CN102071213B - 分泌型碱性磷酸酶示踪ta克隆真核表达载体及其构建方法 - Google Patents
分泌型碱性磷酸酶示踪ta克隆真核表达载体及其构建方法 Download PDFInfo
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Abstract
本发明公开了一种分泌型碱性磷酸酶示踪TA克隆真核表达载体,其是在真核表达启动子(CMV)下游和BGH多聚腺嘌呤转录终止子的上游插入碱性磷酸酶示踪TA克隆位点。本发明所述载体可利用培养基上清进行检测;可利用化学发光仪器进行检测,不仅灵敏度高而且可以根据标准碱性磷酸酶的对照进行定量;发明人把目的基因插入位点改造成TA克隆载体位点,因此可以把PCR产物直接插入到报告基因载体,既节省了时间又节省了科研成本。
Description
技术领域
本发明涉及一种真核表达载体,具体地说是一种分泌型碱性磷酸酶示踪TA克隆真核表达载体。
背景技术
真核基因在细胞内的临时表达对于研究基因在细胞内的功能具有重要意义,在当今大多数高水平的论著中多用真核表达质粒来实现,然而由于质粒转染真核细胞的效率与转染试剂、细胞类型、细胞状态等有很大的关系。其转染效率从低到小于1%到高达90%以上不等。而转染效率又直接影响到后续的研究结果。一般低于10%的转染效率就很难达到预期的效果,不仅浪费了材料而且导致研究失败。
目前现阶段转染效率很难估计,通常需要转染大量荧光蛋白基因用以分选转染细胞。同时由于在同一实验中不同的转染效率差异直接影响到结果的可比性,因此构建一个具有示踪作用的真核转染质粒具有重要意义。
分泌型碱性磷酸酶在科研上被广泛用于启动子研究的报告基因,是一个很好的真核转染示踪蛋白。目前被广泛应用的基因表达示踪蛋白为EGFP,可通过荧光显微镜直接观测转染效率,但不能进行定量分析。另外还有β-半乳糖苷酶基因,通过化学显色或ELISA进行半定量且操作复杂,还有荧光蛋白酶等。但所有上述现有技术都需要基于细胞或细胞裂解物进行研究。还有通过示踪基因载体和目的基因载体共转染的办法来评估细胞转染效率,而这种方法很不稳定。
发明内容
本发明目的在于提供一种分泌型碱性磷酸酶示踪TA克隆真核表达载体。
本发明的另一目的在于提供上述示踪载体的构建方法。
本发明的发明思路基于下述两方面的考虑。第一,在科学研究中,广泛使用真核表达载体进行瞬间转染来研究基因的功能,而载体的转染效率直接影响到研究的结果。好多真实的基因功能因为转染效率低或不一致而被掩盖,因此选择一个能清楚得知转染效率的示踪物非常重要。分泌型碱性磷酸酶作为示踪物正好能满足这一要求;第二,多数研究的基因是通过RT-PCR直接从组织细胞获得,而普通Taq酶具有3’末端腺嘌呤转移酶活性,因此PCR产物往往在其3’末端突出一个A,结果导致PCR产物不能直接插入平端质粒载体,而需要插入3’末端有T突出的载体即“T载体”。我们把分泌型碱性磷酸酶示踪真核表达载体构建成一个能把目的基因插入到由CMV启动子调控的TA克隆位点,从而构成一个分泌型碱性磷酸酶示踪TA克隆真核表达载体。为实现这一目标,我们将含双AhdI酶切位点的寡核苷酸插入到CMV启动子的下游,从而构建了一个分泌型碱性磷酸酶示踪的T载体。该TA克隆位点直接位于真核表达启动子(CMV)的下游和BGH多聚腺嘌呤转录终止子的上游,因此当质粒被转入细胞后,CMV启动子可直接启动插入PCR序列全长的转录而实现目的基因的表达。
为了达成本发明上述目的,本发明采用如下具体技术方案:
本发明提供了一种分泌型碱性磷酸酶示踪TA克隆真核表达载体,所述载体是在真核表达启动子下游和BGH多聚腺嘌呤转录终止子的上游插入碱性磷酸酶示踪TA克隆位点。
本发明所述载体的碱基序列如SEQ ID No.10所示。
本发明分泌型碱性磷酸酶示踪TA克隆真核表达载体是利用下述方法构建的,步骤为:
1、原核TA克隆载体:把TA位点引入pUC19载体构建成pUC19-TA载体;
所述步骤具体为:
1.1首先合成两条含AhdI酶切位点的寡核苷酸,退火形成双链DNA;
TA-oligo-s:
5’P-AATTCACTAGTGGACATTATGTCTCGAGACATAATGTCGG-3’;
TA-oligo-as:
5’P-AATTCCGACATTATGTCTCGAGACATAATGTCCACTAGTG-3’
EcoRI酶切pUC19质粒,与上述DNA双链连接形成前T载体;序列如SEQ IDNo.1所示;
1.2制备间隔DNA
引物:aspp2-xhoI-s:CCATCTCGAGCCCATCATACCCATCCAAG
aspp2-xhoI-as:TTCCTCGAGCGATACGCTCTGAGCCAG
以本实验室构建的pcDNA4-ASPP2模板扩增,pcDNA4-ASPP2模板序列如SEQ ID No.11所示,扩增片段中不含常用的限制性酶切位点,扩增序列如SEQ IDNo.2所示;
1.3XhoI酶切步骤1.2制备的间隔DNA后,插入到前T载体中;
1.4设计突变引物AhdI-mut-s:TGCCTGACTCCCCGTGGTGTAGAT,AhdI-mut-as:TCGTAGTTATCTACACCACGGGGAGT把ampicilin抗性基因中的AhdI酶切位点GACTCCCCGTC突变为GACTCCCCGTG,形成pUC19-TA前T载体,序列如SEQ ID No.3所示。
1.5AhdI酶切pUC19-TA前T载体,回收大片段,即为pUC19-TA载体,第89碱基处为开放性T末端,序列如SEQ ID No.4所示。
2、把pUC19-TA载体的TA序列克隆入pcDNA4 His载体中得到pcDNA5-TA克隆载体;
所述步骤具体为:
2.1以pUC19-TA前T载体为模板,扩增片段如SEQ ID No.5所示,引物为:
TA-HindIII-S:GCCGCGGGAAGCTtATATCACTAGTGAATTC
TA-XbaI-AS:GGTCGACCTCTAGACGGCCGCGAATTCCGAC
2.2HindIII和XbaI双酶切SEQ ID No.5,定向连接到pcDNA4 His vector上,用引物
AhdI-mut-s:TGCCTGACTCCCCGTGGTGTAGAT,
AhdI-mut-as:TCGTAGTTATCTACACCACGGGGAGT
把ampicilin抗性基因中的AhdI酶切位点GACTCCCCGTC突变为GACTCCCCGTG,形成pcDNA5-TA前T载体,如SEQ ID No.6所示;
2.3AhdI酶切上述pcDNA5-TA前T载体,回收大片段即为pcDNA5-TA载体,第946碱基处为开放性T末端,如SEQ ID No.7所示。
3、把pcDNA5-TA克隆载体上的CMV-TA-poly A序列经PCR得到后克隆到pSEAP2-control载体中构建成具有真核表达功能并外分泌碱性磷酸酶(AP)的TA载体pcDNA5AP-TA。
3.1以pcDNA5-TA前T载体为模板,扩增片段,如SEQ ID No.8所示,引物为:
CMV-NotI:GCATGAAGCGGCCGCTTAGGGTTAGGCGTT
polyA-NotI:AGCTGGTTGCGGCCGCCTCAGAAGCCAT
3.2NotI酶切后,插入到pSEAP2-control载体上,测序验证反向插入的为正确克降,引物为
AhdI-mut-s:TGCCTGACTCCCCGTGGTGTAGAT
AhdI-mut-as:TCGTAGTTATCTACACCACGGGGAGT
3.3把ampicilin抗性基因中的AhdI酶切位点GACTCCCCGTC突变为GACTCCCCGTG,形成pcDNA5AP-TA前T载体,如SEQ ID No.9所示;
3.4AhdI酶切回收大片段,即为pcDNA5AP-TA载体,第5245碱基处为开放性T末端,如SEQ ID No.10所示。
本发明可广泛用于基因功能的研究,使研究易于操作,结果更可靠,同时可潜在用于药物作用基因筛查的功能。
本发明的优点及有益效果:
(1)本发明提供的是一个分泌型碱性磷酸酶示踪载体,可利用培养基上清进行检测;
(2)可利用化学发光仪器进行检测,不仅灵敏度高而且可以根据标准碱性磷酸酶的对照进行定量;
(3)发明人把目的基因插入位点改造成TA克隆载体位点,因此可以把PCR产物直接插入到报告基因载体,既节省了时间又节省了科研成本。
附图说明
图1为将两条含AhdI酶切位点的寡核苷酸退火形成的双链DNA酶切示意图;
图2为puc19-TA载体结构图;
图3为pcDNA5-TA载体结构图;
图4为pcDNA5AP-TA载体结构图;
图5为EGFP绿色荧光水平与SEAP-表达水平。
具体实施方式
实施例1pcDNA5AP-TA质粒的构建
pcDNA5AP-TA载体的构建步骤主要产生一个3’末端突出“T”的线性载体。传统的方法是在平末端的双链DNA 3’-末端通过DNA末端转移酶加入“T”。为了降低成本和便于操作,我们利用AhdI切割GACNNN/NNGTC的特点,引入正向重复序列,通过AhdI切割产生3’末端突出“T”的线性载体。但是由于在载体的氨苄抗性筛选基因的下游存在一个AhdI切点,我们首先通过DNA点突变的方法在不改变氨基酸编码的情况下突变掉AhdI切割位点。
具体步骤如下:
1、原核TA克隆载体:把TA位点引入pUC19载体构建成pUC19-TA载体;
1.1首先合成两条含AhdI酶切位点的寡核苷酸,退火形成双链DNA;
TA-oligo-s:5’-P-AATTCACTAGTGGACATT/ATGTCTCGAGACATA/ATGTCGG-3’
TA-oligo-as:5’P-AATTCCGACATTATGTCTCGAGACATAATGTCCACTAGTG-3’
EcoRI酶切pUC19质粒,与上述DNA双链连接形成前T载体;序列如SEQ IDNo.1所示;如图1所示。
1.2制备间隔DNA
引物:aspp2-xhoI-s:CCATCTCGAGCCCATCATACCCATCCAAG
aspp2-xhoI-as:TTCCTCGAGCGATACGCTCTGAGCCAG
以本实验室构建的pcDNA4-ASPP2(SEQ ID No.11所示)为模板扩增,扩增片段中不含常用的限制性酶切位点(BamH1,EcoRI,HindIII,NotI,XhoI,XbaI,ApaI等),扩增序列如SEQ ID No.2所示;反应条件:94℃15秒,55℃30秒,72℃30秒扩增30个循环。
1.3XhoI酶切步骤1.2制备的间隔DNA后,插入到前T载体的XhoI位点,该位点位于两个AhdI切点的中间(CTCGAG);
1.4设计突变引物AhdI-mut-s:TGCCTGACTCCCCGTGGTGTAGAT,AhdI-mut-as:TCGTAGTTATCTACACCACGGGGAGT把ampicilin抗性基因中的AhdI酶切位点GACTCCCCGTC突变为GACTCCCCGTG,形成pUC19-TA前T载体,序列如SEQ ID No.3所示。
1.5AhdI酶切pUC19-TA前T载体,回收大片段,即为pUC19-TA载体,第89碱基处为开放性T末端,序列如SEQ ID No.4所示。如图2所示。
2、把pUC19-TA载体的TA序列克隆入pcDNA4 His载体中得到pcDNA5-TA克隆载体;
2.1以pUC19-TA前T载体(SEQ ID No.3)为模板,扩增片段如SEQ ID No.5所示,引物为:
TA-HindIII-S:GCCGCGGGAAGCTtATATCACTAGTGAATTC
TA-XbaI-AS:GGTCGACCTCTAGACGGCCGCGAATTCCGAC
2.2HindIII和XbaI双酶切SEQ ID No.5,定向连接到pcDNA4 His vector上,用引物
AhdI-mut-s:TGCCTGACTCCCCGTGGTGTAGAT
AhdI-mut-as:TCGTAGTTATCTACACCACGGGGAGT
把ampicilin抗性基因中的AhdI酶切位点GACTCCCCGTC突变为GACTCCCCGTG,形成pcDNA5-TA前T载体,如SEQ ID No.6所示;
2.3AhdI酶切上述pcDNA5-TA前T载体(SEQ ID No.6),回收大片段即为pcDNA5-TA载体,第946碱基处为开放性T末端,如SEQ ID No.7所示,结构如图3所示。
3、把pcDNA5-TA前T载体(SEQ ID No.6)上的CMV-TA-poly A序列经PCR(94℃15秒,55℃30秒,72℃1分30秒扩增30个循环)得到后克隆到pSEAP载体中构建成具有真核表达功能并外分泌碱性磷酸酶(AP)的TA载体pcDNA5AP-TA,具体步骤:
3.1以pcDNA5-TA前T载体为模板,扩增片段,如SEQ ID No.8所示,引物为:
CMV-NotI:GCATGAAGCGGCCGCTTAGGGTTAGGCGTT
polyA-NotI:AGCTGGTTGCGGCCGCCTCAGAAGCCAT
3.2NotI酶切后,插入到pSEAP2-control载体上,测序验证反向插入的为正确克隆,引物为
AhdI-mut-s:TGCCTGACTCCCCGTGGTGTAGAT
AhdI-mut-as:TCGTAGTTATCTACACCACGGGGAGT
3.3把ampicilin抗性基因中的AhdI酶切位点GACTCCCCGTC突变为GACTCCCCGTG,形成pcDNA5AP-TA前T载体,如SEQ ID No.9所示;
3.4AhdI酶切回收大片段,即为pcDNA5AP-TA载体,第5245碱基处为开放性T末端,如SEQ ID No.10所示,结构如图4所示。
实施例2载体应用验证
1、所用引物:EGFP-s:TAGTGGAAGCTTGACCATGGTGAGCAAGGGCGAG,
EGFP-as:GCTTGGGATCCAATTCTTACTTGTACAGCTCG
2、方法:应用rTaq酶,以pCMS-EGFP载体(clontech)为模板扩增EGFP编码片段。应用实施例1构建的pcDNA5AP-TA载体实现TA克隆连接,测序验证方向。转染293T细胞后,荧光倒置显微镜观察绿色荧光,可见光分析仪检测碱性磷酸酶分泌水平,发现两者水平呈正相关。
3、结果:如图5所示,图5为EGFP绿色荧光水平与SEAP-表达水平。从结果看出上清的SEAP化学发光水平与目的基因EGFP(细胞显绿色荧光)呈显著正相关。当DNA转染浓度为800ng时,60%的细胞有绿色荧光,同时SEAP的化学发光值较阴性对照升高了近24倍(5932288/250395)。
序列表
<110>首都医科大学附属北京佑安医院
<120>分泌型碱性磷酸酶示踪的TA克隆真核表达载体及其构建方法
<130>
<160>11
<170>PatentIn version 3.5
<210>1
<211>3055
<212>DNA
