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基本信息

質粒圖譜和多克隆位點信息

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載體描述

pBAD/His和PBAD/Myc-His載體質粒是衍生于pBR322載體。載體設計用來在大腸桿菌中進行可調節，劑量依賴性的表達和純化重組目的蛋白。使用大腸桿菌araBAD啟動子（pBAD）增強了大腸桿菌重組蛋白可溶性表達的水平。pBAD/His和pBAD/Myc His載體上的調節蛋白AraC能夠調控pBad啟動子。

pBAD/His A,B,C 載體介紹

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:

The araBAD promoter for tightly regulated expression

Translation initiation signals optimized for E. coliexpression

 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-阿拉伯糖調控表達

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.

載體序列

LOCUS       pBAD/His A    4102 bp     DNA    circular    SYN

DEFINITION  pBAD/His A

ACCESSION   

KEYWORDS

FEATURES             Location/Qualifiers

     source          1..4102

                     /organism="pBAD/His A"

                     /mol_type="other DNA"

     misc_feature    4..19

                     /label="araO2"

     misc_feature    161..172

                     /label="araO1"

     misc_feature    203..216

                     /label="CAP_BS"

     misc_feature    208..227

                     /label="pBAD_fwd_primer"

     misc_feature    213..251

                     /label="AraI1I2"

     promoter        248..276

                     /label="ARA_promoter"

     misc_feature    351..369

                     /label="Xpress_fwd_primer"

     misc_feature    352..384

                     /label="T7_leader"

     misc_feature    388..411

                     /label="Xpress_EK"

     misc_feature    complement(503..520)

                     /label="pBAD_rev_primer"

     misc_feature    complement(503..520)

                     /label="pTrcHis_rev_primer"

     terminator      553..710

                     /label="rrnB_terminator"

     terminator      676..719

                     /label="rrnB_T1_terminator"

     terminator      851..878

                     /label="rrnB_T2_terminator"

     promoter        919..947

                     /label="AmpR_promoter"

     gene            989..1849

                     /label="Ampicillin"

                     /gene="Ampicillin"

     CDS             989..1849

                     /label="ORF frame 2"

     rep_origin      2004..2623

                     /label="pBR322_origin"

     misc_feature    3020..3042

                     /label="pGEX_3_primer"

     misc_feature    complement(3198..4076)

                     /label="AraC"

     CDS             complement(3198..25)

                     /label="ORF frame 1"

     CDS             complement(3198..4085)

                     /label="ORF frame 3"

