pcDNA4/myc-His A

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/myc-His A
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: 5075 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 A 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 A载体图谱



pcDNA4-myc-His A 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       pcDNA4/myc-His A	5075 bp 	DNA	SYN
DEFINITION  pcDNA4/myc-His A
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..5075
                     /organism="pcDNA4/myc-His A"
                     /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(1082..1099)
                     /label="BGH_rev_primer"
     terminator      1085..1312
                     /label="bGH_PA_terminator"
     rep_origin      1375..1681
                     /label="f1_origin"
     misc_feature    complement(1795..1815)
                     /label="pBABE_3_primer"
     misc_feature    complement(1801..2017)
                     /label="SV40_enhancer"
     promoter        1813..2082
                     /label="SV40_promoter"
     rep_origin      1981..2058
                     /label="SV40_origin"
     misc_feature    2043..2062
                     /label="SV40pro_F_primer"
     promoter        2176..2243
                     /label="EM7_promoter"
     gene            2244..2615
                     /label="bleo"
                     /gene="bleo"
     misc_feature    2244..2618
                     /label="sh_ble"
     terminator      2751..2870
                     /label="SV40_PA_terminator"
     misc_feature    2839..2858
                     /label="EBV_rev_primer"
     promoter        complement(2914..2932)
                     /label="M13_reverse_primer"
     misc_feature    complement(2931..2953)
                     /label="M13_pUC_rev_primer"
     promoter        complement(2967..2996)
                     /label="lac_promoter"
     rep_origin      complement(3305..3924)
                     /label="pBR322_origin"
     gene            complement(4079..4939)
                     /label="Ampicillin"
                     /gene="Ampicillin"
     CDS             complement(4079..4939)
                     /label="ORF frame 2"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
     promoter        complement(4981..5009)
                     /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 CCCTTCGAAC AAAAACTCAT CTCAGAAGAG
 1021 GATCTGAATA TGCATACCGG TCATCATCAC CATCACCATT GAGTTTAAAC CCGCTGATCA
 1081 GCCTCGACTG TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC
 1141 TTGACCCTGG AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG
 1201 CATTGTCTGA GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG
 1261 GAGGATTGGG AAGACAATAG CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGCTTCTGAG
 1321 GCGGAAAGAA CCAGCTGGGG CTCTAGGGGG TATCCCCACG CGCCCTGTAG CGGCGCATTA
 1381 AGCGCGGCGG GTGTGGTGGT TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG
 1441 CCCGCTCCTT TCGCTTTCTT CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA
 1501 GCTCTAAATC GGGGCATCCC TTTAGGGTTC CGATTTAGTG CTTTACGGCA CCTCGACCCC
 1561 AAAAAACTTG ATTAGGGTGA TGGTTCACGT AGTGGGCCAT CGCCCTGATA GACGGTTTTT
 1621 CGCCCTTTGA CGTTGGAGTC CACGTTCTTT AATAGTGGAC TCTTGTTCCA AACTGGAACA
 1681 ACACTCAACC CTATCTCGGT CTATTCTTTT GATTTATAAG GGATTTTGGG GATTTCGGCC
 1741 TATTGGTTAA AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTAATT CTGTGGAATG
 1801 TGTGTCAGTT AGGGTGTGGA AAGTCCCCAG GCTCCCCAGG CAGGCAGAAG TATGCAAAGC
 1861 ATGCATCTCA ATTAGTCAGC AACCAGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA
 1921 AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCATAG TCCCGCCCCT AACTCCGCCC
 1981 ATCCCGCCCC TAACTCCGCC CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT
 2041 TTTATTTATG CAGAGGCCGA GGCCGCCTCT GCCTCTGAGC TATTCCAGAA GTAGTGAGGA
 2101 GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA AAGCTCCCGG GAGCTTGTAT ATCCATTTTC
 2161 GGATCTGATC AGCACGTGTT GACAATTAAT CATCGGCATA GTATATCGGC ATAGTATAAT
 2221 ACGACAAGGT GAGGAACTAA ACCATGGCCA AGTTGACCAG TGCCGTTCCG GTGCTCACCG
 2281 CGCGCGACGT CGCCGGAGCG GTCGAGTTCT GGACCGACCG GCTCGGGTTC TCCCGGGACT
 2341 TCGTGGAGGA CGACTTCGCC GGTGTGGTCC GGGACGACGT GACCCTGTTC ATCAGCGCGG
 2401 TCCAGGACCA GGTGGTGCCG GACAACACCC TGGCCTGGGT GTGGGTGCGC GGCCTGGACG
 2461 AGCTGTACGC CGAGTGGTCG GAGGTCGTGT CCACGAACTT CCGGGACGCC TCCGGGCCGG
 2521 CCATGACCGA GATCGGCGAG CAGCCGTGGG GGCGGGAGTT CGCCCTGCGC GACCCGGCCG
 2581 GCAACTGCGT GCACTTCGTG GCCGAGGAGC AGGACTGACA CGTGCTACGA GATTTCGATT
 2641 CCACCGCCGC CTTCTATGAA AGGTTGGGCT TCGGAATCGT TTTCCGGGAC GCCGGCTGGA
 2701 TGATCCTCCA GCGCGGGGAT CTCATGCTGG AGTTCTTCGC CCACCCCAAC TTGTTTATTG
 2761 CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT
 2821 TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAT CATGTCTGTA
 2881 TACCGTCGAC CTCTAGCTAG AGCTTGGCGT AATCATGGTC ATAGCTGTTT CCTGTGTGAA
 2941 ATTGTTATCC GCTCACAATT CCACACAACA TACGAGCCGG AAGCATAAAG TGTAAAGCCT
 3001 GGGGTGCCTA ATGAGTGAGC TAACTCACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC
 3061 AGTCGGGAAA CCTGTCGTGC CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG
 3121 GTTTGCGTAT TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC
 3181 GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG
 3241 GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA
 3301 AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC
 3361 GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC
 3421 CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG
 3481 CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCAATGCTC ACGCTGTAGG TATCTCAGTT
 3541 CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC
 3601 GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC
 3661 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG
 3721 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG
 3781 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA
 3841 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG
 3901 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT
 3961 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA
 4021 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT
 4081 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG
 4141 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA
 4201 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC
 4261 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT
 4321 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG
 4381 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA
 4441 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG
 4501 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA
 4561 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG
 4621 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT
 4681 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA
 4741 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA
 4801 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG
 4861 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC
 4921 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT
 4981 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC
 5041 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTC
//

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