pcDNA4/HisMax A

pcDNA4/HisMax A

pcDNA4/HisMax A载体基本信息

pcDNA4/HisMax A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 A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 AGGTACCTAG GATCCAGTGT GGTGGAATTC

 1201 TGCAGATATC CAGCACAGTG GCGGCCGCTC GAGTCTAGAG GGCCCGTTTA AACCCGCTGA

 1261 TCAGCCTCGA CTGTGCCTTC TAGTTGCCAG CCATCTGTTG TTTGCCCCTC CCCCGTGCCT

 1321 TCCTTGACCC TGGAAGGTGC CACTCCCACT GTCCTTTCCT AATAAAATGA GGAAATTGCA

 1381 TCGCATTGTC TGAGTAGGTG TCATTCTATT CTGGGGGGTG GGGTGGGGCA GGACAGCAAG

 1441 GGGGAGGATT GGGAAGACAA TAGCAGGCAT GCTGGGGATG CGGTGGGCTC TATGGCTTCT

 1501 GAGGCGGAAA GAACCAGCTG GGGCTCTAGG GGGTATCCCC ACGCGCCCTG TAGCGGCGCA

 1561 TTAAGCGCGG CGGGTGTGGT GGTTACGCGC AGCGTGACCG CTACACTTGC CAGCGCCCTA

 1621 GCGCCCGCTC CTTTCGCTTT CTTCCCTTCC TTTCTCGCCA CGTTCGCCGG CTTTCCCCGT

 1681 CAAGCTCTAA ATCGGGGCAT CCCTTTAGGG TTCCGATTTA GTGCTTTACG GCACCTCGAC

 1741 CCCAAAAAAC TTGATTAGGG TGATGGTTCA CGTAGTGGGC CATCGCCCTG ATAGACGGTT

 1801 TTTCGCCCTT TGACGTTGGA GTCCACGTTC TTTAATAGTG GACTCTTGTT CCAAACTGGA

 1861 ACAACACTCA ACCCTATCTC GGTCTATTCT TTTGATTTAT AAGGGATTTT GGGGATTTCG

 1921 GCCTATTGGT TAAAAAATGA GCTGATTTAA CAAAAATTTA ACGCGAATTA ATTCTGTGGA

 1981 ATGTGTGTCA GTTAGGGTGT GGAAAGTCCC CAGGCTCCCC AGGCAGGCAG AAGTATGCAA

 2041 AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC

 2101 AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG

 2161 CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT

 2221 TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCTGCCTCTG AGCTATTCCA GAAGTAGTGA

 2281 GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAAAGCTCC CGGGAGCTTG TATATCCATT

 2341 TTCGGATCTG ATCAGCACGT GTTGACAATT AATCATCGGC ATAGTATATC GGCATAGTAT

 2401 AATACGACAA GGTGAGGAAC TAAACCATGG CCAAGTTGAC CAGTGCCGTT CCGGTGCTCA

 2461 CCGCGCGCGA CGTCGCCGGA GCGGTCGAGT TCTGGACCGA CCGGCTCGGG TTCTCCCGGG

 2521 ACTTCGTGGA GGACGACTTC GCCGGTGTGG TCCGGGACGA CGTGACCCTG TTCATCAGCG

 2581 CGGTCCAGGA CCAGGTGGTG CCGGACAACA CCCTGGCCTG GGTGTGGGTG CGCGGCCTGG

 2641 ACGAGCTGTA CGCCGAGTGG TCGGAGGTCG TGTCCACGAA CTTCCGGGAC GCCTCCGGGC

 2701 CGGCCATGAC CGAGATCGGC GAGCAGCCGT GGGGGCGGGA GTTCGCCCTG CGCGACCCGG

 2761 CCGGCAACTG CGTGCACTTC GTGGCCGAGG AGCAGGACTG ACACGTGCTA CGAGATTTCG

 2821 ATTCCACCGC CGCCTTCTAT GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG GACGCCGGCT

 2881 GGATGATCCT CCAGCGCGGG GATCTCATGC TGGAGTTCTT CGCCCACCCC AACTTGTTTA

 2941 TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC AAATTTCACA AATAAAGCAT

 3001 TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT CAATGTATCT TATCATGTCT

 3061 GTATACCGTC GACCTCTAGC TAGAGCTTGG CGTAATCATG GTCATAGCTG TTTCCTGTGT

 3121 GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC CGGAAGCATA AAGTGTAAAG

 3181 CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCA CTGCCCGCTT

 3241 TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC GCGGGGAGAG

 3301 GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG CGCTCGGTCG

 3361 TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA TCCACAGAAT

 3421 CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTA

 3481 AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA

 3541 ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC CAGGCGTTTC

 3601 CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC GGATACCTGT

 3661 CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCAATG CTCACGCTGT AGGTATCTCA

 3721 GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG

 3781 ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT

 3841 CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA GGCGGTGCTA

 3901 CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGGACAGTA TTTGGTATCT

 3961 GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC

 4021 AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA

 4081 AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA

 4141 ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT

 4201 TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA

 4261 GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT CTGTCTATTT CGTTCATCCA

 4321 TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC

 4381 CCAGTGCTGC AATGATACCG CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA

 4441 ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC GCCTCCATCC

 4501 AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA

 4561 ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC GTCGTTTGGT ATGGCTTCAT

 4621 TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG

 4681 CGGTTAGCTC CTTCGGTCCT CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC

 4741 TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA AGATGCTTTT

 4801 CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA GTGTATGCGG CGACCGAGTT

 4861 GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA TAGCAGAACT TTAAAAGTGC

 4921 TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG GATCTTACCG CTGTTGAGAT

 4981 CCAGTTCGAT GTAACCCACT CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA

 5041 GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA

 5101 CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA TTATTGAAGC ATTTATCAGG

 5161 GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA GAAAAATAAA CAAATAGGGG

 5221 TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CTGACGTC

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