pcDNA4/HisMax A

pcDNA4/HisMax A

载体基本信息

出品公司:	Ybscience
载体名称:	pcDNA4/HisMax 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:	5258 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 (N-端）, Xpress Epitope Tag（N-端）
载体抗性:	氨苄青霉素
筛选标记:	Zeocin
克隆菌株:	TOP10F´, DH5a
宿主细胞（系）:	常规细胞系，如293、Hela等
备注:	pcDNA4/HisMax A 载体是哺乳动物表达载体，适用于cDNA的表达与克隆； QBI SP163增强子，使得目的基因的高水平表达提高了3~5倍； pcDNA4/HisMax A,B,C的区别仅在于多克隆位点处； 含EK (Enterokinase)切割位点
产品目录号:	V864-20
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

载体简介

 

pcDNA4/HisMax A, B, and C载体介绍：

pcDNA4/HisMax is specifically designed to maximize protein expression in a variety of mammalian cells. The vector contains the QBI SP163 translational enhancer to increase expression levels two- to five-fold above those seen with the promoter alone . In addition to SP163-enhanced expression, pcDNA4/HisMax includes a cleavable N-terminal Xpress tag for rapid detection of recombinant protein with an Anti-Xpress Antibody. pcDNA4/HisMax is available TOPO Cloning-ready for five-minute cloning of Taqamplified PCR products.

pcDNA4/HisMax A, B, and C are 5.3 kb vectors derived from pcDNA4/His and designed for overproduction of recombinant proteins in mammalian cell lines. Features of the vectors allow purification and detection of expressed proteins. 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
 QBI SP163 translational enhancer for increased levels of recombinant protein expression (Stein et al., 1998) (see page 4 for more information)
 Three reading frames to facilitate in-frame cloning with an N-terminal peptide encoding the Xpress epitope and a polyhistidine metal-binding tag
 Zeocin resistance gene for selection of stable cell lines 
 Episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g. COS-1, COS-7)
The control plasmid, pcDNA4/HisMax/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/HisMax.
 Consult the multiple cloning sites described on pages 5-7 to determine which vector (A, B, or C) should be used to clone your gene in frame with the N-terminal Xpress epitope and the polyhistidine tag.
 Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 50 to 100 μg/ml ampicillin or 25-50 μg/ml Zeocin.
 Analyze your transformants for the presence of insert by restriction digestion.
 Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in frame with the N-terminal peptide.
 Transfect your construct into the cell line of choice using your own method of transfection. Generate a stable cell line, if desired.
 Test for expression of your recombinant gene by western blot analysis or functional assay. 
 To purify your recombinant protein, you may use metal-chelating resin such as ProBond. ProBond resin is available separately.

表达目的基因：

We have a wide variety of mammalian expression vectors utilizing the CMV or EF-1α promoter. Vectors are available with the Xpress (N-terminal), c-myc (C-terminal), or V5 (C-terminal) epitope for detection and either the neomycin, blasticidin, or Zeocin resistance genes. All vectors utilize the polyhistidine tag for purification using ProBond resin. 

The pcDNA4/HisMax vectors are fusion vectors. To ensure proper expression of your recombinant protein, you must clone your gene in frame with the ATG at base pairs 1080-1082. This will create a fusion with the N-terminal polyhistidine tag, Xpress epitope, and the enterokinase cleavage site. The vector is supplied with the multiple cloning site in three reading frames relative to the N-terminal peptide to facilitate cloning.

If you wish to clone your gene as close as possible to the enterokinase cleavage site, follow the guidelines below:
 Digest pcDNA4/HisMax A, B, or C with Kpn I.
 Create blunt ends with T4 DNA polymerase and dNTPs.
 Clone your blunt-ended insert in frame with the lysine codon (AAG) of the enterokinase recognition site.

 

