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1、九种载体的多克隆位点及质粒图谱九种载体的多克隆位点及质粒图谱 这九种载体的多克隆位点均为 GGA TCC GCA GAA TTC AGC GCT AGC CAC CAT CAT BamHI EcoRI NheI His His His CAT CAT CAT TAA GCTT His His His HindIII 我们建议您扩增基因5 端选BamHI或BglII，3端选NheI或XbaI或SpeI，同时3端不要带终止密码子，注意与GGA TCC表达框架正确，载体用NheIBamHI双酶切。BamHI和BglII为同裂酶，NheI、XbaI和SpeI为同裂酶。 如您目的基因无法满足上述要求，如
2、同时含有BamHI和BglII，5端可选择EcoRI，但须注意与表达框架GAA TTC对齐。如您目的基因同时含有XbaI、NheI和SpeI，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XbaI、NheI和SpeI位点5端保护碱基须在5个左右才容易被酶切割。 pLLp-OmpA Size: 7.5Kb 多克隆位点： Plpp lac operator RBS CGAGGCGCAAAAA ATG AAA AAG ACA GCT ATC GC
3、G Met Lys Lys Thr Ala Ile Ala OmpA signal ATT GCA GTG GCA CTG GCA GGT TTC GCT ACC GTC GCT CAG GCT GGA TCC GCA Ile Ala Val Ala Leu Ala Gly Phe Ala Thr Val Ala Gln Ala BamHI GAA TTC AGC GCT AGC CAC CAT CAT CAT CAT CAT TAAGCTT EcoRI NheI 6His-tag HindIII BamHI EcoRI NheI 6His- stop HindIII Plpp lac ope
4、rator RBSATGOmpA signalMCS 基因克隆表达功能区序列如下，经DNA双向测序验证ctcactttatctaagatgaatccgatggaagcatcctgttttctctcaatttttttatctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggat
5、aacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagataacgaggcgcaaaaaatgaaaaagacagctatcgcgattgcagtggcactggcaggtttcgctaccgtcgctcaggctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagt
6、aatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtttaccgctactgcgtcacgcgtaacatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta 载体其它部分序列见Genebank Locus ASAK3121 (plasmid pIN-III-ompA1) pLLp-STII Size: 7.5kb 多克隆住点： Pl
7、pp lac operator RBS GAAGGAGATATACAT ATG AAA AAG AAC ATC GCA TTC CTC Met Lys Lys Asn Ile Ala Phe Leu STII signal CTG GCA TCT ATG TTT GTT TTC TCT ATC GCT ACC AAC GCT TAC GCT GGA TCC Leu Ala Ser Met Phe Val Phe Ser Ile Ala Thr Asn Ala Tyr Ala BammHI GCA GAA TTC AGC GCT AGC CAC CAT CAT CAT CAT CAT TAA G
8、CTT NheI 6His-tag HindIII BamHI EcoRI NheI 6His- stop HindIII Plpp lac operator ATGSTII signal peptideMCS 基因克隆表达功能区序列如下，经DNA双向测序验证 ctcactttatctaagatgaatccgatggaagcatcctgttttctctcaatttttttatctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctaca
9、tggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagaaataattttgtttaactttaagaaggagatatacatatgaaaaagaacatcgcattcctcctggcatctatgtttgttttctctatcgctaccaacgcttacgctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccgg
10、ctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtttaccgctactgcgtcacgcgtaacatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgata
11、caacgctttatcgacta 载体其它部分序列见Genebank Locus ASAK3121 (plasmid pIN-III-ompA1) STII signal peptide见Genebank Locus HSTI_ECOLI (p22542) (三)pMBP-p Vector size: 6.7kb Ptac lac operator 分泌型maltose binding protein AAC AAC AAC AAC AAC AAC AAC AAT Thrombin cut site AAC AAC CTG GGT ATC GAT ACT ACT GAG AAC TAT CC
12、G CGT GGA TCC GCA GAA Asn Tyr Pro Arg BamHI EcoRI TTC AGC GCT AGC CAC CAT CAT CAT CAT CAT TAA GCTT NheI 6His-tag HindIII BamHI EcoRI NheI 6His- stop HindIII Ptac lac operator 分泌型 MBPthrombin cut site MCS 基因克隆表达功能区序列如下，经DNA双向测序验证 Gccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgc
13、cgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagctt HindIII 载体其它部分序列见pMal-p2 Maltose binding protein(E.coli)编码氨基酸序列见Genebank MBP MW=42KD (四)pMBP-c Vector
