pTYB2

pTYB2

pTYB2载体基本信息

pTYB2载体质粒图谱和多克隆位点信息

pTYB2载体简介

pTYB2 is an E. coli cloning and expression vector (7474 bp) used in the IMPACT™ Kit (NEB #E6901) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag (1,2). This C-terminal fusion vector is designed for the in-frame insertion of a target gene into the polylinker upstream of an intein tag (the Sce VMA intein/chitin binding domain, 55 kDa) (1,2). This results in the fusion of the C-terminus of the target protein to the N-terminus of the intein tag. Thiol-induced self-cleavage of the intein releases the target protein from the chitin-bound intein tag, resulting in a single column purification of the target protein.

For fusion of the N-terminus of the target protein to the intein tag, use pTYB21 (NEB #N6709) or pTYB22 (NEB #N6710). This vector can be used in conjuction with pTYB22 (NEB #N6710) to test which fusion constructin (N-terminal or C-terminal) maximizes the expression and yield of a target protein (3).

pTYB2 Features

Þ      The NdeI site in the polylinker contains an ATG sequence for translation initiation.

Þ      The SmaI site is used for cloning the 3´ end of the target gene and will yield a target protein with a single glycine residue added to its C-terminus after cleavage of the intein.

Þ      A pBR322 derivative with a ColE1 replication origin. Expression of the fusion gene is under the control of an IPTG-inducible T7 promoter (4).

Þ      Expression requires an E. coli host that carries the T7 RNA Polymerase gene [e.g., T7 Express Competent E. coli (High Efficiency) NEB #C2566), BL21(DE3) Competent E. coli (NEB #C2527) and derivatives].

Þ      Ampicillin resistance.

Þ      M13 allows for the production of single-stranded DNA by helper phage superinfection of cells bearing the plasmid.

Þ      M13K07 helper phage is available from (NEB #N0315).

Other IMPACT vectors are available which allow for fusion of a target gene to N- or C- terminus of an intein, the cleavage reaction may be induced by thiol reagent or temperature/pH shift. The sites in the polylinker region are identical to or compatible with (i.e., NheI of pTYB2 and SpeI of pTYB12) those of pTYB12 (NEB #N6902). This allows the same amplified target gene to be cloned into either vector for optimizing protein expression. Vector derived residues may be present at the N- and/or C-termini of the target proteins.

pTYB2载体序列

ORIGIN

    1 AACTACGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT 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 GCCGGCATGC CGCCCTTTCG TCTTCAAGAA TTAATTCCCA ATTCCCCAGG

