc agaggctcac cgctcaagag gtccgctaat 840
tctggagcga ttgttattgt tttttctttt cttcttctat tcgaaaccca gtttttgatt 900
tgaatgcgag ataaactggt attcttcatt agattctcta ggcccttggt atctagatat 960
gggttctcga tgttctttgc aaaccaactt tctagtattc ggacattttc ttttgtaaac 1020
cggtgtcctc tgtaaggttt agtacttttg tttatcatat cttgagttac cacattaaat 1080
accaacccat ccgccgattt atttttctgt gtaagttgat aattacttct atcgttttct 1140
atgctgcgca tttctttgag taatacagta atggtagtag tgagttgaga tgttgtttgc 1200
aacaacttct tctcctcatc actaatctta cggtttttgt tggccctaga taagaatcct 1260
aatatatccc ttaattcaac ttcttcttct gttgttacac tctctggtaa cttaggtaaa 1320
ttacagcaaa tagaaaagag ctttttattc ttgatttttg ttctttcggg gaaactgtat 1380
aaaacttcca aaaaggaaaa gtaaaacaat acatctcctt atatcaaaga aaatcaagaa 1440
ggacaacatg gatgatattt gtagtatggc ggaaaacata aacagaactc tgtttaacat 1500
tctaggtact gagattgatg aaatcaatct caatactaat aatctttata atgtatgttt 1560
tcatttcaag gatagccttt gaatcaattt actaacaata cttcagttta taatggaaag 1620
taatttgact aaagtagagc aacatacatt acacaaaaat atttctaaca ataggttaga 1680
aatataccac cacattaaaa aagagaagag cccaaaggga aaatcatcaa tatcacccca 1740
agcacgggca tttttagaac aggtttttag aagaaagcaa agccttaatt ccaaggaaaa 1800
agaagaagtt gcaaagaaat gtggcattac tccacttcaa gtaagagttt gggtatgtaa 1860
tatgagaatc aaacttaaat atatcctata ctaacaattt gtagttcata aataaacgta 1920
tgagatctaa ataaattcgt tttcaatgat taaaatagca tagtcgggtt tttcttttag 1980
tttcagcttt ccgcaacagt aaaattttat aaaccctggt tttggttttg tagagtggtt 2040
gacgaataat tatgctgaag tacgtggtga cggatattgg gaagatgtgt ttgtacattt 2100
ggccttatag agtgtggtcg tggcggaggt tgtttatctt tcgagtactg aatgttgtca 2160
gtatagctat cctatttgaa actccccatc gtcttgctct tgttctcaat gtttgtttat 2220
atactcatat ttctatgtgt ttatacaatt gctattgttt atataatgta gtgacatttt 2280
ctcttaatct tatactaatt tctatgacat ttatataaga agagacttat gatcaacata 2340
attttgcaaa ctttgagaga aatatgtctt tctactgcga taaagttatt atttagatta 2400
catgtcacca acattttcgt atatggcgat ataatttatc atgttttggt atgataattt 2460
aatttttaaa aaaacaaatt taattgacct cattaattaa tatttattaa tacctttaaa 2520
tgttgaggta aatagctatt ttctctcttc ttttccttta gttggaattt gcacaagaaa 2580
atgtttttcc acacacttta gcgttttttc ctaaatgttg gaataaaaaa caactatcat 2640
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
acaaaaatat ttctaacaat 20
<210> 22
<211> 8743
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg 60
ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt 120
tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt 180
taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc 240
cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc 300
atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata 360
gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat 420
atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt 480
tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag 540
gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat 600
agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc 660
aatattttag tagcccgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca 720
gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagag aataggaact 780
tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc 840
tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata 900
tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtatat gtgttatgta 960
gtatactctt tcttcaacaa ttaaatactc tcggtagcca agttggttta aggcgcaaga 1020
ctgtaattta tcactacgaa atcttgagat cgggcgttcg actcgccccc gggagagatg 1080
gccggcatgg tcccagcctc ctcgctggcg ccggctgggc aacaccttcg ggtggcgaat 1140
gggactttac aaaaatattt ctaacaatgt tttagagcta gaaatagcaa gttaaaataa 1200
