pcDNA4/HisMax C

价格:1500元

联系方式:I47-825O-882O

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载体基本信息

出品公司: Invitrogen
载体名称: pcDNA4/HisMax C
质粒类型: 哺乳动物表达载体;cDNA表达载体
高拷贝/低拷贝: 高拷贝
克隆方法: 多克隆位点,限制性内切酶
启动子: CMV
载体大小: 5257 bp
5' 测序引物及序列: T7 Forward: 5’-TAATACGACTCACTATAGGG-3’
3' 测序引物及序列: BGH Reverse: 5-TAGAAGGCACAGTCGAGG-3
载体标签: His Tag (N-端), Xpress Epitope Tag(N-端)
载体抗性: 氨苄青霉素
筛选标记: Zeocin
克隆菌株: TOP10F´, DH5a
宿主细胞(系): 常规细胞系,如293、Hela等
备注: pcDNA4/HisMax C 载体是哺乳动物表达载体,适用于cDNA的表达与克隆;
QBI SP163增强子,使得目的基因的高水平表达提高了3~5倍;
pcDNA4/HisMax A,B,C的区别仅在于多克隆位点处;
含EK (Enterokinase)切割位点
产品目录号: V864-20
稳定性: 瞬表达 或 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

载体质粒图谱和多克隆位点信息

pcDNA4-HisMax C载体图谱



pcDNA4-HisMax C 多克隆位点

pcDNA4-HisMax 载体特征1
pcDNA4-HisMax 载体特征2

载体简介

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. For antibody to the Xpress epitope, please see the next page.
 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.

