pMal-p4X

pMalp4X载体基本信息

pMalp4X载体质粒图谱和多克隆位点信息

pMalp4X载体简介

The pMAL™-4 vectors provide a method for expressing and purifying a protein produced from a cloned gene or open reading frame. The cloned gene is inserted downstream from the malE gene of E. coli, which encodes maltose-binding protein (MBP), resulting in the expression of an MBP fusion protein (1,2). The MBP in these vectors has been engineered for tighter binding to amylose. The method uses the strong “tac” promoter and the malE translation initiation signals to give high-level expression of the cloned sequences (3,4), and a one-step purification of the fusion protein using MBP’s affinity for maltose (5). The vectors express the malE gene (with or without its signal sequence) fused to the lacZα gene. Restriction sites between malE and lacZα are available for inserting the coding sequence of interest. Insertion inactivates the β-galactosidase α-fragment activity of the malE-lacZα fusion, which results in a blue to white color change on Xgal plates when the construction is transformed into an α-complementing host such as TB1, JM107 or NEB 5-alpha Competent E. coli (6,7). When present, the signal peptide on pre-MBP directs fusion proteins to the periplasm. For fusion proteins that can be successfully exported, this allows folding and disulfide bond formation to take place in the periplasm of E. coli, as well as allowing purification of the protein from the periplasm (8). The vectors carry the lacIq gene, which codes for the Lac repressor. This keeps expression from Ptac low in the absence of IPTG induction. The pMAL-4 vectors also contain the sequence coding for the recognition site of a specific protease, located just 5´ to the polylinker insertion sites. This allows MBP to be cleaved from the protein of interest after purification. The pMAL-c4X and pMAL-p4X vectors that are included in the system encode the site for Factor Xa (9, 10). Factor Xa cleaves after its four amino acid recognition sequence, so that few or no vector-derived residues are attached to the protein of interest, depending on the site used for cloning. pMAL vectors containing sites for alternative proteases are also available. The vectors pMAL-c4G (NEB #N8106) and pMAL-p4G encode the site for Genenase™I , which cleaves following the sequence His-Tyr. The vectors pMAL-c4E and pMAL-p4E encode the site for Enterokinase (NEB #P8070), which cleaves following the sequence Asp-Asp-Asp-Asp-Lys.

