| 出品公司: | Invitrogen |
|---|---|
| 载体名称: | pcDNA6/V5-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: | 限制性内切酶，多克隆位点 |
| 启动子: | CMV |
| 载体大小: | 5148 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: | V5 Epitope(C-端）；His tag(C-端） |
| 载体抗性: | 氨苄青霉素 |
| 筛选标记: | Blasticidin |
| 克隆菌株: | TOP10F´, JM109 and INVaF´ |
| 宿主细胞（系）: | 常规细胞系，如293、Hela等 |
| 备注: |
pcDNA6/V5-His B载体是cDNA的表达与克隆载体； CMV启动子驱动目的基因的过表达； 易于克隆到其它载体中去； pcDNA6/V5-His A,B,C仅阅读框不同。 |
| 产品目录号: | V220-01 |
| 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
pcDNA6/V5-His B载体 pcDNA6/V5-HisB质粒基本信息
pcDNA6/V5-His B质粒图谱 pcDNA6/V5-HisB载体图谱和pcDNA6/V5-His B载体序列 pcDNA6/V5-HisB质粒序列多克隆位点信息
pcDNA6/V5-His B质粒 pcDNA6/V5-HisB载体简介
pcDNA6/V5-His A, B, and C are 5.1 kb vectors derived from pcDNA3.1(+) and designed for high-level expression, selection on blasticidin, purification, and detection of recombinant proteins in mammalian hosts. High-level stable and non-replicative transient expression can be carried out in most mammalian cells.
pcDNA6/V5-His A, B, and C 含有以下元件:
• 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 tag encoding the V5 epitope and a polyhistidine metal-binding peptide
• Blasticidin resistance gene (BSD) from Aspergillus terreus for selection in prokaryotic and eukaryotic cells (Kimura et al., 1994)
• 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, pcDNA6/V5-His/lacZ, is the pcDNA6/V5-His A vector with a 3.2 kb fragment containing the β-galactosidase gene cloned in frame with the C-terminal peptide (see page 11). It is included for use as a positive control for transfection, expression, purification, and detection in the cell line of choice.
实验流程
使用pcDNA.6/V5-His 载体表达目的基因的实验流程：
1. Consult the multiple cloning sites described on pages 3–5 to determine which vector (A, B, or C) should be used to clone your gene in frame with the C-terminal V5 epitope and polyhistidine tag.
2. Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 100 μg/mL blasticidin or 50 to 100 μg/mL ampicillin.
3. Analyze your transformants for the presence of insert by restriction digestion.
4. Select a transformant with the correct restriction pattern and confirm that your gene is in frame with the C-terminal peptide by sequencing.
5. Transfect your construct into the cell line of choice using your own method of transfection.
6. Test for expression of your recombinant gene by western blot analysis or functional assay. If you do not have an antibody to your protein, Life Technologies offers the Anti-V5 Antibody or the Anti-His(C-term) Antibody to detect your recombinant fusion protein.
7. To purify your recombinant fusion protein, you may use metal-chelating resin such as ProBond.. ProBond. resin is available separately.
pcDNA6/V5-His B质粒序列 pcDNA6/V5-HisB载体序列
LOCUS pcDNA6/V5-His B 5148 bp DNA SYN
DEFINITION pcDNA6/V5-His B
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
FEATURES Location/Qualifiers
source 1..5148
/organism="pcDNA6/V5-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(1104..1121)
/label="BGH_rev_primer"
terminator 1107..1334
/label="bGH_PA_terminator"
rep_origin 1397..1703
/label="f1_origin"
misc_feature complement(1817..1837)
/label="pBABE_3_primer"
misc_feature complement(1823..2038)
/label="SV40_enhancer"
promoter 1835..2103
/label="SV40_promoter"
rep_origin 2002..2079
/label="SV40_origin"
misc_feature 2064..2083
/label="SV40pro_F_primer"
promoter 2197..2264
/label="EM7_promoter"
gene 2265..2663
/label="blast"
/gene="blast"
terminator 2824..2943
/label="SV40_PA_terminator"
misc_feature 2912..2931
/label="EBV_rev_primer"
promoter complement(2987..3005)
/label="M13_reverse_primer"
misc_feature complement(3004..3026)
/label="M13_pUC_rev_primer"
promoter complement(3040..3069)
/label="lac_promoter"
gene complement(4152..5012)
/label="Ampicillin"
/gene="Ampicillin"
CDS complement(4152..5012)
/label="ORF frame 2"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
promoter complement(5054..5082)
/label="AmpR_promoter"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
ORIGIN
1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT 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 ATGGGTGGAG 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 AAGCTTGGTA CCGAGCTCGG ATCCACTAGT CCAGTGTGGT GGAATTCTGC
961 AGATATCCAG CACAGTGGCG GCCGCTCGAG TCTAGAGGGC CCGCGGTTCG AAGGTAAGCC
1021 TATCCCTAAC CCTCTCCTCG GTCTCGATTC TACGCGTACC GGTCATCATC ACCATCACCA
1081 TTGAGTTTAA ACCCGCTGAT CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC CATCTGTTGT
1141 TTGCCCCTCC CCCGTGCCTT CCTTGACCCT GGAAGGTGCC ACTCCCACTG TCCTTTCCTA
1201 ATAAAATGAG GAAATTGCAT CGCATTGTCT GAGTAGGTGT CATTCTATTC TGGGGGGTGG