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 GGATCGATGG

  421 GGATCCGAGC TCGAGATCTG CAGCTGGTAC CATATGGGAA TTCGAAGCTT GGCTGTTTTG

  481 GCGGATGAGA GAAGATTTTC AGCCTGATAC AGATTAAATC AGAACGCAGA AGCGGTCTGA

  541 TAAAACAGAA TTTGCCTGGC GGCAGTAGCG CGGTGGTCCC ACCTGACCCC ATGCCGAACT

  601 CAGAAGTGAA ACGCCGTAGC GCCGATGGTA GTGTGGGGTC TCCCCATGCG AGAGTAGGGA

  661 ACTGCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT TCGTTTTATC

  721 TGTTGTTTGT CGGTGAACGC TCTCCTGAGT AGGACAAATC CGCCGGGAGC GGATTTGAAC

  781 GTTGCGAAGC AACGGCCCGG AGGGTGGCGG GCAGGACGCC CGCCATAAAC TGCCAGGCAT

  841 CAAATTAAGC AGAAGGCCAT CCTGACGGAT GGCCTTTTTG CGTTTCTACA AACTCTTTTG

  901 TTTATTTTTC TAAATACATT CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT

  961 GCTTCAATAA TATTGAAAAA GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT

 1021 TCCCTTTTTT GCGGCATTTT GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT

 1081 AAAAGATGCT GAAGATCAGT TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG

 1141 CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA

 1201 AGTTCTGCTA TGTGGCGCGG TATTATCCCG TGTTGACGCC GGGCAAGAGC AACTCGGTCG

 1261 CCGCATACAC TATTCTCAGA ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT

 1321 TACGGATGGC ATGACAGTAA GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC

 1381 TGCGGCCAAC TTACTTCTGA CAACGATCGG AGGACCGAAG GAGCTAACCG CTTTTTTGCA

 1441 CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT

 1501 ACCAAACGAC GAGCGTGACA CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT

 1561 ATTAACTGGC GAACTACTTA CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC

 1621 GGATAAAGTT GCAGGACCAC TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA

 1681 TAAATCTGGA GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG

 1741 TAAGCCCTCC CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG

 1801 AAATAGACAG ATCGCTGAGA TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA

 1861 AGTTTACTCA TATATACTTT AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA

 1921 GGTGAAGATC CTTTTTGATA ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA

 1981 CTGAGCGTCA GACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG

 2041 CGTAATCTGC TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA

 2101 TCAAGAGCTA CCAACTCTTT TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA

 2161 TACTGTCCTT CTAGTGTAGC CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC

 2221 TACATACCTC GCTCTGCTAA TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG

 2281 TCTTACCGGG TTGGACTCAA GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC

 2341 GGGGGGTTCG TGCACACAGC CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT

 2401 ACAGCGTGAG CTATGAGAAA GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC

 2461 GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG

 2521 GTATCTTTAT AGTCCTGTCG GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG

 2581 CTCGTCAGGG GGGCGGAGCC TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT

 2641 GGCCTTTTGC TGGCCTTTTG CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA

 2701 TAACCGTATT ACCGCCTTTG AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG

 2761 CAGCGAGTCA GTGAGCGAGG AAGCGGAAGA GCGCCTGATG CGGTATTTTC TCCTTACGCA

 2821 TCTGTGCGGT ATTTCACACC GCATATGGTG CACTCTCAGT ACAATCTGCT CTGATGCCGC

 2881 ATAGTTAAGC CAGTATACAC TCCGCTATCG CTACGTGACT GGGTCATGGC TGCGCCCCGA

 2941 CACCCGCCAA CACCCGCTGA CGCGCCCTGA CGGGCTTGTC TGCTCCCGGC ATCCGCTTAC

 3001 AGACAAGCTG TGACCGTCTC CGGGAGCTGC ATGTGTCAGA GGTTTTCACC GTCATCACCG

 3061 AAACGCGCGA GGCAGCAGAT CAATTCGCGC GCGAAGGCGA AGCGGCATGC ATAATGTGCC

 3121 TGTCAAATGG ACGAAGCAGG GATTCTGCAA ACCCTATGCT ACTCCGTCAA GCCGTCAATT

 3181 GTCTGATTCG TTACCAATTA TGACAACTTG ACGGCTACAT CATTCACTTT TTCTTCACAA

 3241 CCGGCACGGA ACTCGCTCGG GCTGGCCCCG GTGCATTTTT TAAATACCCG CGAGAAATAG

 3301 AGTTGATCGT CAAAACCAAC ATTGCGACCG ACGGTGGCGA TAGGCATCCG GGTGGTGCTC

 3361 AAAAGCAGCT TCGCCTGGCT GATACGTTGG TCCTCGCGCC AGCTTAAGAC GCTAATCCCT

 3421 AACTGCTGGC GGAAAAGATG TGACAGACGC GACGGCGACA AGCAAACATG CTGTGCGACG

 3481 CTGGCGATAT CAAAATTGCT GTCTGCCAGG TGATCGCTGA TGTACTGACA AGCCTCGCGT

 3541 ACCCGATTAT CCATCGGTGG ATGGAGCGAC TCGTTAATCG CTTCCATGCG CCGCAGTAAC

 3601 AATTGCTCAA GCAGATTTAT CGCCAGCAGC TCCGAATAGC GCCCTTCCCC TTGCCCGGCG

 3661 TTAATGATTT GCCCAAACAG GTCGCTGAAA TGCGGCTGGT GCGCTTCATC CGGGCGAAAG

 3721 AACCCCGTAT TGGCAAATAT TGACGGCCAG TTAAGCCATT CATGCCAGTA GGCGCGCGGA

 3781 CGAAAGTAAA CCCACTGGTG ATACCATTCG CGAGCCTCCG GATGACGACC GTAGTGATGA

 3841 ATCTCTCCTG GCGGGAACAG CAAAATATCA CCCGGTCGGC AAACAAATTC TCGTCCCTGA

 3901 TTTTTCACCA CCCCCTGACC GCGAATGGTG AGATTGAGAA TATAACCTTT CATTCCCAGC

 3961 GGTCGGTCGA TAAAAAAATC GAGATAACCG TTGGCCTCAA TCGGCGTTAA ACCCGCCACC

 4021 AGATGGGCAT TAAACGAGTA TCCCGGCAGC AGGGGATCAT TTTGCGCTTC AGCCATACTT

 4081 TTCATACTCC CGCCATTCAG AG

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