基因敲出质粒图谱

（1）PKD46载体基本信息出品公司:--载体名称:pKD46质粒类型:细菌表达；基因敲除系统高拷贝/低拷贝:--启动子:--克隆方法:多克隆位点，限制性内切酶载体大小:6329bp5'测序引物及序列:--3'测序引物及序列:--载体标签:--载体抗性:--筛选标记:--备注:pKD46ismoreefficientthanpKD20formakinggenedisruptions.产品目录号:--稳定性:--组成型:--病毒/非病毒:--LOCUSpKD466329bpDNAcircularSYN11-SEP-2001DEFINITIONRedrecombinaseplasmidpKD46,completesequence.ACCESSIONAY048746VERSIONAY048746.1GI:15554345KEYWORDS.SOURCERedrecombinaseplasmidpKD46ORGANISMRedrecombinaseplasmidpKD46othersequences;artificialsequences;vectors.REFERENCE1(bases1to6329)AUTHORSDatsenko,K.A.andWanner,B.L.TITLEOne-stepinactivationofchromosomalgenesinEscherichiacoliK-12usingPCRproductsJOURNALProc.Natl.Acad.Sci.U.S.A.97(12),6640-6645(2000)PUBMED10829079REFERENCE2(bases1to6329)AUTHORSDatsenko,K.A.andWanner,B.L.TITLEDirectSubmissionJOURNALSubmitted(29-JUL-2001)BiologicalSciences,PurdueUniversity,LillyHallofLifeSciences,WestLafayette,IN47907,USAFEATURESLocation/Qualifierssource1..6329/organism="RedrecombinaseplasmidpKD46"/mol_type="genomicDNA"/db_xref="taxon:170495"CDScomplement(11..889)/codon_start=1/transl_table=11/product="araCprotein"/protein_id="AAL02045.1"/db_xref="GI:15554346"misc_featurecomplement(1040..1068)/note="Pc"misc_feature1165..1192/note="ParaB"CDS1244..1660/note="recombination;138"/codon_start=1/transl_table=11/product="gamprotein"/protein_id="AAL02046.1"/db_xref="GI:15554347"CDS1666..2451/note="recombination;261"/codon_start=1/transl_table=11/product="betprotein"/protein_id="AAL02047.1"/db_xref="GI:15554348"CDS2448..3128/note="exo"/codon_start=1/transl_table=11/product="exonuclease"/protein_id="AAL02048.1"/db_xref="GI:15554349"misc_feature3118..3387/note="tL3"CDS3125..3307/note="60a"/codon_start=1/transl_table=11/product="orf60aprotein"/protein_id="AAL02051.1"/db_xref="GI:15554352"CDScomplement(3496..4446)/codon_start=1/transl_table=11/product="repA101protein"/protein_id="AAL02049.1"/db_xref="GI:15554350"rep_origin4450..5190/note="oriR101"CDScomplement(5338..6198)/note="bla;ampicillinresistance"/codon_start=1/transl_table=11/product="beta-lactamase"/protein_id="AAL02050.1"/db_xref="GI:15554351"ORIGIN1catcgatttattatgacaacttgacggctacatcattcactttttcttcacaaccggcac61ggaactcgctcgggctggccccggtgcattttttaaatacccgcgagaaatagagttgat121cgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtgctcaaaagca181gcttcgcctggctgatacgttggtcctcgcgccagcttaagacgctaatccctaactgct241ggcggaaaagatgtgacagacgcgacggcgacaagcaaacatgctgtgcgacgctggcga301tatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcctcgcgtacccgat361tatccatcggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgct421caagcagatttatcgccagcagctccgaatagcgcccttccccttgcccggcgttaatga481tttgcccaaacaggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaccccg541tattggcaaatattgacggccagttaagccattcatgccagtaggcgcgcggacgaaagt601aaacccactggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctctc661ctggcgggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttca721ccaccccctgaccgcgaatggtgagattgagaatataacctttcattcccagcggtcggt781cgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacccgccaccagatggg841cattaaacgagtatcccggcagcaggggatcattttgcgcttcagccatacttttcatac901tcccgccattcagagaagaaaccaattgtccatattgcatcagacattgccgtcactgcg961tcttttactggctcttctcgctaaccaaaccggtaaccccgcttattaaaagcattctgt1021aacaaagcgggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggca1081gaaaagtccacattgattatttgcacggcgtcacactttgctatgccatagcatttttat1141ccataagattagcggatcctacctgacgctttttatcgcaactctctactgtttctccat1201acccgtttttttgggaattcgagctctaaggaggttataaaaaatggatattaatactga1261aactgagatcaagcaaaagcattcactaaccccctttcctgttttcctaatcagcccggc1321atttcgcgggcgatattttcacagctatttcaggagttcagccatgaacgcttattacat1381tcaggatcgtcttgaggctcagagctgggcgcgtcactaccagcagctcgcccgtgaaga1441gaaagaggcagaactggcagacgacatggaaaaaggcctgccccagcacctgtttgaatc1501gctatgcatcgatcatttgcaacgccacggggccagcaaaaaatccattacccgtgcgtt1561tgatgacgatgttgagtttcaggagcgcatggcagaacacatccggtacatggttgaaac1621cattgctcaccaccaggttgatattgattcagaggtataaaacgaatgagtactgcactc1681gcaacgctggctgggaagctggctgaacgtgtcggcatggattctgtcgacccacaggaa1741ctgatcaccactcttcgccagacggcatttaaaggtgatgccagcgatgcgcagttcatc1801gcattactgatcgttgccaaccagtacggccttaatccgtggacgaaagaaatttacgcc1861tttcctgataagcagaatggcatcgttccggtggtgggcgttgatggctggtcccgcatc1921atcaatgaaaaccagcagtttgatggcatggactttgagcaggacaatgaatcctgtaca1981tgccggatttaccgcaaggaccgtaatcatccgatctgcgttaccgaatggatggatgaa2041tgccgccgcgaaccattcaaaactcgcgaaggcagagaaatcacggggccgtggcagtcg2101catcccaaacggatgttacgtcataaagccatgattcagtgtgcccgtctggccttcgga2161tttgctggtatctatgacaaggatgaagccgagcgcattgtcgaaaatactgcatacact