<213>人工序列
<400>1
gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 60
atcactagtg aattcactag tggacattat gtctcgagac ataatgtcgg aattcgcggc 120
cgcctgcagg tcgaccatat gggagagctc ccaacgcgtt ggatgcatag cttgagtatt 180
ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 240
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 300
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 360
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 420
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 480
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 540
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 600
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 660
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 720
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 780
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 840
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 900
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 960
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 1020
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 1080
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 1140
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 1200
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 1260
cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 1320
taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 1380
caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 1440
gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 1500
gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 1560
ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 1620
attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 1680
gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 1740
tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 1800
agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 1860
gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 1920
actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 1980
tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 2040
attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 2100
tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 2160
tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 2220
aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 2280
tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 2340
cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag 2400
gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat 2460
ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa 2520
tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta 2580
ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca 2640
ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat 2700
cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg 2760
agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc 2820
acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt 2880
cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac 2940
gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt 3000
cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca ctata 3055
<210>2
<211>469
<212>DNA
<213>人工序列
<400>2
ccatcctcga gcccatcata cccatccaag tcagcttctg tgactgccag ctcagaaagc 60
ccagtagaaa tccagaatcc atatttacat gtggagcccg aaaaggaggt ggtctctctg 120
gttcctgaat cattgtcccc agaggatgtg gggaatgcca gtacagagaa cagtgacatg 180
ccagctcctt ctccaggcct tgattatgag cctgagggag tcccagacaa cagcccaaat 240
ctccagaata acccagaaga accaaatcca gaggctccac atgtgctaga tgtgtacctg 300
gaggagtacc ctccataccc acccccacca tacccatctg gggagcctga agggcccgga 360
gaagactcgg tgagcatgcg cccgcctgaa atcaccgggc aggtctctct gcctcctggt 420
aaaaggacaa acttgcgtaa aactggctca gagcgtatcg ctcgaggaa 469
<210>3
<211>3510
<212>DNA
<213>人工序列
<400>3
gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 60
atcactagtg aattcactag tggacattat gtctcgagcc catcataccc atccaagtca 120
gcttctgtga ctgccagctc agaaagccca gtagaaatcc agaatccata tttacatgtg 180
gagcccgaaa aggaggtggt ctctctggtt cctgaatcat tgtccccaga ggatgtgggg 240
aatgccagta cagagaacag tgacatgcca gctccttctc caggccttga ttatgagcct 300
gagggagtcc cagacaacag cccaaatctc cagaataacc cagaagaacc aaatccagag 360
gctccacatg tgctagatgt gtacctggag gagtaccctc catacccacc cccaccatac 420
ccatctgggg agcctgaagg gcccggagaa gactcggtga gcatgcgccc gcctgaaatc 480
accgggcagg tctctctgcc tcctggtaaa aggacaaact tgcgtaaaac tggctcagag 540
cgtatcgctc gagacataat gtcggaattc gcggccgcct gcaggtcgac catatgggag 600
agctcccaac gcgttggatg catagcttga gtattctata gtgtcaccta aatagcttgg 660
cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 720
acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 780
cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 840
attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 900
cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 960
caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 1020
caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 1080
ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1140
cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 1200
ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 1260
tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 1320
gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 1380
ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 1440
ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 1500
gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 1560
aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 1620
tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 1680
ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 1740
tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 1800
aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 1860
tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtg gtgtagataa 1920
ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 1980
gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 2040
gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 2100
taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 2160
tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 2220
ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 2280
tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 2340
ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 2400
tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 2460
ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 2520
aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 2580
actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 2640
aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 2700
tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 2760
aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 2820
ctgatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg 2880
taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 2940
accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt 3000
tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 3060
aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa 3120
gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat 3180
ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag 3240
gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg 3300
ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg 3360
ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc 3420
tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 3480
ggccagtgaa ttgtaatacg actcactata 3510
<210>4
<211>3041
<212>DNA
<213>人工序列
<400>4
gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 60
atcactagtg aattcactag tggacattaa tgtcggaatt cgcggccgcc tgcaggtcga 120
ccatatggga gagctcccaa cgcgttggat gcatagcttg agtattctat agtgtcacct 180
aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 240
aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 300
gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 360
gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 420
ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 480
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 540
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 600
gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 660
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 720
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 780
aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 840
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 900
taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 960
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 1020
gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 1080
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 1140
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 1200
tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 1260
ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 1320
taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 1380
tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 1440
ggtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 1500
gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 1560
cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 1620
ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 1680
aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 1740
atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 1800
tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 1860
gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 1920
aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 1980
acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 2040
ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 2100
tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 2160
aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 2220
catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 2280
atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 2340
aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 2400
tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 2460
ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 2520
cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 2580
ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 2640
accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 2700
gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 2760
gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 2820
caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg 2880
cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 2940
gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 3000
tgtaaaacga cggccagtga attgtaatac gactcactat a 3041
<210>5
<211>547
<212>DNA
<213>人工序列
<400>5
gccgcgggaa gcttatatca ctagtgaatt cactagtgga cattatgtct cgagcccatc 60
atacccatcc aagtcagctt ctgtgactgc cagctcagaa agcccagtag aaatccagaa 120
tccatattta catgtggagc ccgaaaagga ggtggtctct ctggttcctg aatcattgtc 180
cccagaggat gtggggaatg ccagtacaga gaacagtgac atgccagctc cttctccagg 240
ccttgattat gagcctgagg gagtcccaga caacagccca aatctccaga ataacccaga 300
agaaccaaat ccagaggctc cacatgtgct agatgtgtac ctggaggagt accctccata 360
cccaccccca ccatacccat ctggggagcc tgaagggccc ggagaagact cggtgagcat 420
gcgcccgcct gaaatcaccg ggcaggtctc tctgcctcct ggtaaaagga caaacttgcg 480
taaaactggc tcagagcgta tcgctcgaga cataatgtcg gaattcgcgg ccgtctagag 540
gtcgacc 547
<210>6
<211>5459
<212>DNA
<213>人工序列
<400>6
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttatat cactagtgaa ttcactagtg gacattatgt ctcgagccca 960
tcatacccat ccaagtcagc ttctgtgact gccagctcag aaagcccagt agaaatccag 1020
aatccatatt tacatgtgga gcccgaaaag gaggtggtct ctctggttcc tgaatcattg 1080
tccccagagg atgtggggaa tgccagtaca gagaacagtg acatgccagc tccttctcca 1140
ggccttgatt atgagcctga gggagtccca gacaacagcc caaatctcca gaataaccca 1200
gaagaaccaa atccagaggc tccacatgtg ctagatgtgt acctggagga gtaccctcca 1260
tacccacccc caccataccc atctggggag cctgaagggc ccggagaaga ctcggtgagc 1320
atgcgcccgc ctgaaatcac cgggcaggtc tctctgcctc ctggtaaaag gacaaacttg 1380
cgtaaaactg gctcagagcg tatcgctcga gacataatgt cggaattcgc ggccgtctag 1440
agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 1500
tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 1560
ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 1620
tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 1680
tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc 1740
ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 1800
cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 1860
cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 1920
tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 1980
gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 2040
tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 2100
ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 2160
taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc 2220
ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag 2280
tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 2340
atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 2400
ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct 2460
gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc 2520
ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg 2580
catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga 2640
ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg 2700
accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg 2760
acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct 2820
gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga 2880
acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg 2940
agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact 3000
gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa 3060
tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct 3120
tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3180
caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3240
tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat 3300
ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 3360
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 3420
cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 3480
tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 3540
ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 3600
taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 3660
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 3720
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 3780
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 3840
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 3900
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 3960
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4020
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4080
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 4140
gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4200
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 4260
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 4320
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 4380
ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 4440
atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 4500
tctgtctatt tcgttcatcc atagttgcct gactccccgt ggtgtagata actacgatac 4560
gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 4620
ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 4680
caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 4740
cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 4800
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 4860
cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 4920
agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 4980
tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 5040
agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 5100
atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 5160
ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 5220
cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 5280
caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 5340
attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 5400
agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc 5459
<210>7
<211>4990
<212>DNA
<213>人工序列
<400>7
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttatat cactagtgaa ttcactagtg gacattaatg tcggaattcg 960
cggccgtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc 1020
cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 1080
actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 1140
attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 1200
catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct 1260
agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 1320
cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 1380
tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 1440
gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 1500
tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 1560
ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 1620
tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 1680
taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt 1740
ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 1800
ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 1860
agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 1920
ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg 1980
cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 2040
gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa 2100
ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat 2160
ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga 2220
gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt 2280
ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa 2340
caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt 2400
cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc 2460
gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga 2520
ggagcaggac tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt 2580
gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat 2640
gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag 2700
caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 2760
gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt 2820
ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 2880
caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 2940
cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 3000
gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 3060
ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 3120
ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 3180
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 3240
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 3300
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 3360
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 3420
gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 3480
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 3540
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 3600
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 3660
cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 3720
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 3780
tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 3840
ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 3900
attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 3960
ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 4020
tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 4080
aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 4140
acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 4200
aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 4260
agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 4320
ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 4380
agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 4440
tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 4500
tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 4560
attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 4620
taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 4680
aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 4740
caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 4800
gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 4860
cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 4920
tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 4980
acctgacgtc 4990
<210>8
<211>1556
<212>DNA
<213>人工序列
<400>8
gcatgaagcg gccgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat 60
atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 120
ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 180
gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 240
caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 300
cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 360
ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 420
tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 480
gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 540
gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 600
tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 660
taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga 720
cccaagctgg ctagcgttta aacttaagct tatatcacta gtgaattcac tagtggacat 780
tatgtctcga gcccatcata cccatccaag tcagcttctg tgactgccag ctcagaaagc 840
ccagtagaaa tccagaatcc atatttacat gtggagcccg aaaaggaggt ggtctctctg 900
gttcctgaat cattgtcccc agaggatgtg gggaatgcca gtacagagaa cagtgacatg 960
ccagctcctt ctccaggcct tgattatgag cctgagggag tcccagacaa cagcccaaat 1020
ctccagaata acccagaaga accaaatcca gaggctccac atgtgctaga tgtgtacctg 1080
gaggagtacc ctccataccc acccccacca tacccatctg gggagcctga agggcccgga 1140
gaagactcgg tgagcatgcg cccgcctgaa atcaccgggc aggtctctct gcctcctggt 1200
aaaaggacaa acttgcgtaa aactggctca gagcgtatcg ctcgagacat aatgtcggaa 1260
ttcgcggccg tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag 1320
ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 1380
tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 1440
ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 1500
caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggccgcaa ccagct 1556
<210>9
<211>6648
<212>DNA
<213>人工序列
<400>9
ggtaccgagc tcttacgcgt gctagcccgg gctcgagatc tgcgatctgc atctcaatta 60
gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 120
cgcccattct ccgccccatc gctgactaat tttttttatt tatgcagagg ccgaggccgc 180
ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 240
caaaaagctt cgaatcgcga attcgcccac catgctgctg ctgctgctgc tgctgggcct 300
gaggctacag ctctccctgg gcatcatccc agttgaggag gagaacccgg acttctggaa 360
ccgcgaggca gccgaggccc tgggtgccgc caagaagctg cagcctgcac agacagccgc 420
caagaacctc atcatcttcc tgggcgatgg gatgggggtg tctacggtga cagctgccag 480
gatcctaaaa gggcagaaga aggacaaact ggggcctgag atacccctgg ccatggaccg 540
cttcccatat gtggctctgt ccaagacata caatgtagac aaacatgtgc cagacagtgg 600
agccacagcc acggcctacc tgtgcggggt caagggcaac ttccagacca ttggcttgag 660
tgcagccgcc cgctttaacc agtgcaacac gacacgcggc aacgaggtca tctccgtgat 720
gaatcgggcc aagaaagcag ggaagtcagt gggagtggta accaccacac gagtgcagca 780
cgcctcgcca gccggcacct acgcccacac ggtgaaccgc aactggtact cggacgccga 840
cgtgcctgcc tcggcccgcc aggaggggtg ccaggacatc gctacgcagc tcatctccaa 900
catggacatt gacgtgatcc taggtggagg ccgaaagtac atgtttcgca tgggaacccc 960
agaccctgag tacccagatg actacagcca aggtgggacc aggctggacg ggaagaatct 1020
ggtgcaggaa tggctggcga agcgccaggg tgcccggtat gtgtggaacc gcactgagct 1080
catgcaggct tccctggacc cgtctgtgac ccatctcatg ggtctctttg agcctggaga 1140
catgaaatac gagatccacc gagactccac actggacccc tccctgatgg agatgacaga 1200
ggctgccctg cgcctgctga gcaggaaccc ccgcggcttc ttcctcttcg tggagggtgg 1260
tcgcatcgac catggtcatc atgaaagcag ggcttaccgg gcactgactg agacgatcat 1320
gttcgacgac gccattgaga gggcgggcca gctcaccagc gaggaggaca cgctgagcct 1380
cgtcactgcc gaccactccc acgtcttctc cttcggaggc taccccctgc gagggagctc 1440
catcttcggg ctggcccctg gcaaggcccg ggacaggaag gcctacacgg tcctcctata 1500
cggaaacggt ccaggctatg tgctcaagga cggcgcccgg ccggatgtta ccgagagcga 1560
gagcgggagc cccgagtatc ggcagcagtc agcagtgccc ctggacgaag agacccacgc 1620
aggcgaggac gtggcggtgt tcgcgcgcgg cccgcaggcg cacctggttc acggcgtgca 1680
ggagcagacc ttcatagcgc acgtcatggc cttcgccgcc tgcctggagc cctacaccgc 1740
ctgcgacctg gcgccccccg ccggcaccac cgacgccgcg cacccgggtt actctagagt 1800
cggggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc 1860
acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 1920
tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 1980
tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt 2040
ggtaaaatcg ataaggatct gaacgatgga gcggagaatg ggcggaactg ggcggagtta 2100
ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc 2160
tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat 2220
tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta 2280
actgacacac attccacagc ggatccgtcg accgatgccc ttgagagcct tcaacccagt 2340
cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt 2400
tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc cgcttcctcg ctcactgact 2460
cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 2520
ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 2580
aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 2640
acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 2700
gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 2760
ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 2820
gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 2880
cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 2940
taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 3000
atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 3060
cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 3120
cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 3180
ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 3240
ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 3300
tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 3360
aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 3420
tatttcgttc atccatagtt gcctgactcc ccgtggtgta gataactacg atacgggagg 3480
gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 3540
atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 3600
tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 3660
ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 3720
ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 3780
tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 3840
ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 3900
ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 3960
tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 4020
gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 4080
taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 4140
cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 4200
agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 4260
gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 4320
ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta 4380
gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 4440
gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 4500
ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 4560
acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 4620
agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 4680
aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 4740
cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 4800
acaaaatatt aacgtttaca atttcccatt cgccattcag gctgcgcaac tgttgggaag 4860
ggcgatcggt gcgggcctct tcgctattac gccagcccaa gctaccatga taagtaagta 4920
atattaaggt acgggaggta cttggagcgg ccgcctcaga agccatagag cccaccgcat 4980
ccccagcatg cctgctattg tcttcccaat cctccccctt gctgtcctgc cccaccccac 5040
cccccagaat agaatgacac ctactcagac aatgcgatgc aatttcctca ttttattagg 5100
aaaggacagt gggagtggca ccttccaggg tcaaggaagg cacgggggag gggcaaacaa 5160
cagatggctg gcaactagaa ggcacagtcg aggctgatca gcgggtttaa acgggccctc 5220
tagacggccg cgaattccga cattatgtct cgagcgatac gctctgagcc agttttacgc 5280
aagtttgtcc ttttaccagg aggcagagag acctgcccgg tgatttcagg cgggcgcatg 5340
ctcaccgagt cttctccggg cccttcaggc tccccagatg ggtatggtgg gggtgggtat 5400
ggagggtact cctccaggta cacatctagc acatgtggag cctctggatt tggttcttct 5460
gggttattct ggagatttgg gctgttgtct gggactccct caggctcata atcaaggcct 5520
ggagaaggag ctggcatgtc actgttctct gtactggcat tccccacatc ctctggggac 5580
aatgattcag gaaccagaga gaccacctcc ttttcgggct ccacatgtaa atatggattc 5640
tggatttcta ctgggctttc tgagctggca gtcacagaag ctgacttgga tgggtatgat 5700
gggctcgaga cataatgtcc actagtgaat tcactagtga tataagctta agtttaaacg 5760
ctagccagct tgggtctccc tatagtgagt cgtattaatt tcgataagcc agtaagcagt 5820
gggttctcta gttagccaga gagctctgct tatatagacc tcccaccgta cacgcctacc 5880
gcccatttgc gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg 5940
gtgccaaaac aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc 6000
aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg taatagcgat 6060
gactaatacg tagatgtact gccaagtagg aaagtcccat aaggtcatgt actgggcata 6120
atgccaggcg ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata 6180
cacttgatgt actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg 6240
gaaagtccct attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg 6300
gtcgttgggc ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat 6360
atatgggcta tgaactaatg accccgtaat tgattactat taataactag tcaataatca 6420
atgtcaacgc gtatatctgg cccgtacatc gcgaagcagc gcaaaacgcc taaccctaag 6480
cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgaatc 6540
gatagtacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac tagcaaaata 6600
ggctgtcccc agtgcaagtg caggtgccag aacatttctc tatcgata 6648
<210>10
<211>4990
<212>DNA
<213>人工序列
<400>10
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttatat cactagtgaa ttcactagtg gacattaatg tcggaattcg 960
cggccgtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc 1020
cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 1080
actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 1140
attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 1200
catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct 1260
agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 1320
cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 1380
tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 1440
gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 1500
tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 1560
ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 1620
tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 1680
taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt 1740
ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 1800
ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 1860
agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 1920
ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg 1980
cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 2040
gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa 2100
ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat 2160
ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga 2220
gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt 2280
ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa 2340
caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt 2400
cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc 2460
gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga 2520
ggagcaggac tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt 2580
gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat 2640
gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag 2700
caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 2760
gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt 2820
ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 2880
caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 2940
cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 3000
gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 3060
ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 3120
ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 3180
