pcDNA4/HisMax B

pcDNA4/HisMax B

pcDNA4/HisMax B载体基本信息

pcDNA4/HisMax B载体质粒图谱和多克隆位点信息

pcDNA4/HisMax A, B, and C载体介绍：

pcDNA4/HisMax is specifically designed to maximize protein expression in a variety of mammalian cells. The vector contains the QBI SP163 translational enhancer to increase expression levels two- to five-fold above those seen with the promoter alone . In addition to SP163-enhanced expression, pcDNA4/HisMax includes a cleavable N-terminal Xpress tag for rapid detection of recombinant protein with an Anti-Xpress Antibody. pcDNA4/HisMax is available TOPO Cloning-ready for five-minute cloning of Taqamplified PCR products.

pcDNA4/HisMax A, B, and C are 5.3 kb vectors derived from pcDNA4/His and designed for overproduction of recombinant proteins in mammalian cell lines. Features of the vectors allow purification and detection of expressed proteins. High-level stable and transient expression can be carried out in most mammalian cells. The vectors contain the following elements:

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

 QBI SP163 translational enhancer for increased levels of recombinant protein expression (Stein et al., 1998) (see page 4 for more information)

 Three reading frames to facilitate in-frame cloning with an N-terminal peptide encoding the Xpress epitope and a polyhistidine metal-binding tag

 Zeocin resistance gene for selection of stable cell lines

 Episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g. COS-1, COS-7)

The control plasmid, pcDNA4/HisMax/lacZ, is included for use as a positive control for transfection, expression, and detection in the cell line of choice.

实验流程：

Use the following outline to clone and express your gene of interest in pcDNA4/HisMax.

 Consult the multiple cloning sites described on pages 5-7 to determine which vector (A, B, or C) should be used to clone your gene in frame with the N-terminal Xpress epitope and the polyhistidine tag.

 Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 50 to 100 μg/ml ampicillin or 25-50 μg/ml Zeocin.

 Analyze your transformants for the presence of 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 N-terminal peptide.

 Transfect your construct into the cell line of choice using your own method of transfection. Generate a stable cell line, if desired.

 Test for expression of your recombinant gene by western blot analysis or functional assay. .

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

表达目的基因：

We have a wide variety of mammalian expression vectors utilizing the CMV or EF-1α promoter. Vectors are available with the Xpress (N-terminal), c-myc (C-terminal), or V5 (C-terminal) epitope for detection and either the neomycin, blasticidin, or Zeocin resistance genes. All vectors utilize the polyhistidine tag for purification using ProBond resin.

The pcDNA4/HisMax vectors are fusion vectors. To ensure proper expression of your recombinant protein, you must clone your gene in frame with the ATG at base pairs 1080-1082. This will create a fusion with the N-terminal polyhistidine tag, Xpress epitope, and the enterokinase cleavage site. The vector is supplied with the multiple cloning site in three reading frames relative to the N-terminal peptide to facilitate cloning.

If you wish to clone your gene as close as possible to the enterokinase cleavage site, follow the guidelines below:

 Digest pcDNA4/HisMax A, B, or C with Kpn I.

 Create blunt ends with T4 DNA polymerase and dNTPs.

 Clone your blunt-ended insert in frame with the lysine codon (AAG) of the enterokinase recognition site.

pcDNA4/HisMax B载体序列

ORIGIN

    1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT 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 ATGGGTGGAC 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 AAGCTTAGCG CAGAGGCTTG GGGCAGCCGA GCGGCAGCCA GGCCCCGGCC

  961 CGGGCCTCGG TTCCAGAAGG GAGAGGAGCC CGCCAAGGCG CGCAAGAGAG CGGGCTGCCT

 1021 CGCAGTCCGA GCCGGAGAGG GAGCGCGAGC CGCGCCGGCC CCGGACGGCC TCCGAAACCA

 1081 TGGGGGGTTC TCATCATCAT CATCATCATG GTATGGCTAG CATGACTGGT GGACAGCAAA

 1141 TGGGTCGGGA TCTGTACGAC GATGACGATA AGGTACCTAA GGATCCAGTG TGGTGGAATT

 1201 CTGCAGATAT CCAGCACAGT GGCGGCCGCT CGAGTCTAGA GGGCCCGTTT AAACCCGCTG

 1261 ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA GCCATCTGTT GTTTGCCCCT CCCCCGTGCC

