pTYB12

pTYB12

pTYB11载体基本信息

pTYB11载体质粒图谱和多克隆位点信息

pTYB11载体简介

pTYB11 is an E. coli plasmid cloning vector designed for recombinant protein expression and purification using the IMPACT? Kit (NEB #E6901) (1). It contains the pMB1 origin of replication from pBR322 and is maintained at a similar copy number to pBR322; in addition, pTYB11 also contains an M13 origin of replication.

The multiple cloning site (MCS) is positioned to allow translational fusion of the Sce VMA intein tag to the N-terminus of the cloned target protein (1). The chitin binding domain (CBD) from B. circulans, embedded in the intein, facilitates purification of the intein-target protein precursor.

Transcription of the gene fusion is controlled by the inducible T7 promoter, requiring E. coli strains containing integrated copies of the T7 RNA polymerase gene [e.g., C2566, C2833 or BL21(DE3)] for expression. Basal expression from the T7 promoter is minimized by the binding of the Lac repressor, encoded by the lacI gene, to the lac operator immediately downstream of the T7 promoter (2). Translation of the fusion utilizes the translation initiation signal (Shine Dalgarno sequence) from the strongly expressed T7 gene 10 protein (φ10).

pTYB12 is identical to pTYB11 except for the MCS regions

Enzymes with unique restriction sites are shown in bold type. Location of sites of all NEB restriction enzymes can be found on the NEB web site (choose Technical Reference > DNA Sequences and Maps). Restriction site coordinates refer to the position of the 5′-most base on the top strand in each recognition sequence.

Open reading frame (ORF) coordinates are in the form "translational start – translational stop”; numbers refer to positions on the top (clockwise) strand, regardless of the direction of transcription and include the start and stop codons. Component genes or regions of fusion ORFs are indented below the ORF itself.

pMB1 origin of replication coordinates include the region from the -35 promoter sequence of the RNAII transcript to the RNA/DNA switch point. For the M13 origin, the arrow shows the direction of synthesis of the (+) strand, which gets packaged into phage particles. bla (ApR) gene coordinates include the signal sequence.