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 3240
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 3300
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 3360
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 3420
gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 3480
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 3540
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 3600
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 3660
cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 3720
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 3780
tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 3840
ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 3900
attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 3960
ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 4020
tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tggtgtagat 4080
aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 4140
acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 4200
aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 4260
agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 4320
ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 4380
agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 4440
tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 4500
tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 4560
attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 4620
taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 4680
aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 4740
caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 4800
gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 4860
cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 4920
tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 4980
acctgacgtc 4990
<210>11
<211>8425
<212>DNA
<213>人工序列
<400>11
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttacca tggggggttc tcatcatcat catcatcatg gtatggctag 960
catgactggt ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggtaccagg 1020
ccgatgtttc ttaccgtgta tctcagtaac aatgagcagc acttcacaga agttccagtt 1080
actccagaaa caatatgcag agacgtggtg gatctgtgca aagaacccgg cgagagtgat 1140
tgccatttgg ctgaagtgtg gtgtggctct gaacgtccag ttgcggataa tgagcgaatg 1200
tttgatgttc ttcaacgatt tggaagtcag aggaacgaag ttcgcttctt ccttcgtcat 1260
gaacgccccc ctggcaggga cattgtgagt ggaccaagat ctcaggatcc aagtttaaaa 1320
agaaatggtg taaaagttcc tggtgaatat cgaagaaagg agaacggtgt taatagtcct 1380
aggatggatc tgactcttgc tgaacttcag gaaatggcat ctcgccagca gcaacagatt 1440
gaagcccagc aacaattgct ggcaactaag gaacagcgct taaagttttt gaaacaacaa 1500
gatcagcgac aacagcaaca agttgctgag caggagaaac ttaaaaggct aaaagaaata 1560
gctgagaatc aggaagctaa gctaaaaaaa gtgagagcac ttaaaggcca cgtggaacag 1620
aagagactaa gcaatgggaa acttgtggag gaaattgaac agatgaataa tttgttccag 1680
caaaaacaga gggagctcgt cctggctgtg tcaaaagtag aagaactgac caggcagcta 1740
gagatgctca agaacggcag gatcgacagc caccatgaca atcagtctgc agtggctgag 1800
cttgatcgcc tctataagga gctgcagcta agaaacaaat tgaatcaaga gcagaatgcc 1860
aagctacaac aacagaggga gtgtttgaat aagcgtaatt cagaagtggc agtcatggat 1920
aagcgtgtta atgagctgag ggaccggctg tggaagaaga aggcagctct acagcaaaaa 1980
gaaaatctac cagtttcatc tgatggaaat cttccccagc aagccgcgtc agccccaagc 2040
cgtgtggctg cagtaggtcc ctatatccag tcgtctacta tgcctcggat gccctcaagg 2100
cctgaattgc tggtgaagcc agccctgccg gatggttcct tggtcattca ggcttcagag 2160
gggccgatga aaatacagac actgcccaac atgagatctg gggctgcttc acaaactaaa 2220
ggctctaaaa tccatccagt tggccctgat tggagtcctt caaatgcaga tcttttccca 2280
agccaaggct ctgcttctgt acctcaaagc actgggaatg ctctggatca agttgatgat 2340
ggagaggttc cgctgaggga gaaagagaag aaagtgcgtc cgttctcaat gtttgatgca 2400
gtagaccagt ccaatgcccc accttccttt ggtactctga ggaagaacca gagcagtgaa 2460
gatatcttgc gggatgctca ggttgcaaat aaaaatgtgg ctaaagtacc acctcctgtt 2520
cctacaaaac caaaacagat taatttgcct tattttggac aaactaatca gccaccttca 2580
gacattaagc cagacggaag ttctcagcag ttgtcaacag ttgttccgtc catgggaact 2640
aaaccaaaac cagcagggca gcagccgaga gtgctgctat ctcccagcat accttcggtt 2700
ggccaagacc agaccctttc tccaggttct aagcaagaaa gtccacctgc tgctgccgtc 2760
cggcccttta ctccccagcc ttccaaagac accttacttc cacccttcag aaaaccccag 2820
accgtggcag caagttcaat atattccatg tatacgcaac agcaggcgcc aggaaaaaac 2880
ttccagcagg ctgtgcagag cgcgttgacc aagactcata ccagagggcc acacttttca 2940
agtgtatatg gtaagcctgt aattgctgct gcccagaatc aacagcagca cccagagaac 3000
atttattcca atagccaggg caagcctggc agtccagaac ctgaaacaga gcctgtttct 3060
tcagttcagg agaaccatga aaacgaaaga attcctcggc cactcagccc aactaaatta 3120
ctgcctttct tatctaatcc ttaccgaaac cagagtgatg ctgacctaga agccttacga 3180
aagaaactgt ctaacgcacc aaggcctcta aagaaacgta gttctattac agagccagag 3240
ggtcctaatg ggccaaatat tcagaagctt ttatatcaga ggaccaccat agcggccatg 3300
gagaccatct ctgtcccatc atacccatcc aagtcagctt ctgtgactgc cagctcagaa 3360
agcccagtag aaatccagaa tccatattta catgtggagc ccgaaaagga ggtggtctct 3420
ctggttcctg aatcattgtc cccagaggat gtggggaatg ccagtacaga gaacagtgac 3480
atgccagctc cttctccagg ccttgattat gagcctgagg gagtcccaga caacagccca 3540
aatctccaga ataacccaga agaaccaaat ccagaggctc cacatgtgct tgatgtgtac 3600
ctggaggagt accctccata cccaccccca ccatacccat ctggggagcc tgaagggccc 3660
ggagaagact cggtgagcat gcgcccgcct gaaatcaccg ggcaggtctc tctgcctcct 3720
ggtaaaagga caaacttgcg taaaactggc tcagagcgta tcgctcatgg aatgagggtg 3780
aaattcaacc cccttgcttt actgctagat tcgtctttgg agggagaatt tgaccttgta 3840
cagagaatta tttatgaggt tgatgaccca agcctcccca atgatgaagg catcacggct 3900
cttcacaatg ctgtgtgtgc aggccacaca gaaatcgtta agttcctggt acagtttggt 3960
gtaaatgtaa atgctgctga tagtgatgga tggactccat tacattgtgc tgcctcatgt 4020
aacaacgtcc aagtgtgtaa gtttttggtg gagtcaggag ccgctgtgtt tgccatgacc 4080
tacagtgaca tgcagactgc tgcagataag tgcgaggaaa tggaggaagg ctacactcag 4140
tgctcccaat ttctttatgg agttcaggag aagatgggca taatgaataa aggagtcatt 4200
tatgcgcttt gggattatga acctcagaat gatgatgagc tgcccatgaa agaaggagac 4260
tgcatgacaa tcatccacag ggaagacgaa gatgaaatcg aatggtggtg ggcgcgcctt 4320
aatgataagg agggatatgt tccacgtaac ttgctgggac tgtacccaag aattaaacca 4380
agacaaagga gcttggcctg atctagaggg cccgtttaaa cccgctgatc agcctcgact 4440
gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 4500
gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 4560
agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 4620
gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga 4680
accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg 4740
ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 4800
ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 4860
cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 4920
gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 4980
acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 5040
cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta 5100
aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt 5160
tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 5220
attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 5280
gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc 5340
taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 5400
cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg 5460
gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc 5520
agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt 5580
gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt 5640
cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga 5700
cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca 5760
ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc 5820
cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga 5880
gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt 5940
gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt ccaccgccgc 6000
cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca 6060
gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa 6120
tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 6180
ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac 6240
ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 6300
gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 6360
atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 6420
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 6480
tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 6540
agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 6600
aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 6660
gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 6720
tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 6780
cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 6840
ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 6900
cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 6960
atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 7020
agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 7080
gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 7140
gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 7200
tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 7260
agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 7320
gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 7380
aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 7440
aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 7500
ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 7560
gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 7620
aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 7680
ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 7740
tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 7800
ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 7860
cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 7920
agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 7980
gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 8040
gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 8100
acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 8160
acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 8220
agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 8280
aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 8340
gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 8400
tccccgaaaa gtgccacctg acgtc 8425
Claims (6)
1.一种分泌型碱性磷酸酶示踪TA克隆真核表达载体,其特征在于,所述载体是在真核表达启动子(CMV)下游和BGH多聚腺嘌呤转录终止子的上游插入碱性磷酸酶示踪TA克隆位点,所述载体的碱基序列如SEQ ID No.10所示。
2.一种分泌型碱性磷酸酶示踪TA克隆真核表达载体,其特征在于,所述载体是在真核表达启动子(CMV)下游和BGH多聚腺嘌呤转录终止子的上游插入碱性磷酸酶示踪TA克隆位点,所述载体结构如图4所示。
3.根据权利要求1所述的分泌型碱性磷酸酶示踪TA克隆真核表达载体的构建方法,其特征在于,所述方法包括下述步骤:
(1)原核TA克隆载体:把TA位点引入pUC19载体构建成pUC19-TA载体;
(2)把pUC19-TA载体的TA序列克隆入pcDNA4His载体中得到pcDNA5-TA克隆载体;
(3)把pcDNA5-TA克隆载体上的CMV-TA-polyA序列经PCR得到后克隆到pSEAP载体中构建成具有真核表达功能并外分泌碱性磷酸酶(AP)的TA载体pcDNA5AP-TA。
4.根据权利要求4所述的分泌型碱性磷酸酶示踪TA克隆真核表达载体的构建方法,其特征在于,所述步骤(1)为:
1)首先合成两条含AhdI酶切位点的寡核苷酸,退火形成双链DNA;
TA-oligo-s:
5’P-AATTCACTAGTGGACATTATGTCTCGAGACATAATGTCGG-3’;
TA-oligo-as:
5’P-AATTCCGACATTATGTCTCGAGACATAATGTCCACTAGTG-3’
EcoRI酶切pUC19质粒,与上述DNA双链连接形成前T载体;序列如SEQ
ID No.1所示;
2)制备间隔DNA
引物:aspp2-xhoI-s:CCATCTCGAGCCCATCATACCCATCCAAG
aspp2-xhoI-as:TTCCTCGAGCGATACGCTCTGAGCCAG
以pcDNA-ASPP2质粒为模板扩增,如SEQ ID No.11所示,扩增片段中不含常用的限制性酶切位点,扩增序列如SEQ ID No.2所示;
3)XhoI酶切步骤2)制备的间隔DNA后,插入到前T载体中;
4)设计突变引物AhdI-mut-s:TGCCTGACTCCCCGTGGTGTAGAT,AhdI-mut-as:TCGTAGTTATCTACACCACGGGGAGT把ampicilin抗性基因中的AhdI酶切位点GACTCCCCGTC突变为GACTCCCCGTG,形成pUC19-TA前T载体,序列如SEQ ID No.3所示;
5)AhdI酶切pUC19-TA前T载体,回收大片段,即为pUC19-TA载体,第89碱基处为开放性T末端,序列如SEQ ID No.4所示。
5.根据权利要求4所述的分泌型碱性磷酸酶示踪TA克隆真核表达载体的构建方法,其特征在于,所述步骤(2)为:
1)以pUC19-TA前T载体为模板,扩增片段如SEQ ID No.5所示,引物为:TA-HindIII-S:GCCGCGGGAAGCTtATATCACTAGTGAATTC
TA-XbaI-AS:GGTCGACCTCTAGACGGCCGCGAATTCCGAC
2)HindIII和XbaI双酶切SEQ ID No.5,定向连接到pcDNA4His vector上,用引物
AhdI-mut-s:TGCCTGACTCCCCGTGGTGTAGAT
AhdI-mut-as:TCGTAGTTATCTACACCACGGGGAGT
把ampicilin抗性基因中的AhdI酶切位点GACTCCCCGTC突变为GACTCCCCGTG,形成pcDNA5-TA前T载体,如SEQ ID No.6所示;
3)AhdI酶切上述pcDNA5-TA前T载体,回收大片段即为pcDNA5-TA载体,第946碱基处为开放性T末端,如SEQ ID No.7所示。
6.根据权利要求4所述的分泌型碱性磷酸酶示踪TA克隆真核表达载体的构建方法,其特征在于,所述步骤(3)为:
1)以pcDNA5-TA前T载体为模板,扩增片段,如SEQ ID No.8所示,引物为:
CMV-NotI:GCATGAAGCGGCCGCTTAGGGTTAGGCGTT
polyA-NotI:AGCTGGTTGCGGCCGCCTCAGAAGCCAT
2)NotI酶切后,插入到pSEAP2-control载体上,测序验证反向插入的为正确克隆,引物为
AhdI-mut-s:TGCCTGACTCCCCGTGGTGTAGAT
AhdI-mut-as:TCGTAGTTATCTACACCACGGGGAGT
3)把ampicilin抗性基因中的AhdI酶切位点GACTCCCCGTC突变为GACTCCCCGTG,形成pcDNA5AP-TA前T载体,如SEQ ID No.9所示;4)AhdI酶切回收大片段,即为pcDNA5AP-TA载体,第5245碱基处为开放性T末端,如SEQ ID No.10所示。
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Non-Patent Citations (3)
Title |
---|
Jeung JU 等.Construction of two pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites for direct cloning of PCR products.《Plasmid》.2002,160-163. * |
金春元 等.PCR产物真核TA克隆法构建基因表达重组体.《中华医学遗传学杂志》.1997,第14卷(第2期),115-116. * |
顾頠 等.基于XcmⅠ酶切的高效TA克隆载体的构建.《生物技术通讯》.2008,第19卷(第3期),386-388. * |
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