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tattttttgt 1260
cactattgtt atgtaaaatg ccacctctga cagtatggaa cgcaaacttc tgtctagtgg 1320
atataggtct agagatctgt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac 1380
atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt 1440
gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat 1500
tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac ctgcgagcag 1560
ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat 1620
ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg 1680
ctaggataca gttctcacat cacatccgaa cataaacaac catgggtaag gaaaagactc 1740
acgtttcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg 1800
ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg 1860
cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga 1920
tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc 1980
gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggcaaaaca gcattccagg 2040
tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc 2100
gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc 2160
tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg 2220
agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt ttgccattct 2280
caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg 2340
ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc 2400
ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc 2460
aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg 2520
agtttttcta atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt 2580
atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt 2640
ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg 2700
cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca 2760
tccagtgtcg aaaacgagct ctcgagaacc cttaatagtc gaacaagaag caggcaaagt 2820
ttagagcact gcccctccgc actcaaaaaa gaaaaaacta ggaggaaaat aaaattctca 2880
accacacaaa cacataaaca catacaaata caaatacaag cttatttact tgacatcgcg 2940
cgatcttcca ctattcagcg ccgtccgccc tctctcgtgt tttttgttta cgcgacaact 3000
atgcgaaatc cggagcaacg ggcaaccgtt tggggaaaga ccacacccac gcgcgatcgc 3060
catggcaacg aggtcgcaca cgccccacac ccagacctcc ctgcgagcgg gcatgggtac 3120
aatgtccccg ttgccacaga caccacttcg tagcacagcg cagagcgtag cgtgttgttg 3180
ctgctgacaa aagaaaattt ttcttagcaa agcaaaggag gggaagcacg ggcagatagc 3240
accgtaccat acccttggaa actcgaaatg aacgaagcag gaaatgagag aatgagagtt 3300
ttgtaggtat atatagcggt agtgtttgcg cgttaccatc atcttctgga tctatctatt 3360
gttcttttcc tcatcacttt cccctttttc gctcttcttc ttgtctttta tttctttctt 3420
ttttttaatt gttccctcga ttggctatct accaaagaat ccaaacttaa tacacgtatt 3480
tatttgtcca attaccatgg ataagaaata ctcaataggc ttagatatcg gcacaaatag 3540
cgtcggatgg gcggtgatca ctgatgaata taaggttccg tctaaaaagt tcaaggttct 3600
gggaaataca gaccgccaca gtatcaaaaa aaatcttata ggggctcttt tatttgacag 3660
tggagagaca gcggaagcga ctcgtctcaa acggacagct cgtagaaggt atacacgtcg 3720
gaagaatcgt atttgttatc tacaggagat tttttcaaat gagatggcga aagtagatga 3780
tagtttcttt catcgacttg aagagtcttt tttggtggaa gaagacaaga agcatgaacg 3840
tcatcctatt tttggaaata tagtagatga agttgcttat catgagaaat atccaactat 3900
ctatcatctg cgaaaaaaat tggtagattc tactgataaa gcggatttgc gcttaatcta 3960
tttggcctta gcgcatatga ttaagtttcg tggtcatttt ttgattgagg gagatttaaa 4020
tcctgataat agtgatgtgg acaaactatt tatccagttg gtacaaacct acaatcaatt 4080
atttgaagaa aaccctatta acgcaagtgg agtagatgct aaagcgattc tttctgcacg 4140
attgagtaaa tcaagacgat tagaaaatct cattgctcag ctccccggtg agaagaaaaa 4200
tggcttattt gggaatctca ttgctttgtc attgggtttg acccctaatt ttaaatcaaa 4260
ttttgatttg gcagaagatg ctaaattaca gctttcaaaa gatacttacg atgatgattt 4320