pTYB11载体序列

ORIGIN

    1 AGAAGGCCAT CCTGACGGAT GGCCTTTTTG CGTTTCTACA AACTCTTTTT GTTTATTTTT

   61 CTAAATACAT TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA

  121 ATATTGAAAA AGGAAGAGTA TGAGTATTCA ACATTTCCGT GTCGCCCTTA TTCCCTTTTT

  181 TGCGGCATTT TGCCTTCCTG TTTTTGCTCA CCCAGAAACG CTGGTGAAAG TAAAAGATGC

  241 TGAAGATCAG TTGGGTGCAC GAGTGGGTTA CATCGAACTG GATCTCAACA GCGGTAAGAT

  301 CCTTGAGAGT TTTCGCCCCG AAGAACGTTC TCCAATGATG AGCACTTTTA AAGTTCTGCT

  361 ATGTGGCGCG GTATTATCCC GTGTTGACGC CGGGCAAGAG CAACTCGGTC GCCGCATACA

  421 CTATTCTCAG AATGACTTGG TTGAGTACTC ACCAGTCACA GAAAAGCATC TTACGGATGG

  481 CATGACAGTA AGAGAATTAT GCAGTGCTGC CATAACCATG AGTGATAACA CTGCGGCCAA

  541 CTTACTTCTG ACAACGATCG GAGGACCGAA GGAGCTAACC GCTTTTTTGC ACAACATGGG

  601 GGATCATGTA ACTCGCCTTG ATCGTTGGGA ACCGGAGCTG AATGAAGCCA TACCAAACGA

  661 CGAGCGTGAC ACCACGATGC CTGTAGCAAT GGCAACAACG TTGCGCAAAC TATTAACTGG

  721 CGAACTACTT ACTCTAGCTT CCCGGCAACA ATTAATAGAC TGGATGGAGG CGGATAAAGT

  781 TGCAGGACCA CTTCTGCGCT CGGCCCTTCC GGCTGGCTGG TTTATTGCTG ATAAATCTGG

  841 AGCCGGTGAG CGTGGGTCTC GCGGTATCAT TGCAGCACTG GGGCCAGATG GTAAGCCCTC

  901 CCGTATCGTA GTTATCTACA CGACGGGGAG TCAGGCAACT ATGGATGAAC GAAATAGACA

  961 GATCGCTGAG ATAGGTGCCT CACTGATTAA GCATTGGTAA CTGTCAGACC AAGTTTACTC

 1021 ATATATACTT TAGATTGATT TACCCCGGTT GATAATCAGA AAAGCCCCAA AAACAGGAAG

 1081 ATTGTATAAG CAAATATTTA AATTGTAAAC GTTAATATTT TGTTAAAATT CGCGTTAAAT

 1141 TTTTGTTAAA TCAGCTCATT TTTTAACCAA TAGGCCGAAA TCGGCAAAAT CCCTTATAAA

 1201 TCAAAAGAAT AGCCCGAGAT AGGGTTGAGT GTTGTTCCAG TTTGGAACAA GAGTCCACTA

 1261 TTAAAGAACG TGGACTCCAA CGTCAAAGGG CGAAAAACCG TCTATCAGGG CGATGGCCCA

 1321 CTACGTGAAC CATCACCCAA ATCAAGTTTT TTGGGGTCGA GGTGCCGTAA AGCACTAAAT

 1381 CGGAACCCTA AAGGGAGCCC CCGATTTAGA GCTTGACGGG GAAAGCCGGC GAACGTGGCG

 1441 AGAAAGGAAG GGAAGAAAGC GAAAGGAGCG GGCGCTAGGG CGCTGGCAAG TGTAGCGGTC

 1501 ACGCTGCGCG TAACCACCAC ACCCGCCGCG CTTAATGCGC CGCTACAGGG CGCGTAAAAG

 1561 GATCTAGGTG AAGATCCTTT TTGATAATCT CATGACCAAA ATCCCTTAAC GTGAGTTTTC

 1621 GTTCCACTGA GCGTCAGACC CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT

 1681 TCTGCGCGTA ATCTGCTGCT TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT

 1741 GCCGGATCAA GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT

 1801 ACCAAATACT GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA ACTCTGTAGC

 1861 ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA

 1921 GTCGTGTCTT ACCGGGTTGG ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG

 1981 CTGAACGGGG GGTTCGTGCA CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG

 2041 ATACCTACAG CGTGAGCTAT GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG

 2101 GTATCCGGTA AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA

 2161 CGCCTGGTAT CTTTATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT

 2221 GTGATGCTCG TCAGGGGGGC GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG

 2281 GTTCCTGGCC TTTTGCTGGC CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC

 2341 TGTGGATAAC CGTATTACCG CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC

 2401 CGAGCGCAGC GAGTCAGTGA GCGAGGAAGC TATGGTGCAC TCTCAGTACA ATCTGCTCTG

 2461 ATGCCGCATA GTTAAGCCAG TATACACTCC GCTATCGCTA CGTGACTGGG TCATGGCTGC

 2521 GCCCCGACAC CCGCCAACAC CCGCTGACGC GCCCTGACGG GCTTGTCTGC TCCCGGCATC

 2581 CGCTTACAGA CAAGCTGTGA CCGTCTCCGG GAGCTGCATG TGTCAGAGGT TTTCACCGTC

 2641 ATCACCGAAA CGCGCGAGGC AGCTGCGGTA AAGCTCATCA GCGTGGTCGT GCAGCGATTC

 2701 ACAGATGTCT GCCTGTTCAT CCGCGTCCAG CTCGTTGAGT TTCTCCAGAA GCGTTAATGT

 2761 CTGGCTTCTG ATAAAGCGGG CCATGTTAAG GGCGGTTTTT TCCTGTTTGG TCACTGATGC

 2821 CTCCGTGTAA GGGGGATTTC TGTTCATGGG GGTAATGATA CCGATGAAAC GAGAGAGGAT

 2881 GCTCACGATA CGGGTTACTG ATGATGAACA TGCCCGGTTA CTGGAACGTT GTGAGGGTAA

 2941 ACAACTGGCG GTATGGATGC GGCGGGACCA GAGAAAAATC ACTCAGGGTC AATGCCAGCC

 3001 GAACGCCAGC AAGACGTAGC CCAGCGCGTC GGCCGCCATG CCGGCGATAA TGGCCTGCTT

 3061 CTCGCCGAAA CGTTTGGTGG CGGGACCAGT GACGAAGGCT TGAGCGAGGG CGTGCAAGAT

 3121 TCCGAATACC GCAAGCGACA GGCCGATCAT CGTCGCGCTC CAGCGAAAGC GGTCCTCGCC

 3181 GAAAATGACC CAGAGCGCTG CCGGCACCTG TCCTACGAGT TGCATGATAA AGAAGACAGT

 3241 CATAAGTGCG GCGACGATAG TCATGCCCCG CGCCCACCGG AAGGAGCTGA CTGGGTTGAA

 3301 GGCTCTCAAG GGCATCGGTC GAGATCCCGG TGCCTAATGA GTGAGCTAAC TTACATTAAT

 3361 TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCAGC TGCATTAATG

 3421 AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCCAGGGTG GTTTTTCTTT

 3481 TCACCAGTGA GACGGGCAAC AGCTGATTGC CCTTCACCGC CTGGCCCTGA GAGAGTTGCA

 3541 GCAAGCGGTC CACGCTGGTT TGCCCCAGCA GGCGAAAATC CTGTTTGATG GTGGTTAACG

 3601 GCGGGATATA ACATGAGCTG TCTTCGGTAT CGTCGTATCC CACTACCGAG ATATCCGCAC

 3661 CAACGCGCAG CCCGGACTCG GTAATGGCGC GCATTGCGCC CAGCGCCATC TGATCGTTGG

 3721 CAACCAGCAT CGCAGTGGGA ACGATGCCCT CATTCAGCAT TTGCATGGTT TGTTGAAAAC

 3781 CGGACATGGC ACTCCAGTCG CCTTCCCGTT CCGCTATCGG CTGAATTTGA TTGCGAGTGA

 3841 GATATTTATG CCAGCCAGCC AGACGCAGAC GCGCCGAGAC AGAACTTAAT GGGCCCGCTA

 3901 ACAGCGCGAT TTGCTGGTGA CCCAATGCGA CCAGATGCTC CACGCCCAGT CGCGTACCGT

 3961 CTTCATGGGA GAAAATAATA CTGTTGATGG GTGTCTGGTC AGAGACATCA AGAAATAACG

 4021 CCGGAACATT AGTGCAGGCA GCTTCCACAG CAATGGCATC CTGGTCATCC AGCGGATAGT

 4081 TAATGATCAG CCCACTGACG CGTTGCGCGA GAAGATTGTG CACCGCCGCT TTACAGGCTT

 4141 CGACGCCGCT TCGTTCTACC ATCGACACCA CCACGCTGGC ACCCAGTTGA TCGGCGCGAG

 4201 ATTTAATCGC CGCGACAATT TGCGACGGCG CGTGCAGGGC CAGACTGGAG GTGGCAACGC

 4261 CAATCAGCAA CGACTGTTTG CCCGCCAGTT GTTGTGCCAC GCGGTTGGGA ATGTAATTCA

 4321 GCTCCGCCAT CGCCGCTTCC ACTTTTTCCC GCGTTTTCGC AGAAACGTGG CTGGCCTGGT

 4381 TCACCACGCG GGAAACGGTC TGATAAGAGA CACCGGCATA CTCTGCGACA TCGTATAACG

 4441 TTACTGGTTT CACATTCACC ACCCTGAATT GACTCTCTTC CGGGCGCTAT CATGCCATAC

 4501 CGCGAAAGGT TTTGCGCCAT TCGATGGTGT CCGGGATCTC GACGCTCTCC CTTATGCGAC

 4561 TCCTGCATTA GGAAGCAGCC CAGTAGTAGG TTGAGGCCGT TGAGCACCGC CGCCGCAAGG

 4621 AATGGTGCAT GCAAGGAGAT GGCGCCCAAC AGTCCCCCGG CCACGGGGCC TGCCACCATA

 4681 CCCACGCCGA AACAAGCGCT CATGAGCCCG AAGTGGCGAG CCCGATCTTC CCCATCGGTG

 4741 ATGTCGGCGA TATAGGCGCC AGCAACCGCA CCTGTGGCGC CGGTGATGCC GGCCACGATG

 4801 CGTCCGGCGT AGAGGATCGA GATCTCGATC CCGCGAAATT AATACGACTC ACTATAGGGG

 4861 AATTGTGAGC GGATAACAAT TCCCCTCTAG AAATAATTTT GTTTAACTTT AAGAAGGAGA