14、size: 6.6kb Ptac lac operator 胞内maltose binding protein AAC AAC AAC AAC AAC AAC AAC AAT Thrombin cut site AAC AAC CTG GGT ATC GAT ACT ACT GAG AAC TAT CCG CGT GGA TCC GCA GAA Asn Tyr Pro Arg BamHI EcoRI TTC AGC GCT AGC CAC CAT CAT CAT CAT CAT TAA GCTT NheI 6His-tag HindIII BamHI EcoRI NheI 6His- stop
15、 HindIII Ptac lac operator 胞内 MBP thrombin cut siteMCS 基因克隆表达功能区序列如下，经DNA双向测序验证 Gccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtg
16、gatccgcagaattcagcgctagccaccatcatcatcatcattaagctt HindIII 载体其它部分序列见pMal-c2 Maltose binding protein(E.coli)编码氨基酸序列见Genebank MBP MW=42KD pET-GST vector size: 3.7kb 多克隆位点： P3C cut site PT7 RBS ATGGST CTC GAA GTT CTG TTT CAG GGT CCA GCA GGA Leu Glu Val Leu Phe Gln Gly Pro BamHI TCC GCA GAA TTC AGC GCT AG
17、C CAC CAT CAT CAT CAT TAA GCTT EcoRI NheI 6His-tag HindII BamHI EcoRI NheI 6Hisstop HindIII PT7 GST P3c cut site MCS 基因克隆表达功能区序列如下，经DNA双向测序验证 XbaI tctagatcacacaggaaacagtattc atg tcc cct ata cta ggt tat tgg aaa att aag ggc ctt gtg caa ccc act cga ctt ctt ttg gaa tat ctt gaa gaa aaa tat gaa gag cat tt
18、g tat gag cgc gat gaa ggt gat aaa tgg cga aac aaa aag ttt gaa ttg ggt ttg gag ttt ccc aat ctt cct tat tat att gat ggt gat gtt aaa tta aca cag tct atg gcc atc ata cgt tat ata gct gac aag cac aac atg ttg ggt ggt tgt cca aaa gag cgt gca gag att tca atg ctt gaa gga gcg gtt ttg gat att aga tac ggt gtt tc
19、g aga att gca tat agt aaa gac ttt gaa act ctc aaa gtt gat ttt ctt agc aag cta cct gaa atg ctg aaa atg ttc gaa gat cgt tta tgt cat aaa aca tat tta aat ggt gat cat gta acc cat cct gac ttc atg ttg tat gac gct ctt gat gtt gtt tta tac atg gac cca atg tgc ctg gat gcg ttc cca aaa tta gtt tgt ttt aaa aaa cg
20、t att gaa gct atc cca caa att gat aag tac ttg aaa tcc agc aag tat ata gca tgg cct ttg cag ggc tgg caa gcc acg ttt ggt ggt ggc gac cat cct cca aaa tcg gac agc atg cca gac tct ctc gaa gtt ctg ttt cag ggt cca gca gga tcc gca gaa ttc agc gct agc cac cat cat cat cat cat taa gctt HindIII 载体其它部分序列见pUC18(不含
21、pUC18多克隆位点) Glutathione S-transferase编码氨基酸序列如下： MSPILGYMKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDSMPDSLEVLFQGPAGSAEFSASHHHHHH 24
22、8 AMINO ACIDS MW 28.7KD pET-Trx vector size: 3.3kb 多克隆位点： P3C cut site PT7 RBS TRX(his-patch CTC GAA GTT CTG TTT CAG GGT CCA GCA GGA Leu Glu Val Leu Phe Gln Gly Pro BamHI TCC GCA GAA TTC AGC GCT AGC TAA CAT CAT CAT CAT TAA GCTT EcoRI NheI stop HindIII BamHI EcoRI NheI stop HindIII PT7 p3c cut site M
23、CS 基因克隆表达功能区序列如下，经DNA双向测序验证 XbaI tctagaaataattttgtttaactttaagaaggagatatacatatgtctgataaaattattcatctgactgatgattcttttgatactgatgtacttaaggcagatggtgcaatcctggttgatttctgggcacactggtgcggtccgtgcaaaatgatcgctccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcacaacccgggcactgcgccgaaatatggcatccgtggt
24、atcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggctggctctagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagctaacatcatcatcatcattaagctt HindIII 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： MSDKIIHLTDDSFDTDVL
25、KADGAILVDFWAHWCGPCKMIAPILDEIADEYQGKLTVAKLNIDHNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSSMPDSLEVLFQGPAGSAEFSAS 133 AMINO ACIDS MW 14KD 3 个His形成立体His-patch pET-His vector size: 2.9kb 多克隆位点： thrombin cut site PT7 RBS ATG His CTG GTG CCG CGC GGA TCC GCA GAA TTC AGC GCT Leu Val Pro Arg BamHI EcoRI