agataattta ttggcgcaaa ttggagatca atatgctgat ttgtttttgg cagctaagaa 4380
tttatcagat gctattttac tttcagatat cctaagagta aatactgaaa taactaaggc 4440
tcccctatca gcttcaatga ttaaacgcta cgatgaacat catcaagact tgactctttt 4500
aaaagcttta gttcgacaac aacttccaga aaagtataaa gaaatctttt ttgatcaatc 4560
aaaaaacgga tatgcaggtt atattgatgg gggagctagc caagaagaat tttataaatt 4620
tatcaaacca attttagaaa aaatggatgg tactgaggaa ttattggtga aactaaatcg 4680
tgaagatttg ctgcgcaagc aacggacctt tgacaacggc tctattcccc atcaaattca 4740
cttgggtgag ctgcatgcta ttttgagaag acaagaagac ttttatccat ttttaaaaga 4800
caatcgtgag aagattgaaa aaatcttgac ttttcgaatt ccttattatg ttggtccatt 4860
ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag tctgaagaaa caattacccc 4920
atggaatttt gaagaagttg tcgataaagg tgcttcagct caatcattta ttgaacgcat 4980
gacaaacttt gataaaaatc ttccaaatga aaaagtacta ccaaaacata gtttgcttta 5040
tgagtatttt acggtttata acgaattgac aaaggtcaaa tatgttactg aaggaatgcg 5100
aaaaccagca tttctttcag gtgaacagaa gaaagccatt gttgatttac tcttcaaaac 5160
aaatcgaaaa gtaaccgtta agcaattaaa agaagattat ttcaaaaaaa tagaatgttt 5220
tgatagtgtt gaaatttcag gagttgaaga tagatttaat gcttcattag gtacctacca 5280
tgatttgcta aaaattatta aagataaaga ttttttggat aatgaagaaa atgaagatat 5340
cttagaggat attgttttaa cattgacctt atttgaagat agggagatga ttgaggaaag 5400
acttaaaaca tatgctcacc tctttgatga taaggtgatg aaacagctta aacgtcgccg 5460
ttatactggt tggggacgtt tgtctcgaaa attgattaat ggtattaggg ataagcaatc 5520
tggcaaaaca atattagatt ttttgaaatc agatggtttt gccaatcgca attttatgca 5580
gctgatccat gatgatagtt tgacatttaa agaagacatt caaaaagcac aagtgtctgg 5640
acaaggcgat agtttacatg aacatattgc aaatttagct ggtagccctg ctattaaaaa 5700
aggtatttta cagactgtaa aagttgttga tgaattggtc aaagtaatgg ggcggcataa 5760
gccagaaaat atcgttattg aaatggcacg tgaaaatcag acaactcaaa agggccagaa 5820
aaattcgcga gagcgtatga aacgaatcga agaaggtatc aaagaattag gaagtcagat 5880
tcttaaagag catcctgttg aaaatactca attgcaaaat gaaaagctct atctctatta 5940
tctccaaaat ggaagagaca tgtatgtgga ccaagaatta gatattaatc gtttaagtga 6000
ttatgatgtc gatcacattg ttccacaaag tttccttaaa gacgattcaa tagacaataa 6060
ggtcttaacg cgttctgata aaaatcgtgg taaatcggat aacgttccaa gtgaagaagt 6120
agtcaaaaag atgaaaaact attggagaca acttctaaac gccaagttaa tcactcaacg 6180
taagtttgat aatttaacga aagctgaacg tggaggtttg agtgaacttg ataaagctgg 6240
ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt 6300
ggatagtcgc atgaatacta aatacgatga aaatgataaa cttattcgag aggttaaagt 6360
gattacctta aaatctaaat tagtttctga cttccgaaaa gatttccaat tctataaagt 6420
acgtgagatt aacaattacc atcatgccca tgatgcgtat ctaaatgccg tcgttggaac 6480
tgctttgatt aagaaatatc caaaacttga atcggagttt gtctatggtg attataaagt 6540
ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa 6600
atatttcttt tactctaata tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg 6660
agagattcgc aaacgccctc taatcgaaac taatggggaa actggagaaa ttgtctggga 6720
taaagggcga gattttgcca cagtgcgcaa agtattgtcc atgccccaag tcaatattgt 6780
caagaaaaca gaagtacaga caggcggatt ctccaaggag tcaattttac caaaaagaaa 6840
ttcggacaag cttattgctc gtaaaaaaga ctgggatcca aaaaaatatg gtggttttga 6900
tagtccaacg gtagcttatt cagtcctagt ggttgctaag gtggaaaaag ggaaatcgaa 6960
gaagttaaaa tccgttaaag agttactagg gatcacaatt atggaaagaa gttcctttga 7020
aaaaaatccg attgactttt tagaagctaa aggatataag gaagttaaaa aagacttaat 7080
cattaaacta cctaaatata gtctttttga gttagaaaac ggtcgtaaac ggatgctggc 7140
tagtgccgga gaattacaaa aaggaaatga gctggctctg ccaagcaaat atgtgaattt 7200
tttatattta gctagtcatt atgaaaagtt gaagggtagt ccagaagata acgaacaaaa 7260
acaattgttt gtggagcagc ataagcatta tttagatgag attattgagc aaatcagtga 7320