pMalp4X载体序列

ORIGIN

    1 CCGACACCAT CGAATGGTGC AAAACCTTTC GCGGTATGGC ATGATAGCGC CCGGAAGAGA

   61 GTCAATTCAG GGTGGTGAAT GTGAAACCAG TAACGTTATA CGATGTCGCA GAGTATGCCG

  121 GTGTCTCTTA TCAGACCGTT TCCCGCGTGG TGAACCAGGC CAGCCACGTT TCTGCGAAAA

  181 CGCGGGAAAA AGTGGAAGCG GCGATGGCGG AGCTGAATTA CATTCCCAAC CGCGTGGCAC

  241 AACAACTGGC GGGCAAACAG TCGTTGCTGA TTGGCGTTGC CACCTCCAGT CTGGCCCTGC

  301 ACGCGCCGTC GCAAATTGTC GCGGCGATTA AATCTCGCGC CGATCAACTG GGTGCCAGCG

  361 TGGTGGTGTC GATGGTAGAA CGAAGCGGCG TCGAAGCCTG TAAAGCGGCG GTGCACAATC

  421 TTCTCGCGCA ACGCGTCAGT GGGCTGATCA TTAACTATCC GCTGGATGAC CAGGATGCCA

  481 TTGCTGTGGA AGCTGCCTGC ACTAATGTTC CGGCGTTATT TCTTGATGTC TCTGACCAGA

  541 CACCCATCAA CAGTATTATT TTCTCCCATG AAGACGGTAC GCGACTGGGC GTGGAGCATC

  601 TGGTCGCATT GGGTCACCAG CAAATCGCGC TGTTAGCGGG CCCATTAAGT TCTGTCTCGG

  661 CGCGTCTGCG TCTGGCTGGC TGGCATAAAT ATCTCACTCG CAATCAAATT CAGCCGATAG

  721 CGGAACGGGA AGGCGACTGG AGTGCCATGT CCGGTTTTCA ACAAACCATG CAAATGCTGA

  781 ATGAGGGCAT CGTTCCCACT GCGATGCTGG TTGCCAACGA TCAGATGGCG CTGGGCGCAA

  841 TGCGCGCCAT TACCGAGTCC GGGCTGCGCG TTGGTGCGGA TATCTCGGTA GTGGGATACG

  901 ACGATACCGA AGACAGCTCA TGTTATATCC CGCCGTTAAC CACCATCAAA CAGGATTTTC

  961 GCCTGCTGGG GCAAACCAGC GTGGACCGCT TGCTGCAACT CTCTCAGGGC CAGGCGGTGA

 1021 AGGGCAATCA GCTGTTGCCC GTCTCACTGG TGAAAAGAAA AACCACCCTG GCGCCCAATA

 1081 CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA CGACAGGTTT

 1141 CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TAAGTTAGCT CACTCATTAG

 1201 GCACAATTCT CATGTTTGAC AGCTTATCAT CGACTGCACG GTGCACCAAT GCTTCTGGCG

 1261 TCAGGCAGCC ATCGGAAGCT GTGGTATGGC TGTGCAGGTC GTAAATCACT GCATAATTCG

 1321 TGTCGCTCAA GGCGCACTCC CGTTCTGGAT AATGTTTTTT GCGCCGACAT CATAACGGTT

 1381 CTGGCAAATA TTCTGAAATG AGCTGTTGAC AATTAATCAT CGGCTCGTAT AATGTGTGGA

 1441 ATTGTGAGCG GATAACAATT TCACACAGGA AACAGCCAGT CCGTTTAGGT GTTTTCACGA

 1501 GCACTTCACC AACAAGGACC ATAGCATATG AAAATAAAAA CAGGTGCACG CATCCTCGCA

 1561 TTATCCGCAT TAACGACGAT GATGTTTTCC GCCTCGGCTC TCGCCAAAAT CGAAGAAGGT

 1621 AAACTGGTAA TCTGGATTAA CGGCGATAAA GGCTATAACG GTCTCGCTGA AGTCGGTAAG

 1681 AAATTCGAGA AAGATACCGG AATTAAAGTC ACCGTTGAGC ATCCGGATAA ACTGGAAGAG

 1741 AAATTCCCAC AGGTTGCGGC AACTGGCGAT GGCCCTGACA TTATCTTCTG GGCACACGAC

 1801 CGCTTTGGTG GCTACGCTCA ATCTGGCCTG TTGGCTGAAA TCACCCCGGA CAAAGCGTTC

 1861 CAGGACAAGC TGTATCCGTT TACCTGGGAT GCCGTACGTT ACAACGGCAA GCTGATTGCT

 1921 TACCCGATCG CTGTTGAAGC GTTATCGCTG ATTTATAACA AAGATCTGCT GCCGAACCCG

 1981 CCAAAAACCT GGGAAGAGAT CCCGGCGCTG GATAAAGAAC TGAAAGCGAA AGGTAAGAGC

 2041 GCGCTGATGT TCAACCTGCA AGAACCGTAC TTCACCTGGC CGCTGATTGC TGCTGACGGG

 2101 GGTTATGCGT TCAAGTATGA AAACGGCAAG TACGACATTA AAGACGTGGG CGTGGATAAC

 2161 GCTGGCGCGA AAGCGGGTCT GACCTTCCTG GTTGACCTGA TTAAAAACAA ACACATGAAT