 4681 CATCAAATAA AACGAAAGGC TCAGTCGAAA GACTGGGCCT TTCGTTTTAT CTGTTGTTTG

 4741 TCGGTGAACG CTCTCCTGAG TAGGACAAAT CCGCCGGGAG CGGATTTGAA CGTTGCGAAG

 4801 CAACGGCCCG GAGGGTGGCG GGCAGGACGC CCGCCATAAA CTGCCAGGAA TTAATTCCCC

 4861 AGGCATCAAA TAAAACGAAA GGCTCAGTCG AAAGACTGGG CCTTTCGTTT TATCTGTTGT

 4921 TTGTCGGTGA ACGCTCTCCT GAGTAGGACA AATCCGCCGG GAGCGGATTT GAACGTTGCG

 4981 AAGCAACGGC CCGGAGGGTG GCGGGCAGGA CGCCCGCCAT AAACTGCCAG GAATTAATTC

 5041 CCCAGGCATC AAATAAAACG AAAGGCTCAG TCGAAAGACT GGGCCTTTCG TTTTATCTGT

 5101 TGTTTGTCGG TGAACGCTCT CCTGAGTAGG ACAAATCCGC CGGGAGCGGA TTTGAACGTT

 5161 GCGAAGCAAC GGCCCGGAGG GTGGCGGGCA GGACGCCCGC CATAAACTGC CAGGAATTAA

 5221 TTCCCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT TCGTTTTATC

 5281 TGTTGTTTGT CGGTGAACGC TCTCCTGAGT AGGACAAATC CGCCGGGAGC GGATTTGAAC

 5341 GTTGCGAAGC AACGGCCCGG AGGGTGGCGG GCAGGACGCC CGCCATAAAC TGCCAGGAAT

 5401 TAATTCCCCA GGCATCAAAT AAAACGAAAG GCTCAGTCGA AAGACTGGGC CTTTCGTTTT

 5461 ATCTGTTGTT TGTCGGTGAA CGCTCTCCTG AGTAGGACAA ATCCGCCGGG AGCGGATTTG

 5521 AACGTTGCGA AGCAACGGCC CGGAGGGTGG CGGGCAGGAC GCCCGCCATA AACTGCCAGG

 5581 AATTGGGGAT CGGAATTAAT TCCCGGTTTA AACCGGGGAT CTCGATCCCG CGAAATTAAT

 5641 ACGACTCACT ATAGGGGAAT TGTGAGCGGA TAACAATTCC CCTCTAGAAA TAATTTTGTT

 5701 TAACTTTAAG AAGGAGATAT ACATATGGCT AGCTCGCGAG TCGACGGCGG CCGCGAATTC

 5761 CTCGAGCCCG GGTGCTTTGC CAAGGGTACC AATGTTTTAA TGGCGGATGG GTCTATTGAA

 5821 TGTATTGAAA ACATTGAGGT TGGTAATAAG GTCATGGGTA AAGATGGCAG ACCTCGTGAG

 5881 GTAATTAAAT TGCCCAGAGG AAGAGAAACT ATGTACAGCG TCGTGCAGAA AAGTCAGCAC

 5941 AGAGCCCACA AAAGTGACTC AAGTCGTGAA GTGCCAGAAT TACTCAAGTT TACGTGTAAT

 6001 GCGACCCATG AGTTGGTTGT TAGAACACCT CGTAGTGTCC GCCGTTTGTC TCGTACCATT

 6061 AAGGGTGTCG AATATTTTGA AGTTATTACT TTTGAGATGG GCCAAAAGAA AGCCCCCGAC

 6121 GGTAGAATTG TTGAGCTTGT CAAGGAAGTT TCAAAGAGCT ACCCAATATC TGAGGGGCCT

 6181 GAGAGAGCCA ACGAATTAGT AGAATCCTAT AGAAAGGCTT CAAATAAAGC TTATTTTGAG

 6241 TGGACTATTG AGGCCAGAGA TCTTTCTCTG TTGGGTTCCC ATGTTCGTAA AGCTACCTAC

 6301 CAGACTTACG CTCCAATTCT TTATGAGAAT GACCACTTTT TCGACTACAT GCAAAAAAGT

 6361 AAGTTTCATC TCACCATTGA AGGTCCAAAA GTACTTGCTT ATTTACTTGG TTTATGGATT

 6421 GGTGATGGAT TGTCTGACAG GGCAACTTTT TCGGTTGATT CCAGAGATAC TTCTTTGATG

 6481 GAACGTGTTA CTGAATATGC TGAAAAGTTG AATTTGTGCG CCGAGTATAA GGACAGAAAA

 6541 GAACCACAAG TTGCCAAAAC TGTTAATTTG TACTCTAAAG TTGTCAGAGG TAATGGTATT

 6601 CGCAATAATC TTAATACTGA GAATCCATTA TGGGACGCTA TTGTTGGCTT AGGATTCTTG

 6661 AAGGACGGTG TCAAAAATAT TCCTTCTTTC TTGTCTACGG ACAATATCGG TACTCGTGAA

 6721 ACATTTCTTG CTGGTCTAAT TGATTCTGAT GGCTATGTTA CTGATGAGCA TGGTATTAAA

 6781 GCAACAATAA AGACAATTCA TACTTCTGTC AGAGATGGTT TGGTTTCCCT TGCTCGTTCT

 6841 TTAGGCTTAG TAGTCTCGGT TAACGCAGAA CCTGCTAAGG TTGACATGAA TGTCACCAAA

 6901 CATAAAATTA GTTATGCTAT TTATATGTCT GGTGGAGATG TTTTGCTTAA CGTTCTTTCG

 6961 AAGTGTGCCG GCTCTAAAAA ATTCAGGCCT GCTCCCGCCG CTGCTTTTGC ACGTGAGTGC

 7021 CGCGGATTTT ATTTCGAGTT ACAAGAATTG AAGGAAGACG ATTATTATGG GATTACTTTA

 7081 TCTGATGATT CTGATCATCA GTTTTTGCTT GGATCCCAGG TTGTCGTCCA TGCATGCGGT

 7141 GGCCTGACCG GTCTGAACTC AGGCCTCACG ACAAATCCTG GTGTATCCGC TTGGCAGGTC

 7201 AACACAGCTT ATACTGCGGG ACAATTGGTC ACATATAACG GCAAGACGTA TAAATGTTTG

 7261 CAGCCCCACA CCTCCTTGGC AGGATGGGAA CCATCCAACG TTCCTGCCTT GTGGCAGCTT

 7321 CAATGACTGC AGGAAGGGGA TCCGGCTGCT AACAAAGCCC GAAAGGAAGC TGAGTTGGCT

 7381 GCTGCCACCG CTGAGCAATA ACTAGCATAA CCCCTTGGGG CCTCTAAACG GGTCTTGAGG

 7441 GGTTTTTTGC TGAAAGGAGG AACTATATCC GGAT

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pTYB2其他大肠杆菌表达载体：