pcDNA6/myc-His C

pcDNA6/myc-His C载体基本信息

pcDNA6/myc-His C载体质粒图谱和多克隆位点信息

pcDNA6/myc-His C载体简介

pcDNA.6/myc-His A, B, and C are 5.1-kb vectors designed for overproduction of recombinant proteins in mammalian cell lines. High-level stable and transient expression can be carried out in most mammalian cells.

pcDNA.6/myc-His载体含有以下元件:

 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 peptide encoding the myc (c-myc) epitope and a polyhistidine (6xHis) metal-binding tag.

 Blasticidin resistance gene (bsd) for selection of stable cell lines (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, pcDNA.6/myc-His/lacZ, is included for use as a positive control for transfection, expression, and detection in the cell line of choice.

实验流程

使用pcDNA6/myc-His 载体克隆和表达目的基因的实验流程如下：

 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 myc epitope and the polyhistidine tag. Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 50–100 μg/ml ampicillin or 50 μg/ml blasticidin.

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

 Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in-frame with the C-terminal peptide.

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

  Test for expression of your recombinant gene by western blot analysis or functional assay. For antibodies to the myc epitope or the C-terminal polyhistidine tag, see the next page.

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

pcDNA6/myc-His C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 GCTGGCTAGT

  901 TAAGCTTGGT ACCGAGCTCG GATCCACTAG TCCAGTGTGG TGGAATTCTG CAGATATCCA

  961 GCACAGTGGC GGCCGCTCGA GGTCACCCAT TCGAACAAAA ACTCATCTCA GAAGAGGATC

 1021 TGAATATGCA TACCGGTCAT CATCACCATC ACCATTGAGT TTAAACCCGC TGATCAGCCT

 1081 CGACTGTGCC TTCTAGTTGC CAGCCATCTG TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA

 1141 CCCTGGAAGG TGCCACTCCC ACTGTCCTTT CCTAATAAAA TGAGGAAATT GCATCGCATT

 1201 GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG GCAGGACAGC AAGGGGGAGG

 1261 ATTGGGAAGA CAATAGCAGG CATGCTGGGG ATGCGGTGGG CTCTATGGCT TCTGAGGCGG

 1321 AAAGAACCAG CTGGGGCTCT AGGGGGTATC CCCACGCGCC CTGTAGCGGC GCATTAAGCG

 1381 CGGCGGGTGT GGTGGTTACG CGCAGCGTGA CCGCTACACT TGCCAGCGCC CTAGCGCCCG

 1441 CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCTTTCCC CGTCAAGCTC

 1501 TAAATCGGGG GCTCCCTTTA GGGTTCCGAT TTAGTGCTTT ACGGCACCTC GACCCCAAAA

 1561 AACTTGATTA GGGTGATGGT TCACGTAGTG GGCCATCGCC CTGATAGACG GTTTTTCGCC

 1621 CTTTGACGTT GGAGTCCACG TTCTTTAATA GTGGACTCTT GTTCCAAACT GGAACAACAC

 1681 TCAACCCTAT CTCGGTCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT TCGGCCTATT

 1741 GGTTAAAAAA TGAGCTGATT TAACAAAAAT TTAACGCGAA TTAATTCTGT GGAATGTGTG

 1801 TCAGTTAGGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA

 1861 TCTCAATTAG TCAGCAACCA GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT

 1921 GCAAAGCATG CATCTCAATT AGTCAGCAAC CATAGTCCCG CCCCTAACTC CGCCCATCCC

 1981 GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT GGCTGACTAA TTTTTTTTAT

 2041 TTATGCAGAG GCCGAGGCCG CCTCTGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT

 2101 TTTTGGAGGC CTAGGCTTTT GCAAAAAGCT CCCGGGAGCT TGTATATCCA TTTTCGGATC

 2161 TGATCAGCAC GTGTTGACAA TTAATCATCG GCATAGTATA TCGGCATAGT ATAATACGAC

 2221 AAGGTGAGGA ACTAAACCAT GGCCAAGCCT TTGTCTCAAG AAGAATCCAC CCTCATTGAA

 2281 AGAGCAACGG CTACAATCAA CAGCATCCCC ATCTCTGAAG ACTACAGCGT CGCCAGCGCA

 2341 GCTCTCTCTA GCGACGGCCG CATCTTCACT GGTGTCAATG TATATCATTT TACTGGGGGA

 2401 CCTTGTGCAG AACTCGTGGT GCTGGGCACT GCTGCTGCTG CGGCAGCTGG CAACCTGACT

 2461 TGTATCGTCG CGATCGGAAA TGAGAACAGG GGCATCTTGA GCCCCTGCGG ACGGTGCCGA

 2521 CAGGTGCTTC TCGATCTGCA TCCTGGGATC AAAGCCATAG TGAAGGACAG TGATGGACAG

 2581 CCGACGGCAG TTGGGATTCG TGAATTGCTG CCCTCTGGTT ATGTGTGGGA GGGCTAAGCA

 2641 CTTCGTGGCC GAGGAGCAGG ACTGACACGT GCTACGAGAT TTCGATTCCA CCGCCGCCTT

 2701 CTATGAAAGG TTGGGCTTCG GAATCGTTTT CCGGGACGCC GGCTGGATGA TCCTCCAGCG

 2761 CGGGGATCTC ATGCTGGAGT TCTTCGCCCA CCCCAACTTG TTTATTGCAG CTTATAATGG

 2821 TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC

 2881 TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTATCAT GTCTGTATAC CGTCGACCTC

 2941 TAGCTAGAGC TTGGCGTAAT CATGGTCATA GCTGTTTCCT GTGTGAAATT GTTATCCGCT

 3001 CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT AAAGCCTGGG GTGCCTAATG

 3061 AGTGAGCTAA CTCACATTAA TTGCGTTGCG CTCACTGCCC GCTTTCCAGT CGGGAAACCT

 3121 GTCGTGCCAG CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG

 3181 GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC

 3241 GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG

 3301 AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT

 3361 GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA

 3421 GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT

 3481 CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC

 3541 GGGAAGCGTG GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT

 3601 TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC

 3661 CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC

 3721 CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG

 3781 GTGGCCTAAC TACGGCTACA CTAGAAGAAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC

 3841 AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG

 3901 CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA

 3961 TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT

 4021 TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG

 4081 TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT

 4141 CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC

 4201 CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT

 4261 ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG

 4321 GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG

 4381 CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC

 4441 TACAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA

 4501 ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG

 4561 TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC

 4621 ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA

 4681 CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC

 4741 AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG

 4801 TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC

 4861 CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC

 4921 AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT

 4981 ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT CAGGGTTATT GTCTCATGAG

 5041 CGGATACATA TTTGAATGTA TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC

 5101 CCGAAAAGTG CCACCTGACG TC

//

其他哺乳动物表达载体：