pBad/His B

pBad/His B

pBadHis B载体基本信息

pBadHis B载体质粒图谱和多克隆位点信息

pBadHis B载体简介

pBAD/His和PBAD/Myc-His载体质粒是衍生于pBR322载体。载体设计用来在大肠杆菌中进行可调节，剂量依赖性的表达和纯化重组目的蛋 白。使用大肠杆菌araBAD启动子（pBAD）增强了大肠杆菌重组蛋白可溶性表达的水平。pBAD/His和pBAD/Myc His载体上的调节蛋白AraC能够调控pBad启动子。

pBAD/His A,B,C 载体介绍

The pBAD/His Kit provides all of the necessary reagents to express your protein in a tightly regulated fashion.

The vector pBAD/His allows you to express your protein with an N-terminal tag. The vector provides:

Þ      The araBAD promoter for tightly regulated expression

Þ      Translation initiation signals optimized for E. coliexpression

Þ      N-terminal polyhistidine (6xHis) tag for purification with nickel-chelating resin and detection with an Anti-

HisG Antibody

 N-terminal Xpress epitope for detection and analysis with an Anti-Xpress Antibody

 Enterokinase cleavage site for removing the N-terminal tag following purification

Three vectors are provided (A, B, and C). Each has the N-terminal tag in a different reading frame relative to the multiple cloning site to simplify in-frame cloning of your gene.

The pBAD/His and pBAD/Myc-His plasmids are pBR322-derived expression vectors designed for regulated, dose-dependent recombinant protein expression and purification in E. coli. Optimum levels of soluble, recombinant protein are possible using the araBAD promoter (PBAD) from E. coli. The regulatory protein, AraC, is provided on the pBAD/His and pBAD/Myc-His vectors allowing regulation of PBAD.

L-阿拉伯糖调控表达

In the presence of L-arabinose, expression from PBAD is turned on while the absence of L-arabinose produces very low levels of transcription from PBAD (Lee, 1980; Lee et al., 1987). Uninduced levels are repressed even further by growth in the presence of glucose. Glucose reduces the levels of 3′,5′-cyclic AMP, thus lowering expression of the catabolite-repressed PBAD promoter (Miyada et al., 1984). By varying the concentration of L-arabinose, protein expression levels can be optimized to ensure maximum expression of soluble protein. In addition, the tight regulation of PBAD by AraC is useful for expression of potentially toxic or essential genes (Carson et al., 1991; Dalbey and Wickner, 1985; Guzman et al., 1992; Kuhn and Wickner, 1985; Russell et al., 1989; San Millan et al., 1989). For more information on the mechanism of expression and repression of the ara regulon, refer to Schleif, 1992.