1261 GGTGGGGCAG GACAGCAAGG GGGAGGATTG GGAAGACAAT AGCAGGCATG CTGGGGATGC
1321 GGTGGGCTCT ATGGCTTCTG AGGCGGAAAG AACCAGCTGG GGCTCTAGGG GGTATCCCCA
1381 CGCGCCCTGT AGCGGCGCAT TAAGCGCGGC GGGTGTGGTG GTTACGCGCA GCGTGACCGC
1441 TACACTTGCC AGCGCCCTAG CGCCCGCTCC TTTCGCTTTC TTCCCTTCCT TTCTCGCCAC
1501 GTTCGCCGGC TTTCCCCGTC AAGCTCTAAA TCGGGGGCTC CCTTTAGGGT TCCGATTTAG
1561 TGCTTTACGG CACCTCGACC CCAAAAAACT TGATTAGGGT GATGGTTCAC GTAGTGGGCC
1621 ATCGCCCTGA TAGACGGTTT TTCGCCCTTT GACGTTGGAG TCCACGTTCT TTAATAGTGG
1681 ACTCTTGTTC CAAACTGGAA CAACACTCAA CCCTATCTCG GTCTATTCTT TTGATTTATA
1741 AGGGATTTTG CCGATTTCGG CCTATTGGTT AAAAAATGAG CTGATTTAAC AAAAATTTAA
1801 CGCGAATTAA TTCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA
1861 GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC
1921 CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA
1981 GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG
2041 CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG AGGCCGCCTC TGCCTCTGAG
2101 CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AAAGCTCCCG
2161 GGAGCTTGTA TATCCATTTT CGGATCTGAT CAGCACGTGT TGACAATTAA TCATCGGCAT
2221 AGTATATCGG CATAGTATAA TACGACAAGG TGAGGAACTA AACCATGGCC AAGCCTTTGT
2281 CTCAAGAAGA ATCCACCCTC ATTGAAAGAG CAACGGCTAC AATCAACAGC ATCCCCATCT
2341 CTGAAGACTA CAGCGTCGCC AGCGCAGCTC TCTCTAGCGA CGGCCGCATC TTCACTGGTG
2401 TCAATGTATA TCATTTTACT GGGGGACCTT GTGCAGAACT CGTGGTGCTG GGCACTGCTG
2461 CTGCTGCGGC AGCTGGCAAC CTGACTTGTA TCGTCGCGAT CGGAAATGAG AACAGGGGCA
2521 TCTTGAGCCC CTGCGGACGG TGCCGACAGG TGCTTCTCGA TCTGCATCCT GGGATCAAAG
2581 CCATAGTGAA GGACAGTGAT GGACAGCCGA CGGCAGTTGG GATTCGTGAA TTGCTGCCCT
2641 CTGGTTATGT GTGGGAGGGC TAAGCACTTC GTGGCCGAGG AGCAGGACTG ACACGTGCTA
2701 CGAGATTTCG ATTCCACCGC CGCCTTCTAT GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG
2761 GACGCCGGCT GGATGATCCT CCAGCGCGGG GATCTCATGC TGGAGTTCTT CGCCCACCCC
2821 AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC AAATTTCACA
2881 AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT CAATGTATCT
2941 TATCATGTCT GTATACCGTC GACCTCTAGC TAGAGCTTGG CGTAATCATG GTCATAGCTG
3001 TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC CGGAAGCATA
3061 AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCA
3121 CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC
3181 GCGGGGAGAG GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG
3241 CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA
3301 TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA GCAAAAGGCC
3361 AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG
3421 CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC
3481 CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC
3541 GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG CTCACGCTGT
3601 AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC
3661 GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA
3721 CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA
3781 GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGAACAGTA
3841 TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA
3901 TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA GCAGATTACG
3961 CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG
4021 TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC
4081 TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT
4141 TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT CTGTCTATTT
4201 CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG GGAGGGCTTA
4261 CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC GCTCACCGGC TCCAGATTTA
4321 TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC
4381 GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT
4441 AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC GTCGTTTGGT
4501 ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC CCCCATGTTG
4561 TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG TCAGAAGTAA GTTGGCCGCA
4621 GTGTTATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA
4681 AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA GTGTATGCGG
4741 CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA TAGCAGAACT
4801 TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG GATCTTACCG
4861 CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA ACTGATCTTC AGCATCTTTT
4921 ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA
4981 ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA TTATTGAAGC
5041 ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA GAAAAATAAA
5101 CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CTGACGTC
//
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pcDNA6/V5-His B质粒 pcDNA6/V5-HisB载体应用举例
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