pBK-CMV

pBK-CMV

pBK-CMV载体基本信息

pBK-CMV载体质粒图谱和多克隆位点信息

pBK-CMV载体简介

pBK-CMV Phagemid Vector

The pBK-CMV vector allows you to express proteins in both E. coli and mammalian model systems and saves you valuable time by not having to subclone into an additional vector.  In mammalian cells, the expression is driven by the CMV promoter and is therefore constitutively expressed.  In E. coli, the lac promoter drives high-level expression in the presence of the inducer IPTG, and allows minimal expression in the absence of inducer.

• Constitutive expression in eukaryotic cells

• Inducible protein expression in prokaryotes

• Rescued from ZAP Express lambda vector

The pBK-CMV phagemid vector is a cloning vector derived from a high-copy-number pUC-based plasmid. This vector allows expression in both eukaryotic and prokaryotic systems. Eukaryotic expression is driven by the cytomegalovirus (CMV) immediate early promoter in the pBK-CMV phagemid vector. Stable clone selection in eukaryotic cells is made possible with G418 by the presence of the neomycin- and kanamycin-resistance gene, which is driven by the SV40 early promoter with thymidine kinase (TK) transcription termination and polyadenylation signals. In the pBK-CMV phagemid vector, prokaryotic expression is driven by the lac promoter, which is repressed in the presence of the LacI protein and is inducible by IPTG. In bacteria expressing the lacZΔM15 mutation and lacI, colonies containing vector without insert will be blue in the presence of X-gal and IPTG. Kanamycin-resistant colonies containing vector with insert will be white and can express the inserted gene as a fusion protein.

Inserts should be cloned within the polylinker cloning sites for prokaryotic expression. For eukaryotic expression one can clone either within the polylinker or between the Nhe I site at the 5´ end of the lac promoter and a polylinker cloning site at the 3´ end. Removing the upstream lac sequences from the eukaryotic transcript by cloning between the Nhe I site and the polylinker sites has been shown to give elevated eukaryotic expression with test inserts. 

The pBK–CMV phagemid vector has an extensive polylinker in an SK orientation (the Sac I site is the closest restriction site to the lacZ promoter, and the Kpn I site is the farthest restriction site from the lacZ promoter). The polylinker contains 17 unique restriction enzyme recognition sites, organized with alternating 5´ and 3´ overhangs to allow serial exonuclease III/mung bean nuclease deletions.2 Sites with compatible restriction overhangs, such as Spe I–Xba I and Sal I–Xho I, have been placed on opposite sides of the EcoR I site to allow unidirectional cloning in both the sense and antisense orientations with the ZAP Express vectors and the pBK–CMV phagemid vector. The (–) orientation of the f1 intergenic (IG) region in pBK-CMV allows rescue of antisense single-stranded DNA (ssDNA) relative to thelacZ transcript. This ssDNA can be used for dideoxynucleotide sequencing (Sanger method) or site-specific mutagenesis. Flanking the polylinker are T3 and T7 RNA polymerase promoters that can be used to synthesize RNA in vitro. The choice of promoter used to initiate transcription determines which strand of the DNA insert will be transcribed.