载体序列

LOCUS       pcDNA™4/HisMax C	5257 bp 	DNA	SYN
DEFINITION  pcDNA™4/HisMax C
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..5257
                     /organism="pcDNA™4/HisMax C"
                     /mol_type="other DNA"
     promoter        236..852
                     /label="CMV_immearly_promoter"
     misc_feature    315..602
                     /label="CAG_enhancer"
     misc_feature    769..789
                     /label="CMV_fwd_primer"
     promoter        863..881
                     /label="T7_promoter"
     misc_feature    1112..1130
                     /label="Xpress_fwd_primer"
     misc_feature    1113..1145
                     /label="T7_leader"
     misc_feature    1149..1172
                     /label="Xpress_EK"
     misc_feature    complement(1264..1281)
                     /label="BGH_rev_primer"
     terminator      1267..1494
                     /label="bGH_PA_terminator"
     rep_origin      1557..1863
                     /label="f1_origin"
     misc_feature    complement(1977..1997)
                     /label="pBABE_3_primer"
     misc_feature    complement(1983..2199)
                     /label="SV40_enhancer"
     promoter        1995..2264
                     /label="SV40_promoter"
     rep_origin      2163..2240
                     /label="SV40_origin"
     misc_feature    2225..2244
                     /label="SV40pro_F_primer"
     promoter        2358..2425
                     /label="EM7_promoter"
     gene            2426..2797
                     /label="bleo"
                     /gene="bleo"
     misc_feature    2426..2800
                     /label="sh_ble"
     terminator      2933..3052
                     /label="SV40_PA_terminator"
     misc_feature    3021..3040
                     /label="EBV_rev_primer"
     promoter        complement(3096..3114)
                     /label="M13_reverse_primer"
     misc_feature    complement(3113..3135)
                     /label="M13_pUC_rev_primer"
     promoter        complement(3149..3178)
                     /label="lac_promoter"
     rep_origin      complement(3487..4106)
                     /label="pBR322_origin"
     gene            complement(4261..5121)
                     /label="Ampicillin"
                     /gene="Ampicillin"
     CDS             complement(4261..5121)
                     /label="ORF frame 2"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
     promoter        complement(5163..5191)
                     /label="AmpR_promoter"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
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 AGGTACCAGG ATCCAGTGTG GTGGAATTCT
 1201 GCAGATATCC AGCACAGTGG CGGCCGCTCG AGTCTAGAGG GCCCGTTTAA ACCCGCTGAT
 1261 CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC CATCTGTTGT TTGCCCCTCC CCCGTGCCTT
 1321 CCTTGACCCT GGAAGGTGCC ACTCCCACTG TCCTTTCCTA ATAAAATGAG GAAATTGCAT
 1381 CGCATTGTCT GAGTAGGTGT CATTCTATTC TGGGGGGTGG GGTGGGGCAG GACAGCAAGG
 1441 GGGAGGATTG GGAAGACAAT AGCAGGCATG CTGGGGATGC GGTGGGCTCT ATGGCTTCTG
 1501 AGGCGGAAAG AACCAGCTGG GGCTCTAGGG GGTATCCCCA CGCGCCCTGT AGCGGCGCAT
 1561 TAAGCGCGGC GGGTGTGGTG GTTACGCGCA GCGTGACCGC TACACTTGCC AGCGCCCTAG
 1621 CGCCCGCTCC TTTCGCTTTC TTCCCTTCCT TTCTCGCCAC GTTCGCCGGC TTTCCCCGTC
 1681 AAGCTCTAAA TCGGGGCATC CCTTTAGGGT TCCGATTTAG TGCTTTACGG CACCTCGACC
 1741 CCAAAAAACT TGATTAGGGT GATGGTTCAC GTAGTGGGCC ATCGCCCTGA TAGACGGTTT
 1801 TTCGCCCTTT GACGTTGGAG TCCACGTTCT TTAATAGTGG ACTCTTGTTC CAAACTGGAA
 1861 CAACACTCAA CCCTATCTCG GTCTATTCTT TTGATTTATA AGGGATTTTG GGGATTTCGG
 1921 CCTATTGGTT AAAAAATGAG CTGATTTAAC AAAAATTTAA CGCGAATTAA TTCTGTGGAA
 1981 TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GGCAGGCAGA AGTATGCAAA
 2041 GCATGCATCT CAATTAGTCA GCAACCAGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA
 2101 GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAT AGTCCCGCCC CTAACTCCGC
 2161 CCATCCCGCC CCTAACTCCG CCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT
 2221 TTTTTATTTA TGCAGAGGCC GAGGCCGCCT CTGCCTCTGA GCTATTCCAG AAGTAGTGAG
 2281 GAGGCTTTTT TGGAGGCCTA GGCTTTTGCA AAAAGCTCCC GGGAGCTTGT ATATCCATTT
 2341 TCGGATCTGA TCAGCACGTG TTGACAATTA ATCATCGGCA TAGTATATCG GCATAGTATA
 2401 ATACGACAAG GTGAGGAACT AAACCATGGC CAAGTTGACC AGTGCCGTTC CGGTGCTCAC
 2461 CGCGCGCGAC GTCGCCGGAG CGGTCGAGTT CTGGACCGAC CGGCTCGGGT TCTCCCGGGA
 2521 CTTCGTGGAG GACGACTTCG CCGGTGTGGT CCGGGACGAC GTGACCCTGT TCATCAGCGC
 2581 GGTCCAGGAC CAGGTGGTGC CGGACAACAC CCTGGCCTGG GTGTGGGTGC GCGGCCTGGA
 2641 CGAGCTGTAC GCCGAGTGGT CGGAGGTCGT GTCCACGAAC TTCCGGGACG CCTCCGGGCC
 2701 GGCCATGACC GAGATCGGCG AGCAGCCGTG GGGGCGGGAG TTCGCCCTGC GCGACCCGGC
 2761 CGGCAACTGC GTGCACTTCG TGGCCGAGGA GCAGGACTGA CACGTGCTAC GAGATTTCGA
 2821 TTCCACCGCC GCCTTCTATG AAAGGTTGGG CTTCGGAATC GTTTTCCGGG ACGCCGGCTG
 2881 GATGATCCTC CAGCGCGGGG ATCTCATGCT GGAGTTCTTC GCCCACCCCA ACTTGTTTAT
 2941 TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA ATAAAGCATT
 3001 TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC AATGTATCTT ATCATGTCTG
 3061 TATACCGTCG ACCTCTAGCT AGAGCTTGGC GTAATCATGG TCATAGCTGT TTCCTGTGTG
 3121 AAATTGTTAT CCGCTCACAA TTCCACACAA CATACGAGCC GGAAGCATAA AGTGTAAAGC
 3181 CTGGGGTGCC TAATGAGTGA GCTAACTCAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT
 3241 CCAGTCGGGA AACCTGTCGT GCCAGCTGCA TTAATGAATC GGCCAACGCG CGGGGAGAGG
 3301 CGGTTTGCGT ATTGGGCGCT CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT
 3361 TCGGCTGCGG CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT CCACAGAATC
 3421 AGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA
 3481 AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA
 3541 TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC
 3601 CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC
 3661 CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCAATGC TCACGCTGTA GGTATCTCAG
 3721 TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA
 3781 CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC
 3841 GCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG GCGGTGCTAC
 3901 AGAGTTCTTG AAGTGGTGGC CTAACTACGG CTACACTAGA AGGACAGTAT TTGGTATCTG
 3961 CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA
 4021 AACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC GCAGAAAAAA
 4081 AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT GGAACGAAAA
 4141 CTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT AGATCCTTTT
 4201 AAATTAAAAA TGAAGTTTTA AATCAATCTA AAGTATATAT GAGTAAACTT GGTCTGACAG
 4261 TTACCAATGC TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC GTTCATCCAT
 4321 AGTTGCCTGA CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC CATCTGGCCC
 4381 CAGTGCTGCA ATGATACCGC GAGACCCACG CTCACCGGCT CCAGATTTAT CAGCAATAAA
 4441 CCAGCCAGCC GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA
 4501 GTCTATTAAT TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA
 4561 CGTTGTTGCC ATTGCTACAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA TGGCTTCATT
 4621 CAGCTCCGGT TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC
 4681 GGTTAGCTCC TTCGGTCCTC CGATCGTTGT CAGAAGTAAG TTGGCCGCAG TGTTATCACT
 4741 CATGGTTATG GCAGCACTGC ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC
 4801 TGTGACTGGT GAGTACTCAA CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG
 4861 CTCTTGCCCG GCGTCAATAC GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCT
 4921 CATCATTGGA AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC TGTTGAGATC
 4981 CAGTTCGATG TAACCCACTC GTGCACCCAA CTGATCTTCA GCATCTTTTA CTTTCACCAG
 5041 CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA AAATGCCGCA AAAAAGGGAA TAAGGGCGAC
 5101 ACGGAAATGT TGAATACTCA TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG
 5161 TTATTGTCTC ATGAGCGGAT ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT
 5221 TCCGCGCACA TTTCCCCGAA AAGTGCCACC TGACGTC
//

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