pcDNA6/V5-His B

价格:1500元

联系方式:I47-825O-882O

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pcDNA6/V5-HisB载体基本信息

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

pcDNA6/V5-HisB载体质粒图谱和多克隆位点信息

pcDNA6-V5-His B 载体图谱



pcDNA6-V5-His B 多克隆位点

pcDNA6-V5-His 载体特征1
pcDNA6-V5-His 载体特征2

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-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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