pBadHis B载体序列

ORIGIN

    1 AAGAAACCAA TTGTCCATAT TGCATCAGAC ATTGCCGTCA CTGCGTCTTT TACTGGCTCT

   61 TCTCGCTAAC CAAACCGGTA ACCCCGCTTA TTAAAAGCAT TCTGTAACAA AGCGGGACCA

  121 AAGCCATGAC AAAAACGCGT AACAAAAGTG TCTATAATCA CGGCAGAAAA GTCCACATTG

  181 ATTATTTGCA CGGCGTCACA CTTTGCTATG CCATAGCATT TTTATCCATA AGATTAGCGG

  241 ATCCTACCTG ACGCTTTTTA TCGCAACTCT CTACTGTTTC TCCATACCCG TTTTTTGGGC

  301 TAACAGGAGG AATTAACCAT GGGGGGTTCT CATCATCATC ATCATCATGG TATGGCTAGC

  361 ATGACTGGTG GACAGCAAAT GGGTCGGGAT CTGTACGACG ATGACGATAA GGATCCGAGC

  421 TCGAGATCTG CAGCTGGTAC CATATGGGAA TTCGAAGCTT GGCTGTTTTG GCGGATGAGA

  481 GAAGATTTTC AGCCTGATAC AGATTAAATC AGAACGCAGA AGCGGTCTGA TAAAACAGAA

  541 TTTGCCTGGC GGCAGTAGCG CGGTGGTCCC ACCTGACCCC ATGCCGAACT CAGAAGTGAA

  601 ACGCCGTAGC GCCGATGGTA GTGTGGGGTC TCCCCATGCG AGAGTAGGGA ACTGCCAGGC

  661 ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT TCGTTTTATC TGTTGTTTGT

  721 CGGTGAACGC TCTCCTGAGT AGGACAAATC CGCCGGGAGC GGATTTGAAC GTTGCGAAGC

  781 AACGGCCCGG AGGGTGGCGG GCAGGACGCC CGCCATAAAC TGCCAGGCAT CAAATTAAGC

  841 AGAAGGCCAT CCTGACGGAT GGCCTTTTTG CGTTTCTACA AACTCTTTTG TTTATTTTTC

  901 TAAATACATT CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA

  961 TATTGAAAAA GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT

 1021 GCGGCATTTT GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT

 1081 GAAGATCAGT TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC

 1141 CTTGAGAGTT TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA

 1201 TGTGGCGCGG TATTATCCCG TGTTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC

 1261 TATTCTCAGA ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC

 1321 ATGACAGTAA GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC

 1381 TTACTTCTGA CAACGATCGG AGGACCGAAG GAGCTAACCG CTTTTTTGCA CAACATGGGG

 1441 GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC

 1501 GAGCGTGACA CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC

 1561 GAACTACTTA CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT

 1621 GCAGGACCAC TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA

 1681 GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC

 1741 CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG

 1801 ATCGCTGAGA TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA

 1861 TATATACTTT AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC

 1921 CTTTTTGATA ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA

 1981 GACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC

 2041 TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA

 2101 CCAACTCTTT TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT

 2161 CTAGTGTAGC CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC

 2221 GCTCTGCTAA TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG

 2281 TTGGACTCAA GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG

 2341 TGCACACAGC CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG

 2401 CTATGAGAAA GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC

 2461 AGGGTCGGAA CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT

 2521 AGTCCTGTCG GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG

 2581 GGGCGGAGCC TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC

 2641 TGGCCTTTTG CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT

 2701 ACCGCCTTTG AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA

 2761 GTGAGCGAGG AAGCGGAAGA GCGCCTGATG CGGTATTTTC TCCTTACGCA TCTGTGCGGT

 2821 ATTTCACACC GCATATGGTG CACTCTCAGT ACAATCTGCT CTGATGCCGC ATAGTTAAGC

 2881 CAGTATACAC TCCGCTATCG CTACGTGACT GGGTCATGGC TGCGCCCCGA CACCCGCCAA

 2941 CACCCGCTGA CGCGCCCTGA CGGGCTTGTC TGCTCCCGGC ATCCGCTTAC AGACAAGCTG

 3001 TGACCGTCTC CGGGAGCTGC ATGTGTCAGA GGTTTTCACC GTCATCACCG AAACGCGCGA

 3061 GGCAGCAGAT CAATTCGCGC GCGAAGGCGA AGCGGCATGC ATAATGTGCC TGTCAAATGG

 3121 ACGAAGCAGG GATTCTGCAA ACCCTATGCT ACTCCGTCAA GCCGTCAATT GTCTGATTCG

 3181 TTACCAATTA TGACAACTTG ACGGCTACAT CATTCACTTT TTCTTCACAA CCGGCACGGA

 3241 ACTCGCTCGG GCTGGCCCCG GTGCATTTTT TAAATACCCG CGAGAAATAG AGTTGATCGT

 3301 CAAAACCAAC ATTGCGACCG ACGGTGGCGA TAGGCATCCG GGTGGTGCTC AAAAGCAGCT

 3361 TCGCCTGGCT GATACGTTGG TCCTCGCGCC AGCTTAAGAC GCTAATCCCT AACTGCTGGC

 3421 GGAAAAGATG TGACAGACGC GACGGCGACA AGCAAACATG CTGTGCGACG CTGGCGATAT

 3481 CAAAATTGCT GTCTGCCAGG TGATCGCTGA TGTACTGACA AGCCTCGCGT ACCCGATTAT

 3541 CCATCGGTGG ATGGAGCGAC TCGTTAATCG CTTCCATGCG CCGCAGTAAC AATTGCTCAA

 3601 GCAGATTTAT CGCCAGCAGC TCCGAATAGC GCCCTTCCCC TTGCCCGGCG TTAATGATTT

 3661 GCCCAAACAG GTCGCTGAAA TGCGGCTGGT GCGCTTCATC CGGGCGAAAG AACCCCGTAT

 3721 TGGCAAATAT TGACGGCCAG TTAAGCCATT CATGCCAGTA GGCGCGCGGA CGAAAGTAAA

 3781 CCCACTGGTG ATACCATTCG CGAGCCTCCG GATGACGACC GTAGTGATGA ATCTCTCCTG

 3841 GCGGGAACAG CAAAATATCA CCCGGTCGGC AAACAAATTC TCGTCCCTGA TTTTTCACCA

 3901 CCCCCTGACC GCGAATGGTG AGATTGAGAA TATAACCTTT CATTCCCAGC GGTCGGTCGA

 3961 TAAAAAAATC GAGATAACCG TTGGCCTCAA TCGGCGTTAA ACCCGCCACC AGATGGGCAT

 4021 TAAACGAGTA TCCCGGCAGC AGGGGATCAT TTTGCGCTTC AGCCATACTT TTCATACTCC

 4081 CGCCATTCAG AG

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