 4921 TATACTTATG AAAATCGAAG AAGGTAAACT GGTAATCGGT TCCCTGGAGG GTTGCTTTGC

 4981 CAAGGGTACC AATGTTTTAA TGGCGGATGG GTCTATTGAA TGTATTGAAA ACATTGAGGT

 5041 TGGTAATAAG GTCATGGGTA AAGATGGCAG ACCTCGTGAG GTAATTAAAT TGCCCAGAGG

 5101 AAGAGAAACT ATGTACAGCG TCGTGCAGAA AAGTCAGCAC AGAGCCCACA AAAGTGACTC

 5161 AAGTCGTGAA GTGCCAGAAT TACTCAAGTT TACGTGTAAT GCGACCCATG AGTTGGTTGT

 5221 TAGAACACCT CGTAGTGTCC GCCGTTTGTC TCGTACCATT AAGGGTGTCG AATATTTTGA

 5281 AGTTATTACT TTTGAGATGG GCCAAAAGAA AGCCCCCGAC GGTAGAATTG TTGAGCTTGT

 5341 CAAGGAAGTT TCAAAGAGCT ACCCAATATC TGAGGGGCCT GAGAGAGCCA ACGAATTAGT

 5401 AGAATCCTAT AGAAAGGCTT CAAATAAAGC TTATTTTGAG TGGACTATTG AGGCCAGAGA

 5461 TCTTTCTCTG TTGGGTTCCC ATGTTCGTAA AGCTACCTAC CAGACTTACG CTCCAATTCT

 5521 TTATGAGAAT GACCACTTTT TCGACTACAT GCAAAAAAGT AAGTTTCATC TCACCATTGA

 5581 AGGTCCAAAA GTACTTGCTT ATTTACTTGG TTTATGGATT GGTGATGGAT TGTCTGACAG

 5641 GGCAACTTTT TCGGTTGATT CCAGAGATAC TTCTTTGATG GAACGTGTTA CTGAATATGC

 5701 TGAAAAGTTG AATTTGTGCG CCGAGTATAA GGACAGAAAA GAACCACAAG TTGCCAAAAC

 5761 TGTTAATTTG TACTCTAAAG TTGTCAGAGG TGCTAGCACA AATCCTGGTG TATCCGCTTG

 5821 GCAGGTCAAC ACAGCTTATA CTGCGGGACA ATTGGTCACA TATAACGGCA AGACGTATAA

 5881 ATGTTTGCAG CCCCACACCT CCTTGGCAGG ATGGGAACCA TCCAACGTTC CTGCCTTGTG

 5941 GCAGCTTCAA GGTGGCCATG GTGGTATTCG CAATAATCTT AATACTGAGA ATCCATTATG

 6001 GGACGCTATT GTTGGCTTAG GATTCTTGAA GGACGGTGTC AAAAATATTC CTTCTTTCTT

 6061 GTCTACGGAC AATATCGGTA CTCGTGAAAC ATTTCTTGCT GGTCTAATTG ATTCTGATGG

 6121 CTATGTTACT GATGAGCATG GTATTAAAGC AACAATAAAG ACAATTCATA CTTCTGTCAG

 6181 AGATGGTTTG GTTTCCCTTG CTCGTTCTTT AGGCTTAGTA GTCTCGGTTA ACGCAGAACC

 6241 TGCTAAGGTT GACATGAATG TCACCAAACA TAAAATTAGT TATGCTATTT ATATGTCTGG

 6301 TGGAGATGTT TTGCTTAACG TTCTTTCGAA GTGTGCCGGC TCTAAAAAAT TCAGGCCTGC

 6361 TCCCGCCGCT GCTTTTGCAC GTGAGTGCCG CGGATTTTAT TTCGAGTTAC AAGAATTGAA

 6421 GGAAGACGAT TATTATGGGA TTACTTTATC TGATGATTCT GATCATCAGT TTTTGCTTGG

 6481 ATCCCAGGTT GTTGTACAGA ATGCTGGTCA TATGACTAGT TCGCGAGTCG ACGGCGGCCG

 6541 CGAATTCCTC GAGCCCGGGT GACTGCAGGA AGGGGATCCG GCTGCTAACA AAGCCCGAAA

 6601 GGAAGCTGAG TTGGCTGCTG CCACCGCTGA GCAATAACTA GCATAACCCC TTGGGGCCTC

 6661 TAAACGGGTC TTGAGGGGTT TTTTGCTGAA AGGAGGAACT ATATCCGGAT ATCCCGCAAG

 6721 AGGCCCGGCA GTACCGGCAT AACCAAGCCT ATGCCTACAG CATCCAGGGT GACGGTGCCG

 6781 AGGATGACGA TGAGCGCATT GTTAGATTTC ATACACGGTG CCTGACTGCG TTAGCAATTT

 6841 AACTGTGATA AACTACCGCA TTAAAGCTTG GCACTGGCCG TCGTTTTACA ACGTCGTGAC

 6901 TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG CACATCCCCC TTTCGCCAGC

 6961 TGGCGTAATA GCGAAGAGGC CCGCACCGAT CGCCCTTCCC AACAGTTGCG CAGCCTGAAT

 7021 GGCGAATGGC AGCTTGGCTG TTTTGGCGGA TGAGATAAGA TTTTCAGCCT GATACAGATT

 7081 AAATCAGAAC GCAGAAGCGG TCTGATAAAA CAGAATTTGC CTGGCGGCAG TAGCGCGGTG

 7141 GTCCCACCTG ACCCCATGCC GAACTCAGAA GTGAAACGCC GTAGCGCCGA TGGTAGTGTG

 7201 GGGTCTCCCC ATGCGAGAGT AGGGAACTGC CAGGCATCAA ATAAAACGAA AGGCTCAGTC

 7261 GAAAGACTGG GCCTTTCGTT TTATCTGTTG TTTGTCGGTG AACGCTCTCC TGAGTAGGAC

 7321 AAATCCGCCG GGAGCGGATT TGAACGTTGC GAAGCAACGG CCCGGAGGGT GGCGGGCAGG

 7381 ACGCCCGCCA TAAACTGCCA GGCATCAAAT TAAGC

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其他大肠杆菌表达载体：