 1321 TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC TAATAAAATG AGGAAATTGC

 1381 ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT GGGGTGGGGC AGGACAGCAA

 1441 GGGGGAGGAT TGGGAAGACA ATAGCAGGCA TGCTGGGGAT GCGGTGGGCT CTATGGCTTC

 1501 TGAGGCGGAA AGAACCAGCT GGGGCTCTAG GGGGTATCCC CACGCGCCCT GTAGCGGCGC

 1561 ATTAAGCGCG GCGGGTGTGG TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT

 1621 AGCGCCCGCT CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG

 1681 TCAAGCTCTA AATCGGGGCA TCCCTTTAGG GTTCCGATTT AGTGCTTTAC GGCACCTCGA

 1741 CCCCAAAAAA CTTGATTAGG GTGATGGTTC ACGTAGTGGG CCATCGCCCT GATAGACGGT

 1801 TTTTCGCCCT TTGACGTTGG AGTCCACGTT CTTTAATAGT GGACTCTTGT TCCAAACTGG

 1861 AACAACACTC AACCCTATCT CGGTCTATTC TTTTGATTTA TAAGGGATTT TGGGGATTTC

 1921 GGCCTATTGG TTAAAAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT AATTCTGTGG

 1981 AATGTGTGTC AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC CAGGCAGGCA GAAGTATGCA

 2041 AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG

 2101 CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC ATAGTCCCGC CCCTAACTCC

 2161 GCCCATCCCG CCCCTAACTC CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT

 2221 TTTTTTTATT TATGCAGAGG CCGAGGCCGC CTCTGCCTCT GAGCTATTCC AGAAGTAGTG

 2281 AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG CAAAAAGCTC CCGGGAGCTT GTATATCCAT

 2341 TTTCGGATCT GATCAGCACG TGTTGACAAT TAATCATCGG CATAGTATAT CGGCATAGTA

 2401 TAATACGACA AGGTGAGGAA CTAAACCATG GCCAAGTTGA CCAGTGCCGT TCCGGTGCTC

 2461 ACCGCGCGCG ACGTCGCCGG AGCGGTCGAG TTCTGGACCG ACCGGCTCGG GTTCTCCCGG

 2521 GACTTCGTGG AGGACGACTT CGCCGGTGTG GTCCGGGACG ACGTGACCCT GTTCATCAGC

 2581 GCGGTCCAGG ACCAGGTGGT GCCGGACAAC ACCCTGGCCT GGGTGTGGGT GCGCGGCCTG

 2641 GACGAGCTGT ACGCCGAGTG GTCGGAGGTC GTGTCCACGA ACTTCCGGGA CGCCTCCGGG

 2701 CCGGCCATGA CCGAGATCGG CGAGCAGCCG TGGGGGCGGG AGTTCGCCCT GCGCGACCCG

 2761 GCCGGCAACT GCGTGCACTT CGTGGCCGAG GAGCAGGACT GACACGTGCT ACGAGATTTC

 2821 GATTCCACCG CCGCCTTCTA TGAAAGGTTG GGCTTCGGAA TCGTTTTCCG GGACGCCGGC

 2881 TGGATGATCC TCCAGCGCGG GGATCTCATG CTGGAGTTCT TCGCCCACCC CAACTTGTTT

 2941 ATTGCAGCTT ATAATGGTTA CAAATAAAGC AATAGCATCA CAAATTTCAC AAATAAAGCA

 3001 TTTTTTTCAC TGCATTCTAG TTGTGGTTTG TCCAAACTCA TCAATGTATC TTATCATGTC

 3061 TGTATACCGT CGACCTCTAG CTAGAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG

 3121 TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA

 3181 GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG CGTTGCGCTC ACTGCCCGCT

 3241 TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA

 3301 GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC

 3361 GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA

 3421 TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT

 3481 AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA

 3541 AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT

 3601 CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG

 3661 TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCAAT GCTCACGCTG TAGGTATCTC

 3721 AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC

 3781 GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA

 3841 TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT

 3901 ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC

 3961 TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA

 4021 CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA

 4081 AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA

 4141 AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT

 4201 TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC

 4261 AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC

 4321 ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC

 4381 CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA

 4441 AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC

 4501 CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC

 4561 AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA

 4621 TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA

 4681 GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA

 4741 CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT

 4801 TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT

 4861 TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG

 4921 CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA

 4981 TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC

 5041 AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG

 5101 ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG

 5161 GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG

 5221 GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTC

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