pBK-CMV载体序列

ORIGIN

    1 CACCTGACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG TGTGGTGGTT ACGCGCAGCG

   61 TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC CCTTCCTTTC

  121 TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGGCTCCCT TTAGGGTTCC

  181 GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA TTAGGGTGAT GGTTCACGTA

  241 GTGGGCCATC GCCCTGATAG ACGGTTTTTC GCCCTTTGAC GTTGGAGTCC ACGTTCTTTA

  301 ATAGTGGACT CTTGTTCCAA ACTGGAACAA CACTCAACCC TATCTCGGTC TATTCTTTTG

  361 ATTTATAAGG GATTTTGCCG ATTTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA

  421 AATTTAACGC GAATTTTAAC AAAATATTAA CGCTTACAAT TTACGCGTTA AGATACATTG

  481 ATGAGTTTGG ACAAACCACA ACTAGAATGC AGTGAAAAAA ATGCTTTATT TGTGAAATTT

  541 GTGATGCTAT TGCTTTATTT GTAACCATTA TAAGCTGCAA TAAACAAGTT AACAACAACA

  601 ATTGCATTCA TTTTATGTTT CAGGTTCAGG GGGAGGTGTG GGAGGTTTTT TAAAGCAAGT

  661 AAAACCTCTA CAAATGTGGT ATGGCTGATT ATGATCATGA ACAGACTGTG AGGACTGAGG

  721 GGCCTGAAAT GAGCCTTGGG ACTGTGAATC TAAAATACAC AAACAATTAG AATCAGTAGT

  781 TTAACACATT ATACACTTAA AAATTGGATC TCCATTCGCC ATTCAGGCTG CGCAACTGTT

  841 GGGAAGGGCG ATCGGTGCGG GCCTCTTCGC TATTACGCCA GCTGGCGAAA GGGGGATGTG

  901 CTGCAAGGCG ATTAAGTTGG GTAACGCCAG GGTTTTCCCA GTCACGACGT TGTAAAACGA

  961 CGGCCAGTGA ATTGTAATAC GACTCACTAT AGGGCGAATT GGGTACACTT ACCTGGTACC

 1021 CCACCCGGGT GGAAAATCGA TGGGCCCGCG GCCGCTCTAG AAGTACTCTC GAGAAGCTTT

 1081 TTGAATTCTT TGGATCCACT AGTGTCGACC TGCAGGCGCG CGAGCTCCAG CTTTTGTTCC

 1141 CTTTAGTGAG GGTTAATTTC GAGCTTGGCG TAATCAAGGT CATAGCTGTT TCCTGTGTGA

 1201 AATTGTTATC CGCTCACAAT TCCACACAAT ATACGAGCCG GAAGTATAAA GTGTAAAGCC

 1261 TGGGGTGCCT AATGAGTGAG CTAACTCACA GTAATTGCGG CTAGCGGATC TGACGGTTCA

 1321 CTAAACCAGC TCTGCTTATA TAGACCTCCC ACCGTACACG CCTACCGCCC ATTTGCGTCA

 1381 ATGGGGCGGA GTTGTTACGA CATTTTGGAA AGTCCCGTTG ATTTTGGTGC CAAAACAAAC

 1441 TCCCATTGAC GTCAATGGGG TGGAGACTTG GAAATCCCCG TGAGTCAAAC CGCTATCCAC

 1501 GCCCATTGAT GTACTGCCAA AACCGCATCA CCATGGTAAT AGCGATGACT AATACGTAGA

 1561 TGTACTGCCA AGTAGGAAAG TCCCATAAGG TCATGTACTG GGCATAATGC CAGGCGGGCC

 1621 ATTTACCGTC ATTGACGTCA ATAGGGGGCG TACTTGGCAT ATGATACACT TGATGTACTG

 1681 CCAAGTGGGC AGTTTACCGT AAATACTCCA CCCATTGACG TCAATGGAAA GTCCCTATTG

 1741 GCGTTACTAT GGGAACATAC GTCATTATTG ACGTCAATGG GCGGGGGTCG TTGGGCGGTC

 1801 AGCCAGGCGG GCCATTTACC GTAAGTTATG TAACGCGGAA CTCCATATAT GGGCTATGAA

 1861 CTAATGACCC CGTAATTGAT TACTATTAAT AACTAATGCA TGGCGGTAAT ACGGTTATCC

 1921 ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG

 1981 AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT

 2041 CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG

 2101 GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA

 2161 TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG

 2221 TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT

 2281 CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC

 2341 GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC

 2401 GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT

 2461 GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC

 2521 GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC

 2581 AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG

 2641 AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG

 2701 ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAACCTGAG

 2761 GCTATGGCAG GGCCTGCCGC CCCGACGTTG GCTGCGAGCC CTGGGCCTTC ACCCGAACTT

 2821 GGGGGGTGGG GTGGGGAAAA GGAAGAAACG CGGGCGTATT GGCCCCAATG GGGTCTCGGT

 2881 GGGGTATCGA CAGAGTGCCA GCCCTGGGAC CGAACCCCGC GTTTATGAAC AAACGACCCA

 2941 ACACCGTGCG TTTTATTCTG TCTTTTTATT GCCGTCATAG CGCGGGTTCC TTCCGGTATT

 3001 GTCTCCTTCC GTGTTTCAGT TAGCCTCCCC CTAGGGTGGG CGAAGAACTC CAGCATGAGA

 3061 TCCCCGCGCT GGAGGATCAT CCAGCCGGCG TCCCGGAAAA CGATTCCGAA GCCCAACCTT

 3121 TCATAGAAGG CGGCGGTGGA ATCGAAATCT CGTGATGGCA GGTTGGGCGT CGCTTGGTCG

 3181 GTCATTTCGA ACCCCAGAGT CCCGCTCAGA AGAACTCGTC AAGAAGGCGA TAGAAGGCGA

 3241 TGCGCTGCGA ATCGGGAGCG GCGATACCGT AAAGCACGAG GAAGCGGTCA GCCCATTCGC

 3301 CGCCAAGCTC TTCAGCAATA TCACGGGTAG CCAACGCTAT GTCCTGATAG CGGTCCGCCA

 3361 CACCCAGCCG GCCACAGTCG ATGAATCCAG AAAAGCGGCC ATTTTCCACC ATGATATTCG

 3421 GCAAGCAGGC ATCGCCATGG GTCACGACGA GATCCTCGCC GTCGGGCATG CTCGCCTTGA

 3481 GCCTGGCGAA CAGTTCGGCT GGCGCGAGCC CCTGATGCTC TTCGTCCAGA TCATCCTGAT

 3541 CGACAAGACC GGCTTCCATC CGAGTACGTG CTCGCTCGAT GCGATGTTTC GCTTGGTGGT

 3601 CGAATGGGCA GGTAGCCGGA TCAAGCGTAT GCAGCCGCCG CATTGCATCA GCCATGATGG

 3661 ATACTTTCTC GGCAGGAGCA AGGTGAGATG ACAGGAGATC CTGCCCCGGC ACTTCGCCCA

 3721 ATAGCAGCCA GTCCCTTCCC GCTTCAGTGA CAACGTCGAG CACAGCTGCG CAAGGAACGC

 3781 CCGTCGTGGC CAGCCACGAT AGCCGCGCTG CCTCGTCTTG CAGTTCATTC AGGGCACCGG

 3841 ACAGGTCGGT CTTGACAAAA AGAACCGGGC GCCCCTGCGC TGACAGCCGG AACACGGCGG

 3901 CATCAGAGCA GCCGATTGTC TGTTGTGCCC AGTCATAGCC GAATAGCCTC TCCACCCAAG

 3961 CGGCCGGAGA ACCTGCGTGC AATCCATCTT GTTCAATCAT GCGAAACGAT CCTCATCCTG

 4021 TCTCTTGATC GATCTTTGCA AAAGCCTAGG CCTCCAAAAA AGCCTCCTCA CTACTTCTGG

 4081 AATAGCTCAG AGGCCGAGGC GGCCTCGGCC TCTGCATAAA TAAAAAAAAT TAGTCAGCCA

 4141 TGGGGCGGAG AATGGGCGGA ACTGGGCGGA GTTAGGGGCG GGATGGGCGG AGTTAGGGGC

 4201 GGGACTATGG TTGCTGACTA ATTGAGATGC ATGCTTTGCA TACTTCTGCC TGCTGGGGAG

 4261 CCTGGGGACT TTCCACACCT GGTTGCTGAC TAATTGAGAT GCATGCTTTG CATACTTCTG

 4321 CCTGCTGGGG AGCCTGGGGA CTTTCCACAC CCTAACTGAC ACACATTCCA CAGCTGGTTC

 4381 TTTCCGCCTC AGGACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC

 4441 TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA

 4501 CATTTCCCCG AAAAGTGC

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