pcDNA6/V5-His B

pcDNA6/V5-His B

pcDNA6/V5-HisB载体基本信息

pcDNA6/V5-HisB载体质粒图谱和多克隆位点信息

pcDNA6/V5-HisB载体简介

pcDNA6/V5-His A, B, and C are 5.1 kb vectors derived from pcDNA3.1(+) and designed for high-level expression, selection on blasticidin, purification, and detection of recombinant proteins in mammalian hosts. High-level stable and non-replicative transient expression can be carried out in most mammalian cells.

pcDNA6/V5-His A, B, and C 含有以下元件:

• Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells

• Three reading frames to facilitate in-frame cloning with a C-terminal tag encoding the V5 epitope and a polyhistidine metal-binding peptide

• Blasticidin resistance gene (BSD) from Aspergillus terreus for selection in prokaryotic and eukaryotic cells (Kimura et al., 1994)

• Episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g. COS7)

The control plasmid, pcDNA6/V5-His/lacZ, is the pcDNA6/V5-His A vector with a 3.2 kb fragment containing the β-galactosidase gene cloned in frame with the C-terminal peptide (see page 11). It is included for use as a positive control for transfection, expression, purification, and detection in the cell line of choice.

实验流程

使用pcDNA.6/V5-His 载体表达目的基因的实验流程：

1. Consult the multiple cloning sites described on pages 3–5 to determine which vector (A, B, or C) should be used to clone your gene in frame with the C-terminal V5 epitope and polyhistidine tag.

2. Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 100 μg/mL blasticidin or 50 to 100 μg/mL ampicillin.

3. Analyze your transformants for the presence of insert by restriction digestion.

4. Select a transformant with the correct restriction pattern and confirm that your gene is in frame with the C-terminal peptide by sequencing.

5. Transfect your construct into the cell line of choice using your own method of transfection.

6. Test for expression of your recombinant gene by western blot analysis or functional assay. If you do not have an antibody to your protein, Life Technologies offers the Anti-V5 Antibody or the Anti-His(C-term) Antibody to detect your recombinant fusion protein.

7. To purify your recombinant fusion protein, you may use metal-chelating resin such as ProBond.. ProBond. resin is available separately.