 2221 GCAGACACCG ATTACTCCAT CGCAGAAGCT GCCTTTAATA AAGGCGAAAC AGCGATGACC

 2281 ATCAACGGCC CGTGGGCATG GTCCAACATC GACACCAGCA AAGTGAATTA TGGTGTAACG

 2341 GTACTGCCGA CCTTCAAGGG TCAACCATCC AAACCGTTCG TTGGCGTGCT GAGCGCAGGT

 2401 ATTAACGCCG CCAGTCCGAA CAAAGAGCTG GCAAAAGAGT TCCTCGAAAA CTATCTGCTG

 2461 ACTGATGAAG GTCTGGAAGC GGTTAATAAA GACAAACCGC TGGGTGCCGT AGCGCTGAAG

 2521 TCTTACGAGG AAGAGTTGGT GAAAGATCCG CGGATTGCCG CCACTATGGA AAACGCCCAG

 2581 AAAGGTGAAA TCATGCCGAA CATCCCGCAG ATGTCCGCTT TCTGGTATGC CGTGCGTACT

 2641 GCGGTGATCA ACGCCGCCAG CGGTCGTCAG ACTGTCGATG AAGCCCTGAA AGACGCGCAG

 2701 ACTAATTCGA GCTCGAACAA CAACAACAAT AACAATAACA ACAACCTCGG GATCGAGGGA

 2761 AGGATTTCAG AATTCGGATC CTCTAGAGTC GACCTGCAGG CAAGCTTGGC ACTGGCCGTC

 2821 GTTTTACAAC GTCGTGACTG GGAAAACCCT GGCGTTACCC AACTTAATCG CCTTGCAGCA

 2881 CATCCCCCTT TCGCCAGCTG GCGTAATAGC GAAGAGGCCC GCACCGATCG CCCTTCCCAA

 2941 CAGTTGCGCA GCCTGAATGG CGAATGGCAG CTTGGCTGTT TTGGCGGATG AGATAAGATT

 3001 TTCAGCCTGA TACAGATTAA ATCAGAACGC AGAAGCGGTC TGATAAAACA GAATTTGCCT

 3061 GGCGGCAGTA GCGCGGTGGT CCCACCTGAC CCCATGCCGA ACTCAGAAGT GAAACGCCGT

 3121 AGCGCCGATG GTAGTGTGGG GTCTCCCCAT GCGAGAGTAG GGAACTGCCA GGCATCAAAT

 3181 AAAACGAAAG GCTCAGTCGA AAGACTGGGC CTTTCGTTTT ATCTGTTGTT TGTCGGTGAA

 3241 CGCTCTCCTG AGTAGGACAA ATCCGCCGGG AGCGGATTTG AACGTTGCGA AGCAACGGCC

 3301 CGGAGGGTGG CGGGCAGGAC GCCCGCCATA AACTGCCAGG CATCAAATTA AGCAGAAGGC

 3361 CATCCTGACG GATGGCCTTT TTGCGTTTCT ACAAACTCTT TTGTTTATTT TTCTAAATAC

 3421 ATTCAAATAT GTATCCGCTC ATGAGACAAT AACCCTGATA AATGCTTCAA TAATATTGAA

 3481 AAAGGAAGAG TATGAGTATT CAACATTTCC GTGTCGCCCT TATTCCCTTT TTTGCGGCAT

 3541 TTTGCCTTCC TGTTTTTGCT CACCCAGAAA CGCTGGTGAA AGTAAAAGAT GCTGAAGATC

 3601 AGTTGGGTGC ACGAGTGGGT TACATCGAAC TGGATCTCAA CAGCGGTAAG ATCCTTGAGA

 3661 GTTTTCGCCC CGAAGAACGT TTCCCAATGA TGAGCACTTT TAAAGTTCTG CTATGTGGCG

 3721 CGGTATTATC CCGTGTTGAC GCCGGGCAAG AGCAACTCGG TCGCCGCATA CACTATTCTC

 3781 AGAATGACTT GGTTGAGTAC TCACCAGTCA CAGAAAAGCA TCTTACGGAT GGCATGACAG

 3841 TAAGAGAATT ATGCAGTGCT GCCATAACCA TGAGTGATAA CACTGCGGCC AACTTACTTC

 3901 TGACAACGAT CGGAGGACCG AAGGAGCTAA CCGCTTTTTT GCACAACATG GGGGATCATG

 3961 TAACTCGCCT TGATCGTTGG GAACCGGAGC TGAATGAAGC CATACCAAAC GACGAGCGTG

 4021 ACACCACGAT GCCTGTAGCA ATGGCAACAA CGTTGCGCAA ACTATTAACT GGCGAACTAC

 4081 TTACTCTAGC TTCCCGGCAA CAATTAATAG ACTGGATGGA GGCGGATAAA GTTGCAGGAC

 4141 CACTTCTGCG CTCGGCCCTT CCGGCTGGCT GGTTTATTGC TGATAAATCT GGAGCCGGTG

 4201 AGCGTGGGTC TCGCGGTATC ATTGCAGCAC TGGGGCCAGA TGGTAAGCCC TCCCGTATCG

 4261 TAGTTATCTA CACGACGGGG AGTCAGGCAA CTATGGATGA ACGAAATAGA CAGATCGCTG

 4321 AGATAGGTGC CTCACTGATT AAGCATTGGT AACTGTCAGA CCAAGTTTAC TCATATATAC

 4381 TTTAGATTGA TTTACCCCGG TTGATAATCA GAAAAGCCCC AAAAACAGGA AGATTGTATA

