九种载体的多克隆位点及质粒图谱

1、九种载体的多克隆位点及质粒图谱(pLLP-OmpA、pLLP-STII、pMBP-P、pMBP-C、pTrc-CKS、pET-GST、pET-Trx、pET-His、pET-DsbA)这九种载体的多克隆位点均为GGATCCGCAGAATTCAGCGCTAGCCAC(TAA)CATCATBamHIEcoRINhelHisHisHisCATCATCATTAAGCTTHisHisHisHindlll我们建议您扩增基因5瑞选BamHI或Bglll,3湍选Nhel或Xbal或Spel,同时3,端不要带终止密码子，注意与GGATCC(GlySer)表达框架正确，载体用NhelBamHI双酶切。BamHI和

2、Bglll为同裂酶，Nhel、Xbal和Spel为同裂酶。如您目的基因无法满足上述要求，如同时含有BamHI和Bglll,5端可选择EcoRI,但须注意与表达框架GAATTC(GluPhe)对齐。如您目的基因同时含有Xbal、Nhel和Spel,3,端可选择EcoRI。如果您的目的基因已带有终止密码子，同样可以进行克隆表达，只是注意有些表达载体是在C端带6个His。PCR引物设计时BamHI和EcoRI位点的5端保护碱基有2至3个GC即可，而Xbal、Nhel和Spel位点5,端保护碱基须在5个左右才容易被酶切割。(一)pLLp-OmpASize:7.5Kb多克隆位点：Plpplacopera

3、torRBSICGAGGCGCAAAAAATGAAAAAGACAGCTATCGCGMetLysLysThrAlaIleAlaOmpAsignalATTGCAGTGGCACTGGCAGGTTTCGCTACCGTCGCTCAGGCTGGATCCGCAIleAlaValAlaLeuAlaGlyPheAlaThrValAlaGlnAlaBamHIGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTEcoRINhel6His-tagHindlllBamHIEcoRINheI6His-stopHindIIIOri基因克隆表达功能区序列如下，经DNA双向测序验证ctcacttta

4、tctaagatgaatccgatggaagcatcctgttttctctcaatttttttatctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagata

5、acgaggcgcaaaaaatgaaaaagacagctatcgcgattgcagtggcactggcaggtttcgctaccgtcgctcaggctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtt

6、taccgctactgcgtcacgcgtaacatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta载体其它部分序列见GenebankLocusASAK3121(plasmidpIN-III-ompAI)(二)pLLp-STIISize:7.5kblacoperatorRBSPlpp多克隆住点:GAAGGAGATATACATATGAAAAAGAACATCGCATTCCTCMetLysLysAsn

7、IleAlaPheLeuSTIIsignalCTGGCATCTATGTTTGTTTTCTCTATCGCTACCAACGCTTACGCTGGATCCLeuAlaSerMetPheValPheSerIleAlaThrAsnAlaTyrAlaBammHIGCAGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNhel6His-tagHindlllBamHIEcoRINhel6His-stopHindlllOri基因克隆表达功能区序列如下，经DNA双向测序验证ctcactttatctaagatgaatccgatggaagcatcctgttttctctcaattttttta

8、tctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagaaataattttgtttaactttaagaaggagatatacatatgaaaaagaacat

9、cgcattcctcctggcatctatgtttgttttctctatcgctaccaacgcttacgctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtttaccgctactgcgtcacgcgta

10、acatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta载体其它部分序列见GenebankLocusASAK3121(plasmidpIN-III-ompAI)STIIsignalpeptide见GenebankLocusHSTI_ECOLI(p22542)(三)pMBP-pVectorsize:6.7kbPtaclacoperator分泌型maltosebindingprotein|AACAA

11、CAACAACAACAACAACAATThrombincutsiteAACAACCTGGGTATCGATACTACTGAGAACTATCCGCGTGgAtCCGCAGAAHindlllTTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNhel6His-tagBamHIEcoRINhel6His-stopHindlllOri基因克隆表达功能区序列如下，经DNA双向测序验证Gccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagc

12、ggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindlll载体其它部分序列见pMal-p2Maltosebindingprotein(E.coli)编码氨基酸序列见GenebankMBPMW=42KD(四)pMBP-cVectorsize:6.6kbPtaclacoperator.1的内maltosebindi

13、ngprotenAACAACAACAACAACAACAACAATThrombincutsiteAACAACCTGGGTATCGATACTACTGAGAACTATCCGCGTGgAtCCGCAGAAHindlllTTCAGCGCTAGCCACCATCATCATCATCATTAAGCTTNhel6His-tagOri基因克隆表达功能区序列如下，经DNA双向测序验证Gccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcga

14、tgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindlll载体其它部分序列见pMal-c2Maltosebindingprotein(E.coli)编码氨基酸序列见GenebankMBPMW=42KD(五)pET-GSTvectorsize:3.7kb多克隆位点：P3CcutsitePT7|RBS|一|ATGGST|CTCGAAGTTCTGTT

15、TCAG%GTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHlTCCGCAGAATTCAGCGCTAGCCACCATCATCATCATTAAGCTT基因克隆表达功能区序列如下，经DNA双向测序验证XbaItctagatcacacaggaaacagtattatgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttccc

16、aatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttc

17、atgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggacagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindlll载

18、体其它部分序列见pUC18（不含pUC18多克隆位点）GlutathioneS-transferas却码氨基酸序列如下（含多克隆位点）：MSPILGYMKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDSMPDSLEVLFQ

19、GPAGSAEFSASHHHHHH248AMINOACIDSMW28.7KD（六）pET-Trxvectorsize:3.3kb多克隆位点：P3CcutsitePT7|RBS|TRX（his-patCCTCGAAGTTCTGTTTCAG*GGTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHITCCGCAGAATTCAGCGCTAGCTAACATCATCATCATTAAGCTTEcoRINhelstopHindlllQri基因克隆表达功能区序列如下，经DNA双向测序验证XbaltctagaaataattttgtttaactttaagaaggagatatacaOtgtc

20、tgataaaattattcatctgactgatgattcttttgatactgatgtacttaaggcagatggtgcaatcctggttgatttctgggcacactggtgcggtccgtgcaaaatgatcgctccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcacaacccgggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaaga

21、gttcctcgacgctaacctggctggctctagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagctaacatcatcatcatcattaagcttHindlll载体其它部分序列见pUC18（不含pUC18多克隆位点）Thioredoxinmut(His-patch)(E.coli)编码氨基酸序列如下(含多克隆位点)MSDKllHLTDDSFDTDVLKADGAlLVDFWAHWCGPCKMlAPlLDElADEYQGKLTVAKLNlDHNPGTAPKYGlRGlPTLLLFKNGEVAATKVGALS

22、KGQLKEFLDANLAGSSMPDSLEVLFQGPAGSAEFSAS133AMlNOAClDSMW14KD3个His形成立体His-patch（七）pET-Hisvectorsize:2.9kb多克隆位点：thrombincutsitePT7RBSATGHisCTGGTGCCGCGC*GGATCCGCAGAATTCAGCGCTLeuValProArgBamHIEcoRIAGCTAACATCATCATCATCATTAAGCTTNhelHindlllBamHIEcoRINhelstopHindlllQri基因克隆表达功能区序列如下，经DNA双向测序验证Xbaltctagaaataattttg

23、tttaactttaagaaggagatatacatgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggatccgcagaattcagcgctagctaacatcatcatcatcattaagcttHindlll载体其它部分序列见pUC18（不含pUC18多克隆位点）（八）PTRC-CKSvectorsize:5.2kb多克隆位点：P3CcutsitePTrc|RBS|AtGCKSmuHCTCGAAGTTCTGTTTCAG%GTCCAGCAGGALeuGluValLeuPheGlnGlyProBamHITCCGCAGAATTCAGCGCTAGC

24、CACCATCATCATCATCATTAAGCTTBamHIEcoRINhel6His-stopHindlllPTrcRBSATGCKSmutp3ccutsiteMCSOri基因克隆表达功能区序列如下，经DNA双向测序验证XbaItctagaaataattttgtttaactttaagaaggagatatacaihtgagttttgtggtcattattcccgcgcgctacgcgtccacgcgtctgcccggtaaaccattggttgatattaacggcaaacccatgattgttcatgttcttgaacgcgcgcgtgaatcaggtgccgagcgcatcatcgtggc

25、aaccgatcatgaggatgttgcccgcgccgttgaagccgctggcggtgaagtatgtatgacgcgcgccgatcatcagtcaggaacagaacgtctggcggaagttgtcgaaaaatgcgcattcagcgacgacacggtgatcgttaatgtgcagggtgatgaaccgatgatccctgcgacaatcatccgtcaggttgctgataacctcgctcagcgtcaggtgggtatggcgactctggcggtgccaatccacaatgcggaagaagcgtttaacccgaatgcggtgaaagtggttctcgacgc

26、tgaagggtatgcactgtacttctctcgcgccaccattccttgggatcgtgatcgttttgcagaaggccttgaaaccgttggcgataacttcctgcgtcatcttggtatttatggctaccgtgcaggctttatccgtcgttacgtcaactggcagccaagtccgttagaacacatcgaaacgttagagcagcttcgtgttctgtggtacggcgaaaaaatccatgttgctgttgctcaggaagttcctggcacaggtgtggatacccctgaggacggtagcatgccagactctctcgaagt

27、tctgtttcagggtccagcaggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttHindIII载体其它部分序列见pTrcHisB（不含pTrcHisB多克隆位点）CKSmut（E.coliCTP:CMP-3-deoxy-D-manno-octulosonatecytidylyltransferaseorCMP-KDOsynthetase编码氨基酸序列如下（含多克隆位点）：MSFVVIIPARYASTRLPGKPLVDINGKPMIVHVLERARESGAERIIVATDHEDVARAVEAAGGEVCMTRADHQSGTERLAEVVEK

28、CAFSDDTVIVNVQGDEPMIPATIIRQVADNLAQRQVGMolecularWeight29KDMATLAVPIHNAEEAFNPNAVKVVLDAEGYALYFSRATIPWDRDRFAEGLETVGDNFLRHLGIYGYRAGFIRRYVNWQPSPLEHIETLEQLRVLWYGEKIHVAVAQEVPGTGVDTPEDGSMPDSLEVLFQGPAGSAEFSASHHHHHH268AminoAcids（九）PET-DsbAvectorsize:3.6kbthrombincutsite多克隆位点PT7RBS|ATG-6HisDsbAmut卜ATGTATCCGCGT%GAMetTyrProArgBamHITCCGCAGAATTCAGCGCTAGCTAACATCATCATCATCATTAAGCTT3EcoRINhelstopHindlllQri基因克隆表达功能区序列如下，经DNA双向测序验证）XbaITctagaaataattttgtttaactttaagaaggagatatacatgggcagctctcatcatcatcatcatcactctagcggtgctcagtacgaagatggtaaaca