attttctaag cgtgttattt tagcagatgc caatttagat aaagttctta gtgcatataa 7380
caaacataga gacaaaccaa tacgtgaaca agcagaaaat attattcatt tatttacgtt 7440
gacgaatctt ggagctcccg ctgcttttaa atattttgat acaacaattg atcgtaaacg 7500
atatacgtct acaaaagaag ttttagatgc cactcttatc catcaatcca tcactggtct 7560
ttatgaaaca cgcattgatt tgagtcagct aggaggtgac ggtggagggc caaaaaagaa 7620
aagaaaagtt gaagatgctt ctgctcatca tcaccatcac catcaccatt aaatccgctc 7680
taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag 7740
ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac 7800
gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga 7860
agtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 7920
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 7980
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 8040
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 8100
tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 8160
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 8220
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 8280
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 8340
actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 8400
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 8460
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 8520
tcttttctac cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc 8580
taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag 8640
cgctatttta ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag 8700
agcgctaatt tttcaaacaa agaatctgag ctgcattttt aca 8743
<210> 23
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gcacggaata tgggactact tcg 23
<210> 24
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
actccacttc aagtaagagt ttg 23
<210> 25
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
agtcacatca agatcgttta tgg 23
<210> 26
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gaattcttgt attagacgag ggacggagtg 30
<210> 27
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gaattcttga ttgtttgctt gagtctgag 29

Claims (2)

1.一种向酿酒酵母中导入线性质粒的方法，其特征在于，所述方法包括向源酿酒酵母GA-Y233菌株的线性质粒pGKL1中整合Trp1筛选标记基因，并使酵母交配时负责核融合的KAR1基因无法行使核融合功能，由此得到改造后的源酿酒酵母；经改造后的源酿酒酵母与目标酿酒酵母交配，使线性质粒pGKL1和pGKL2导入目标酿酒酵母菌株中;当源酿酒酵母与目标酿酒酵母的交配型相同时，所述方法还包括交配前，先转换改造后的源酿酒酵母的交配型;
所述源酿酒酵母为GA-Y233菌株，该菌株由酿酒酵母F102-2菌株经URA3基因和Trp1基因失活处理而得，所述酿酒酵母F102-2菌株来源于全球生物资源中心，保存目录号为200585；所述目标酿酒酵母为AH109菌株；
所述方法具体包括：
（1）在源酿酒酵母GA-Y233菌株的pGKL1质粒上整合Trp1筛选标记基因；
（2）用CRISPR-Cas9介导同源重组的方法敲掉KAR1基因编码区第289-573位之间的共285个碱基；
（3）克隆MATα基因序列，并用CRISPR-Cas9介导同源重组的方法将步骤（2）获得的改造后的源酿酒酵母GA-Y233的交配型修改为MATα；
（4）将步骤（3）获得的改造后的酿酒酵母GA-Y233菌株和目标酿酒酵母AH109交配后，在不含尿嘧啶和色氨酸的缺陷型培养基上培养，PCR筛选含有pGKL1和pGKL2质粒的AH109菌株,
所述源酿酒酵母中存在pGKL1和pGKL2两个线性质粒；
所述筛选标记基因为目标酵母菌株的缺陷基因；
所述目标酿酒酵母中不存在线性质粒pGKL1或pGKL2；
所述使酵母交配时负责核融合的KAR1基因无法行使核融合功能是指用CRISPR-Cas9介导同源重组的方法敲掉KAR1基因编码区第289-573位之间的共285个碱基，同源重组所用DNA序列如SEQ ID No.11所示；
所述转换改造后的源酿酒酵母的交配型是指当源酿酒酵母与目标酿酒酵母的交配型相同时，通过同源重组的方式将MATα基因序列导入改造后的源酿酒酵母菌株中，使改造后的源酿酒酵母的交配型变为MATα。
2.根据权利要求1所述的向酿酒酵母中导入线性质粒的方法，其特征在于，包括下述（a）-（g）：
（a）所述步骤（1）中整合的Trp1筛选标记基因编码序列如SEQ ID No.2所示；
（b）所述步骤（1）中检测所用引物对为F-P1-Trp和R-P1-Trp；
（c）所述步骤（2）中CRISPR-Cas9介导同源重组采用的靶位点为KAR1基因的编辑载体pCAS-KAR1的序列如SEQ ID No.13所示；
（d）所述步骤（2）中检测所用引物对为F-KAR1-JC和R-KAR1-JC；
（e）所述步骤（3）中CRISPR-Cas9介导同源重组采用的靶位点为MATa基因的编辑载体pCAS-MATa的序列如SEQ ID No.22所示；
（f）所述步骤（3）中检测所用引物为F-α-JC、F-a-JC和R-α/a-JC；
（g）所述步骤（4）中检测所用引物对为F-P1-Trp和R-P1-Trp、F-KAR1-JC和R-KAR1-JC、F-α-JC和R-α/a-JC、F-a-JC和R-α/a-JC；
其中，F-P1-Trp的序列如SEQ ID No.4所示，R-P1-Trp的序列如SEQ ID No.5所示，F-KAR1-JC的序列如SEQ ID No.14所示，R-KAR1-JC的序列如SEQ ID No.15所示，F-α-JC的序列如SEQ ID No.23所示，F-a-JC的序列如SEQ ID No.24所示，R-α/a-JC的序列如SEQ IDNo.25所示。
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