2221gcagaacgtcagccggaacgcgacatcactccggttaacgatgaaaccatgcaggagatt2281aacactctgctgatcgccctggataaaacatgggatgacgacttattgccgctctgttcc2341cagatatttcgccgcgacattcgtgcatcgtcagaactgacacaggccgaagcagtaaaa2401gctcttggattcctgaaacagaaagccgcagagcagaaggtggcagcatgacaccggaca2461ttatcctgcagcgtaccgggatcgatgtgagagctgtcgaacagggggatgatgcgtggc2521acaaattacggctcggcgtcatcaccgcttcagaagttcacaacgtgatagcaaaacccc2581gctccggaaagaagtggcctgacatgaaaatgtcctacttccacaccctgcttgctgagg2641tttgcaccggtgtggctccggaagttaacgctaaagcactggcctggggaaaacagtacg2701agaacgacgccagaaccctgtttgaattcacttccggcgtgaatgttactgaatccccga2761tcatctatcgcgacgaaagtatgcgtaccgcctgctctcccgatggtttatgcagtgacg2821gcaacggccttgaactgaaatgcccgtttacctcccgggatttcatgaagttccggctcg2881gtggtttcgaggccataaagtcagcttacatggcccaggtgcagtacagcatgtgggtga2941cgcgaaaaaatgcctggtactttgccaactatgacccgcgtatgaagcgtgaaggcctgc3001attatgtcgtgattgagcgggatgaaaagtacatggcgagttttgacgagatcgtgccgg3061agttcatcgaaaaaatggacgaggcactggctgaaattggttttgtatttggggagcaat3121ggcgatgacgcatcctcacgataatatccgggtaggcgcaatcactttcgtctactccgt3181tacaaagcgaggctgggtatttcccggcctttctgttatccgaaatccactgaaagcaca3241gcggctggctgaggagataaataataaacgaggggctgtatgcacaaagcatcttctgtt3301gagttaagaacgagtatcgagatggcacatagccttgctcaaattggaatcaggtttgtg3361ccaataccagtagaaacagacgaagaatccatgggtatggacagttttccctttgatatg3421taacggtgaacagttgttctacttttgtttgttagtcttgatgcttcactgatagataca3481agagccataagaacctcagatccttccgtatttagccagtatgttctctagtgtggttcg3541ttgtttttgcgtgagccatgagaacgaaccattgagatcatacttactttgcatgtcact3601caaaaattttgcctcaaaactggtgagctgaatttttgcagttaaagcatcgtgtagtgt3661ttttcttagtccgttacgtaggtaggaatctgatgtaatggttgttggtattttgtcacc3721attcatttttatctggttgttctcaagttcggttacgagatccatttgtctatctagttc3781aacttggaaaatcaacgtatcagtcgggcggcctcgcttatcaaccaccaatttcatatt3841gctgtaagtgtttaaatctttacttattggtttcaaaacccattggttaagccttttaaa3901ctcatggtagttattttcaagcattaacatgaacttaaattcatcaaggctaatctctat3961atttgccttgtgagttttcttttgtgttagttcttttaataaccactcataaatcctcat4021agagtatttgttttcaaaagacttaacatgttccagattatattttatgaatttttttaa4081ctggaaaagataaggcaatatctcttcactaaaaactaattctaatttttcgcttgagaa4141cttggcatagtttgtccactggaaaatctcaaagcctttaaccaaaggattcctgatttc4201cacagttctcgtcatcagctctctggttgctttagctaatacaccataagcattttccct4261actgatgttcatcatctgagcgtattggttataagtgaacgataccgtccgttctttcct4321tgtagggttttcaatcgtggggttgagtagtgccacacagcataaaattagcttggtttc4381atgctccgttaagtcatagcgactaatcgctagttcatttgctttgaaaacaactaattc4441agacatacatctcaattggtctaggtgattttaatcactataccaattgagatgggctag4501tcaatgataattactagtccttttcctttgagttgtgggtatctgtaaattctgctagac4561ctttgctggaaaacttgtaaattctgctagaccctctgtaaattccgctagacctttgtg4621tgttttttttgtttatattcaagtggttataatttatagaataaagaaagaataaaaaaa4681gataaaaagaatagatcccagccctgtgtataactcactactttagtcagttccgcagta4741ttacaaaaggatgtcgcaaacgctgtttgctcctctacaaaacagaccttaaaaccctaa4801aggcttaagtagcaccctcgcaagctcggttgcggccgcaatcgggcaaatcgctgaata4861ttccttttgtctccgaccatcaggcacctgagtcgctgtctttttcgtgacattcagttc4921gctgcgctcacggctctggcagtgaatgggggtaaatggcactacaggcgccttttatgg4981attcatgcaaggaaactacccataatacaagaaaagcccgtcacgggcttctcagggcgt5041tttatggcgggtctgctatgtggtgctatctgactttttgctgttcagcagttcctgccc5101tctgattttccagtctgaccacttcggattatcccgtgacaggtcattcagactggctaa5161tgcacccagtaaggcagcggtatcatcaacggggtctgacgctcagtggaacgaaaactc5221acgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaa5281ttaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagtta5341ccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagt5401tgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccag5461tgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaacca5521gccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtc5581tattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgt5641tgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcag5701ctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggt5761tagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcat5821ggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgt5881gactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctc5941ttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcat6001cattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccag6061ttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgt6121ttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacg6181gaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggtta6241ttgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttcc6301gcgcacatttccccgaaaagtgccacctg摘要：目前应用最广泛的Red同源重组系统质粒是pKD46，该质粒含有受阿拉伯糖启动子调控的exo、bet、gam基因，具有氨苄青霉素抗性，其复制子为低拷贝温敏型复制子，便于质粒的消除。pKD46质粒序列全长6329bp，详细序列见GenBank进入号：AY048746。关键词：