26、AGC TAA CAT CAT CAT CAT CAT TAA GCTT NheI HindIII BamHI EcoRI NheI stop HindIII PT7 ATG-6His Thrombin cut site MCS 基因克隆表达功能区序列如下，经DNA双向测序验证 XbaI tctagaaataattttgtttaactttaagaaggagatataccatgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggatccgcagaattcagcgctagctaacatcatcatcatcattaagctt HindIII 载体其它部
27、分序列见pUC18 (不含pUC18多克隆位点) (八)PTRC-CKS vector size: 5.2kb 多克隆位点： P3C cut site PTrc RBS ATGCKSmut CTC GAA GTT CTG TTT CAG GGT CCA GCA GGA Leu Glu Val Leu Phe Gln Gly Pro BamHI TCC GCA GAA TTC AGC GCT AGC CAC CAT CAT CAT CAT CATTAA GCTT EcoRI NheI 6His-tag HindIII BamHI EcoRI NheI 6His-stop HindIII PTrc
28、 RBSATGCKSmutp3C cut siteMCS 基因克隆表达功能区序列如下，经DNA双向测序验证 XbaI tctagaaataattttgtttaactttaagaaggagatatacatatgagttttgtggtcattattcccgcgcgctacgcgtccacgcgtctgcccggtaaaccattggttgatattaacggcaaacccatgattgttcatgttcttgaacgcgcgcgtgaatcaggtgccgagcgcatcatcgtggcaaccgatcatgaggatgttgcccgcgccgttgaagccgctggcggtgaagtatgta
29、tgacgcgcgccgatcatcagtcaggaacagaacgtctggcggaagttgtcgaaaaatgcgcattcagcgacgacacggtgatcgttaatgtgcagggtgatgaaccgatgatccctgcgacaatcatccgtcaggttgctgataacctcgctcagcgtcaggtgggtatggcgactctggcggtgccaatccacaatgcggaagaagcgtttaacccgaatgcggtgaaagtggttctcgacgctgaagggtatgcactgtacttctctcgcgccaccattccttgggatcgtgatcgtt
30、ttgcagaaggccttgaaaccgttggcgataacttcctgcgtcatcttggtatttatggctaccgtgcaggctttatccgtcgttacgtcaactggcagccaagtccgttagaacacatcgaaacgttagagcagcttcgtgttctgtggtacggcgaaaaaatccatgttgctgttgctcaggaagttcctggcacaggtgtggatacccctgaggacggtagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagccaccatcatcatc
31、atcattaagctt HindIII 载体其它部分序列见pTrcHisB(不含pTrcHisB多克隆位点) CKSmut(E.coli CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase or CMP-KDO synthetase)编码氨基酸序列如下(含多克隆位点)： MSFVVIIPARYASTRLPGKPLVDINGKPMIVHVLERARESGAERIIVATDHEDVARAVEAAGGEVCMTRADHQSGTERLAEVVEKCAFSDDTVIVNVQGDEPMIPATIIRQVADNLAQRQVGMATLAVPIH
32、NAEEAFNPNAVKVVLDAEGYALYFSRATIPWDRDRFAEGLETVGDNFLRHLGIYGYRAGFIRRYVNWQPSPLEHIETLEQLRVLWYGEKIHVAVAQEVPGTGVDTPEDGSMPDSLEVLFQGPAGSAEFSASHHHHHH 268 Amino Acids Molecular Weight 29KD PET-DsbA vector size: 3.6kb 多克隆位点 thrombin cut site PT7 RBS ATG-6His DsbA mut ATG TAT CCG CGT GGA Met Tyr Pro Arg BamHI TCC
33、GCA GAA TTC AGC GCT AGC TAA CAT CAT CAT CAT CAT TAA GCTT 3 EcoRI NheI stop HindIII BamHI EcoRI NheI stop HindIII PT7 RBS ATG-6His DsbAmut Thrombin cut siteMCS 基因克隆表达功能区序列如下，经DNA双向测序验证） XbaI Tctagaaataattttgtttaactttaagaaggagatataccatgggcagctctcatcatcatcatcatcactctagcggtgctcagtacgaagatggtaaacagtacact
34、accctggaaaaaccggtagctggcgcgccgcaagtgctggagtttttctcyyycttcagcccgcattcctatcagtttgaagaagttctgcatatttctgataatgtgaagaaaaaactgccggaaggcgtgaagatgactaaataccacgtcaacttcatgggtggtgacctgggcaaagatctgactcaggcatgggctgtggcgatggcgctgggcgtggaagacaaagtgactgttccgctgtttgaaggcgtacagaaaacccagaccattcgttctgcttctgatatccgcgat
35、gtatttatcaacgcaggtattaaaggtgaagagtacgacgcggcgtggaacagcttcgtggtgaaatctctggtcgctcagcaggaaaaagctgcagctgacgtgcaattgcgtggcgttccggcgatgtttgttaacggtaaatatcagctgaatccgcagggtatggataccagcaatatggatgtttttgttcagcagtatgctgatacagtgaaatatctgtccgagaaaaaaggtgatgaagatgaagatgaagatagcatgtatccgcgtggatccgcagaattcagcgctagc
36、taacatcatcatcatcattaagctt HindIII 载体其它部分序列见pUC18(不含pUC18多克隆位点) DsbAmut(E.coli)编码氨基酸序列如下(含多克隆位点)： MGSSHHHHHHSSGAQYEDGKQYTTLEKPVAGAPQV;EFFSFFSPHSYQFEEVLHISDNVKKKLPEGVKMTKYHVNFMGGDLGKDLTQAWAVAMALGVEDKVTVPLFEGVQKTQTI RSAS DIRDVFINAGIKGEEYDAAWNSFVVKSLVAQQEKAAADVQLRGVPAMFVNGKYQLNPQGMDTSNMDVFVQQYADTVKYLSEKKGDEDEDEDSMYPRGSAEFSAS 223 Amino Acids Molecular Weight 24.7KD
