pcDNA4/myc-His B

价格:1350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/myc-His B
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: 5079 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 (C-端), c-Myc Epitope Tag(C-端)
载体抗性: 氨苄青霉素
筛选标记: Zeocin
克隆菌株: TOP10F´, DH5a, JM109, TOP10
宿主细胞(系): 常规细胞系,如293、Hela等
备注: pcDNA4/myc-His B 载体是哺乳动物表达载体,适用于cDNA的表达与克隆;
CMV启动子驱动目的基因的高水平表达;
pcDNA4/myc-His A,B,C的 区别仅在于多克隆位点处;
产品目录号: V863-20
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-myc-His B载体图谱



pcDNA4-myc-His B 多克隆位点

pcDNA4-myc-His 载体特征

载体简介

载体描述

pcDNA4/myc-His A, B, and C are 5.1 kb vectors designed for overproduction of recombinant proteins in mammalian cell lines. Features of the vectors allow purification and detection of expressed proteins (see pages 11-12 for more information). 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

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

Zeocin resistance gene for selection of stable cell lines (Mulsant et al., 1988) (see page 14 for more information).

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, pcDNA4/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:

Use the following outline to clone and express your gene of interest in pcDNA4/myc-His:
1.Consult the multiple cloning sites described on pages 3-4 to determine which vector (A, B, or C) to use for cloning your gene in frame with the C-terminal myc epitope and the polyhistidine tag.
2.Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 50 to 100 μg/mL ampicillin or 25 to 50g/mL Zeocin in Low Salt LB. For more information.
3.Analyze your transformants for the presence of insert by restriction digestion.
4.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.
5.Transfect your construct into the cell line of choice using your own method of transfection. Generate a stable cell line, if desired.
6.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.
7.To purify your recombinant protein, you may use metal-chelating resin such as ProBond. ProBond resin is available separately