Red

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同源重组

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基因敲除

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质粒图谱pKD46质粒是目前应用最广泛的\o"ViewallpostsinRed"Red\o"同源重组是指发生在含有同源序列的DNA分子之间或分子之内的重新组合，依赖大范围的DNA同源序列的联分，重组过程中，两个DNA分子交换对等的部分。"同源重组质粒，该质粒含有受阿拉伯糖启动子调控的exo、bet、gam基因，具有氨苄青霉素抗性，其复制子为低拷贝温敏型复制子，便于质粒的消除。pKD46质粒序列全长6329bp，详细序列见GenBank进入号：AY048746。质粒类型：Red\o"Viewallpostsin同源重组"同源重组\o"基因敲除是指对一个结构已知的基因，从分子水平上设计实验，将该基因从宿主染色体中去除或用其它基因取代。"基因敲除载体克隆方法：多克隆位点，限制性内切酶拷贝数：低拷贝载体大小：6329bp（GenBank进入号：AY048746）载体抗性：Ampicillin(氨苄青霉素)备注：30°C培养与同源重组\o"Viewallpostsin基因敲除"基因敲除相关的3个酶：1、Exo蛋白Exo蛋白是一种核酸外切酶，单亚基的分子量为24kD，Exo蛋白的活性形式是一种环状三聚物分子，中间有一中空的通道，通道的一端可容纳双链DNA分子，另一端只可容纳单链DNA。Exo蛋白可结合在双链DNA的末端，从DNA双链的5’端向3’端降解DNA，使DNA分子形成3’粘性末端。2、Beta蛋白Beta蛋白在Red同源重组中起着决定性作用，具有介导互补单链DNA退火的功能，是一种退火蛋白，单亚基的分子量为25.8kD。在溶液中，Beta蛋白自发地形成环状结构，紧紧地结合在单链DNA的3’突出端，防止DNA被单链核酸酶降解，并介导互补单链DNA的退火。双链DNA退火完成后，Beta蛋白从DNA双链上解离下来。3、Gam蛋白Gam蛋白为16kD的多肽分子，可与RecBCD核酸外切酶结合，抑制其对外源DNA的降解作用，防止外源DNA进入细胞后被宿主降解。参考文献：[1]DatsenkoKA1,WannerBL.One-stepinactivationofchromosomalgenesin

EscherichiacoliK-1