| 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 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
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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