载体序列

LOCUS       pcDNA™4/myc-His B	5079 bp 	DNA	SYN
DEFINITION  pcDNA™4/myc-His B
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..5079
                     /organism="pcDNA™4/myc-His B"
                     /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    complement(1086..1103)
                     /label="BGH_rev_primer"
     terminator      1089..1316
                     /label="bGH_PA_terminator"
     rep_origin      1379..1685
                     /label="f1_origin"
     misc_feature    complement(1799..1819)
                     /label="pBABE_3_primer"
     misc_feature    complement(1805..2021)
                     /label="SV40_enhancer"
     promoter        1817..2086
                     /label="SV40_promoter"
     rep_origin      1985..2062
                     /label="SV40_origin"
     misc_feature    2047..2066
                     /label="SV40pro_F_primer"
     promoter        2180..2247
                     /label="EM7_promoter"
     gene            2248..2619
                     /label="bleo"
                     /gene="bleo"
     misc_feature    2248..2622
                     /label="sh_ble"
     terminator      2755..2874
                     /label="SV40_PA_terminator"
     misc_feature    2843..2862
                     /label="EBV_rev_primer"
     promoter        complement(2918..2936)
                     /label="M13_reverse_primer"
     misc_feature    complement(2935..2957)
                     /label="M13_pUC_rev_primer"
     promoter        complement(2971..3000)
                     /label="lac_promoter"
     rep_origin      complement(3309..3928)
                     /label="pBR322_origin"
     gene            complement(4083..4943)
                     /label="Ampicillin"
                     /gene="Ampicillin"
     CDS             complement(4083..4943)
                     /label="ORF frame 2"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
     promoter        complement(4985..5013)
                     /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 GCTGGCTAGT
  901 TAAGCTTGGT ACCGAGCTCG GATCCACTAG TCCAGTGTGG TGGAATTCTG CAGATATCCA
  961 GCACAGTGGC GGCCGCTCGA GTCTAGAGGG CCCGCGGTTC GAACAAAAAC TCATCTCAGA
 1021 AGAGGATCTG AATATGCATA CCGGTCATCA TCACCATCAC CATTGAGTTT AAACCCGCTG
 1081 ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA GCCATCTGTT GTTTGCCCCT CCCCCGTGCC
 1141 TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC TAATAAAATG AGGAAATTGC
 1201 ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT GGGGTGGGGC AGGACAGCAA
 1261 GGGGGAGGAT TGGGAAGACA ATAGCAGGCA TGCTGGGGAT GCGGTGGGCT CTATGGCTTC
 1321 TGAGGCGGAA AGAACCAGCT GGGGCTCTAG GGGGTATCCC CACGCGCCCT GTAGCGGCGC
 1381 ATTAAGCGCG GCGGGTGTGG TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT
 1441 AGCGCCCGCT CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG
 1501 TCAAGCTCTA AATCGGGGCA TCCCTTTAGG GTTCCGATTT AGTGCTTTAC GGCACCTCGA
 1561 CCCCAAAAAA CTTGATTAGG GTGATGGTTC ACGTAGTGGG CCATCGCCCT GATAGACGGT
 1621 TTTTCGCCCT TTGACGTTGG AGTCCACGTT CTTTAATAGT GGACTCTTGT TCCAAACTGG
 1681 AACAACACTC AACCCTATCT CGGTCTATTC TTTTGATTTA TAAGGGATTT TGGGGATTTC
 1741 GGCCTATTGG TTAAAAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT AATTCTGTGG
 1801 AATGTGTGTC AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC CAGGCAGGCA GAAGTATGCA
 1861 AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG
 1921 CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC ATAGTCCCGC CCCTAACTCC
 1981 GCCCATCCCG CCCCTAACTC CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT
 2041 TTTTTTTATT TATGCAGAGG CCGAGGCCGC CTCTGCCTCT GAGCTATTCC AGAAGTAGTG
 2101 AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG CAAAAAGCTC CCGGGAGCTT GTATATCCAT
 2161 TTTCGGATCT GATCAGCACG TGTTGACAAT TAATCATCGG CATAGTATAT CGGCATAGTA
 2221 TAATACGACA AGGTGAGGAA CTAAACCATG GCCAAGTTGA CCAGTGCCGT TCCGGTGCTC
 2281 ACCGCGCGCG ACGTCGCCGG AGCGGTCGAG TTCTGGACCG ACCGGCTCGG GTTCTCCCGG
 2341 GACTTCGTGG AGGACGACTT CGCCGGTGTG GTCCGGGACG ACGTGACCCT GTTCATCAGC
 2401 GCGGTCCAGG ACCAGGTGGT GCCGGACAAC ACCCTGGCCT GGGTGTGGGT GCGCGGCCTG
 2461 GACGAGCTGT ACGCCGAGTG GTCGGAGGTC GTGTCCACGA ACTTCCGGGA CGCCTCCGGG
 2521 CCGGCCATGA CCGAGATCGG CGAGCAGCCG TGGGGGCGGG AGTTCGCCCT GCGCGACCCG
 2581 GCCGGCAACT GCGTGCACTT CGTGGCCGAG GAGCAGGACT GACACGTGCT ACGAGATTTC
 2641 GATTCCACCG CCGCCTTCTA TGAAAGGTTG GGCTTCGGAA TCGTTTTCCG GGACGCCGGC
 2701 TGGATGATCC TCCAGCGCGG GGATCTCATG CTGGAGTTCT TCGCCCACCC CAACTTGTTT
 2761 ATTGCAGCTT ATAATGGTTA CAAATAAAGC AATAGCATCA CAAATTTCAC AAATAAAGCA
 2821 TTTTTTTCAC TGCATTCTAG TTGTGGTTTG TCCAAACTCA TCAATGTATC TTATCATGTC
 2881 TGTATACCGT CGACCTCTAG CTAGAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG
 2941 TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA
 3001 GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG CGTTGCGCTC ACTGCCCGCT
 3061 TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA
 3121 GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC
 3181 GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA
 3241 TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT
 3301 AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA
 3361 AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT
 3421 CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG
 3481 TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCAAT GCTCACGCTG TAGGTATCTC
 3541 AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC
 3601 GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA
 3661 TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT
 3721 ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC
 3781 TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA
 3841 CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA
 3901 AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA
 3961 AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT
 4021 TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC
 4081 AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC
 4141 ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC
 4201 CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA
 4261 AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC
 4321 CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC
 4381 AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA
 4441 TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA
 4501 GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA
 4561 CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT
 4621 TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT
 4681 TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG
 4741 CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA
 4801 TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC
 4861 AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG
 4921 ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG
 4981 GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG
 5041 GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTC
//

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