pcDNA6/V5-HisB载体序列

ORIGIN

    1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG

   61 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG

  121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGC

  181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT

  241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA

  301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC

  361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC

  421 ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT

  481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT

  541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA

  601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG

  661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC

  721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG

  781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA

  841 CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGC

  901 GTTTAAACTT AAGCTTGGTA CCGAGCTCGG ATCCACTAGT CCAGTGTGGT GGAATTCTGC

  961 AGATATCCAG CACAGTGGCG GCCGCTCGAG TCTAGAGGGC CCGCGGTTCG AAGGTAAGCC

 1021 TATCCCTAAC CCTCTCCTCG GTCTCGATTC TACGCGTACC GGTCATCATC ACCATCACCA

 1081 TTGAGTTTAA ACCCGCTGAT CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC CATCTGTTGT

 1141 TTGCCCCTCC CCCGTGCCTT CCTTGACCCT GGAAGGTGCC ACTCCCACTG TCCTTTCCTA

 1201 ATAAAATGAG GAAATTGCAT CGCATTGTCT GAGTAGGTGT CATTCTATTC TGGGGGGTGG

 1261 GGTGGGGCAG GACAGCAAGG GGGAGGATTG GGAAGACAAT AGCAGGCATG CTGGGGATGC

 1321 GGTGGGCTCT ATGGCTTCTG AGGCGGAAAG AACCAGCTGG GGCTCTAGGG GGTATCCCCA

 1381 CGCGCCCTGT AGCGGCGCAT TAAGCGCGGC GGGTGTGGTG GTTACGCGCA GCGTGACCGC

 1441 TACACTTGCC AGCGCCCTAG CGCCCGCTCC TTTCGCTTTC TTCCCTTCCT TTCTCGCCAC

 1501 GTTCGCCGGC TTTCCCCGTC AAGCTCTAAA TCGGGGGCTC CCTTTAGGGT TCCGATTTAG

 1561 TGCTTTACGG CACCTCGACC CCAAAAAACT TGATTAGGGT GATGGTTCAC GTAGTGGGCC

 1621 ATCGCCCTGA TAGACGGTTT TTCGCCCTTT GACGTTGGAG TCCACGTTCT TTAATAGTGG

 1681 ACTCTTGTTC CAAACTGGAA CAACACTCAA CCCTATCTCG GTCTATTCTT TTGATTTATA

 1741 AGGGATTTTG CCGATTTCGG CCTATTGGTT AAAAAATGAG CTGATTTAAC AAAAATTTAA

 1801 CGCGAATTAA TTCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA

 1861 GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC

 1921 CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA

 1981 GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG

 2041 CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG AGGCCGCCTC TGCCTCTGAG

 2101 CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AAAGCTCCCG

 2161 GGAGCTTGTA TATCCATTTT CGGATCTGAT CAGCACGTGT TGACAATTAA TCATCGGCAT

 2221 AGTATATCGG CATAGTATAA TACGACAAGG TGAGGAACTA AACCATGGCC AAGCCTTTGT

 2281 CTCAAGAAGA ATCCACCCTC ATTGAAAGAG CAACGGCTAC AATCAACAGC ATCCCCATCT

 2341 CTGAAGACTA CAGCGTCGCC AGCGCAGCTC TCTCTAGCGA CGGCCGCATC TTCACTGGTG

 2401 TCAATGTATA TCATTTTACT GGGGGACCTT GTGCAGAACT CGTGGTGCTG GGCACTGCTG

 2461 CTGCTGCGGC AGCTGGCAAC CTGACTTGTA TCGTCGCGAT CGGAAATGAG AACAGGGGCA

 2521 TCTTGAGCCC CTGCGGACGG TGCCGACAGG TGCTTCTCGA TCTGCATCCT GGGATCAAAG

 2581 CCATAGTGAA GGACAGTGAT GGACAGCCGA CGGCAGTTGG GATTCGTGAA TTGCTGCCCT

 2641 CTGGTTATGT GTGGGAGGGC TAAGCACTTC GTGGCCGAGG AGCAGGACTG ACACGTGCTA

 2701 CGAGATTTCG ATTCCACCGC CGCCTTCTAT GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG

 2761 GACGCCGGCT GGATGATCCT CCAGCGCGGG GATCTCATGC TGGAGTTCTT CGCCCACCCC

 2821 AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC AAATTTCACA

 2881 AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT CAATGTATCT

 2941 TATCATGTCT GTATACCGTC GACCTCTAGC TAGAGCTTGG CGTAATCATG GTCATAGCTG

 3001 TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC CGGAAGCATA

 3061 AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCA

 3121 CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC

 3181 GCGGGGAGAG GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG

 3241 CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA

 3301 TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA GCAAAAGGCC

 3361 AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG

 3421 CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC

 3481 CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC

 3541 GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG CTCACGCTGT

 3601 AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC

 3661 GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA

 3721 CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA

 3781 GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGAACAGTA

 3841 TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA

 3901 TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA GCAGATTACG

 3961 CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG

 4021 TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC

 4081 TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT

 4141 TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT CTGTCTATTT

 4201 CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG GGAGGGCTTA

 4261 CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC GCTCACCGGC TCCAGATTTA

 4321 TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC

 4381 GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT

 4441 AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC GTCGTTTGGT

 4501 ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC CCCCATGTTG

 4561 TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG TCAGAAGTAA GTTGGCCGCA

 4621 GTGTTATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA

 4681 AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA GTGTATGCGG

 4741 CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA TAGCAGAACT

 4801 TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG GATCTTACCG

 4861 CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA ACTGATCTTC AGCATCTTTT

 4921 ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA

 4981 ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA TTATTGAAGC

 5041 ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA GAAAAATAAA

 5101 CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CTGACGTC

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