 4441 AGCAAATATT TAAATTGTAA ACGTTAATAT TTTGTTAAAA TTCGCGTTAA ATTTTTGTTA

 4501 AATCAGCTCA TTTTTTAACC AATAGGCCGA AATCGGCAAA ATCCCTTATA AATCAAAAGA

 4561 ATAGACCGAG ATAGGGTTGA GTGTTGTTCC AGTTTGGAAC AAGAGTCCAC TATTAAAGAA

 4621 CGTGGACTCC AACGTCAAAG GGCGAAAAAC CGTCTATCAG GGCGATGGCC CACTACGTGA

 4681 ACCATCACCC AAATCAAGTT TTTTGGGGTC GAGGTGCCGT AAAGCACTAA ATCGGAACCC

 4741 TAAAGGGAGC CCCCGATTTA GAGCTTGACG GGGAAAGCCG GCGAACGTGG CGAGAAAGGA

 4801 AGGGAAGAAA GCGAAAGGAG CGGGCGCTAG GGCGCTGGCA AGTGTAGCGG TCACGCTGCG

 4861 CGTAACCACC ACACCCGCCG CGCTTAATGC GCCGCTACAG GGCGCGTAAA AGGATCTAGG

 4921 TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA ACGTGAGTTT TCGTTCCACT

 4981 GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG

 5041 TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC

 5101 AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA

 5161 CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA

 5221 CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC

 5281 TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG

 5341 GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC

 5401 AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG

 5461 TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT

 5521 ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT

 5581 CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG

 5641 CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA

 5701 ACCGTATTAC CGCCTTTGAG TGAGCTGATA CCGCTCGCCG CAGCCGAACG ACCGAGCGCA

 5761 GCGAGTCAGT GAGCGAGGAA GCGGAAGAGC GCCTGATGCG GTATTTTCTC CTTACGCATC

 5821 TGTGCGGTAT TTCACACCGC ATATATGGTG CACTCTCAGT ACAATCTGCT CTGATGCCGC

 5881 ATAGTTAAGC CAGTATACAC TCCGCTATCG CTACGTGACT GGGTCATGGC TGCGCCCCGA

 5941 CACCCGCCAA CACCCGCTGA CGCGCCCTGA CGGGCTTGTC TGCTCCCGGC ATCCGCTTAC

 6001 AGACAAGCTG TGACCGTCTC CGGGAGCTGC ATGTGTCAGA GGTTTTCACC GTCATCACCG

 6061 AAACGCGCGA GGCAGCTGCG GTAAAGCTCA TCAGCGTGGT CGTGCAGCGA TTCACAGATG

 6121 TCTGCCTGTT CATCCGCGTC CAGCTCGTTG AGTTTCTCCA GAAGCGTTAA TGTCTGGCTT

 6181 CTGATAAAGC GGGCCATGTT AAGGGCGGTT TTTTCCTGTT TGGTCACTGA TGCCTCCGTG

 6241 TAAGGGGGAT TTCTGTTCAT GGGGGTAATG ATACCGATGA AACGAGAGAG GATGCTCACG

 6301 ATACGGGTTA CTGATGATGA ACATGCCCGG TTACTGGAAC GTTGTGAGGG TAAACAACTG

 6361 GCGGTATGGA TGCGGCGGGA CCAGAGAAAA ATCACTCAGG GTCAATGCCA GCGCTTCGTT

 6421 AATACAGATG TAGGTGTTCC ACAGGGTAGC CAGCAGCATC CTGCGATGCA GATCCGGAAC

 6481 ATAATGGTGC AGGGCGCTGA CTTCCGCGTT TCCAGACTTT ACGAAACACG GAAACCGAAG

 6541 ACCATTCATG TTGTTGCTCA GGTCGCAGAC GTTTTGCAGC AGCAGTCGCT TCACGTTCGC

 6601 TCGCGTATCG GTGATTCATT CTGCTAACCA GTAAGGCAAC CCCGCCAGCC TAGCCGGGTC

 6661 CTCAACGACA GGAGCACGAT CATGCGCACC CGTGGCCAGG ACCCAACGCT GCCCGAAATT

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其他大肠杆菌表达载体：