载体序列

LOCUS       pcDNA4/His-Max A	5258 bp 	DNA	SYN
DEFINITION  pcDNA4/His-Max A
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..5258
                     /organism="pcDNA4/His-Max 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    1112..1130
                     /label="Xpress_fwd_primer"
     misc_feature    1113..1145
                     /label="T7_leader"
     misc_feature    1149..1172
                     /label="Xpress_EK"
     misc_feature    complement(1265..1282)
                     /label="BGH_rev_primer"
     terminator      1268..1495
                     /label="bGH_PA_terminator"
     rep_origin      1558..1864
                     /label="f1_origin"
     misc_feature    complement(1978..1998)
                     /label="pBABE_3_primer"
     misc_feature    complement(1984..2200)
                     /label="SV40_enhancer"
     promoter        1996..2265
                     /label="SV40_promoter"
     rep_origin      2164..2241
                     /label="SV40_origin"
     misc_feature    2226..2245
                     /label="SV40pro_F_primer"
     promoter        2359..2426
                     /label="EM7_promoter"
     gene            2427..2798
                     /label="bleo"
                     /gene="bleo"
     misc_feature    2427..2801
                     /label="sh_ble"
     terminator      2934..3053
                     /label="SV40_PA_terminator"
     misc_feature    3022..3041
                     /label="EBV_rev_primer"
     promoter        complement(3097..3115)
                     /label="M13_reverse_primer"
     misc_feature    complement(3114..3136)
                     /label="M13_pUC_rev_primer"
     promoter        complement(3150..3179)
                     /label="lac_promoter"
     rep_origin      complement(3488..4107)
                     /label="pBR322_origin"
     gene            complement(4262..5122)
                     /label="Ampicillin"
                     /gene="Ampicillin"
     CDS             complement(4262..5122)
                     /label="ORF frame 2"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
     promoter        complement(5164..5192)
                     /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 GCTGGCTAGC
  901 GTTTAAACTT AAGCTTAGCG CAGAGGCTTG GGGCAGCCGA GCGGCAGCCA GGCCCCGGCC
  961 CGGGCCTCGG TTCCAGAAGG GAGAGGAGCC CGCCAAGGCG CGCAAGAGAG CGGGCTGCCT
 1021 CGCAGTCCGA GCCGGAGAGG GAGCGCGAGC CGCGCCGGCC CCGGACGGCC TCCGAAACCA
 1081 TGGGGGGTTC TCATCATCAT CATCATCATG GTATGGCTAG CATGACTGGT GGACAGCAAA
 1141 TGGGTCGGGA TCTGTACGAC GATGACGATA AGGTACCTAG GATCCAGTGT GGTGGAATTC
 1201 TGCAGATATC CAGCACAGTG GCGGCCGCTC GAGTCTAGAG GGCCCGTTTA AACCCGCTGA
 1261 TCAGCCTCGA CTGTGCCTTC TAGTTGCCAG CCATCTGTTG TTTGCCCCTC CCCCGTGCCT
 1321 TCCTTGACCC TGGAAGGTGC CACTCCCACT GTCCTTTCCT AATAAAATGA GGAAATTGCA
 1381 TCGCATTGTC TGAGTAGGTG TCATTCTATT CTGGGGGGTG GGGTGGGGCA GGACAGCAAG
 1441 GGGGAGGATT GGGAAGACAA TAGCAGGCAT GCTGGGGATG CGGTGGGCTC TATGGCTTCT
 1501 GAGGCGGAAA GAACCAGCTG GGGCTCTAGG GGGTATCCCC ACGCGCCCTG TAGCGGCGCA
 1561 TTAAGCGCGG CGGGTGTGGT GGTTACGCGC AGCGTGACCG CTACACTTGC CAGCGCCCTA
 1621 GCGCCCGCTC CTTTCGCTTT CTTCCCTTCC TTTCTCGCCA CGTTCGCCGG CTTTCCCCGT
 1681 CAAGCTCTAA ATCGGGGCAT CCCTTTAGGG TTCCGATTTA GTGCTTTACG GCACCTCGAC
 1741 CCCAAAAAAC TTGATTAGGG TGATGGTTCA CGTAGTGGGC CATCGCCCTG ATAGACGGTT
 1801 TTTCGCCCTT TGACGTTGGA GTCCACGTTC TTTAATAGTG GACTCTTGTT CCAAACTGGA
 1861 ACAACACTCA ACCCTATCTC GGTCTATTCT TTTGATTTAT AAGGGATTTT GGGGATTTCG
 1921 GCCTATTGGT TAAAAAATGA GCTGATTTAA CAAAAATTTA ACGCGAATTA ATTCTGTGGA
 1981 ATGTGTGTCA GTTAGGGTGT GGAAAGTCCC CAGGCTCCCC AGGCAGGCAG AAGTATGCAA
 2041 AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC
 2101 AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG
 2161 CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT
 2221 TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCTGCCTCTG AGCTATTCCA GAAGTAGTGA
 2281 GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAAAGCTCC CGGGAGCTTG TATATCCATT
 2341 TTCGGATCTG ATCAGCACGT GTTGACAATT AATCATCGGC ATAGTATATC GGCATAGTAT
 2401 AATACGACAA GGTGAGGAAC TAAACCATGG CCAAGTTGAC CAGTGCCGTT CCGGTGCTCA
 2461 CCGCGCGCGA CGTCGCCGGA GCGGTCGAGT TCTGGACCGA CCGGCTCGGG TTCTCCCGGG
 2521 ACTTCGTGGA GGACGACTTC GCCGGTGTGG TCCGGGACGA CGTGACCCTG TTCATCAGCG
 2581 CGGTCCAGGA CCAGGTGGTG CCGGACAACA CCCTGGCCTG GGTGTGGGTG CGCGGCCTGG
 2641 ACGAGCTGTA CGCCGAGTGG TCGGAGGTCG TGTCCACGAA CTTCCGGGAC GCCTCCGGGC
 2701 CGGCCATGAC CGAGATCGGC GAGCAGCCGT GGGGGCGGGA GTTCGCCCTG CGCGACCCGG
 2761 CCGGCAACTG CGTGCACTTC GTGGCCGAGG AGCAGGACTG ACACGTGCTA CGAGATTTCG
 2821 ATTCCACCGC CGCCTTCTAT GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG GACGCCGGCT
 2881 GGATGATCCT CCAGCGCGGG GATCTCATGC TGGAGTTCTT CGCCCACCCC AACTTGTTTA
 2941 TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC AAATTTCACA AATAAAGCAT
 3001 TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT CAATGTATCT TATCATGTCT
 3061 GTATACCGTC GACCTCTAGC TAGAGCTTGG CGTAATCATG GTCATAGCTG TTTCCTGTGT
 3121 GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC CGGAAGCATA AAGTGTAAAG
 3181 CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCA CTGCCCGCTT
 3241 TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC GCGGGGAGAG
 3301 GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG CGCTCGGTCG
 3361 TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA TCCACAGAAT
 3421 CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTA
 3481 AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA
 3541 ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC CAGGCGTTTC
 3601 CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC GGATACCTGT
 3661 CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCAATG CTCACGCTGT AGGTATCTCA
 3721 GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG
 3781 ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT
 3841 CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA GGCGGTGCTA
 3901 CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGGACAGTA TTTGGTATCT
 3961 GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC
 4021 AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA
 4081 AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA
 4141 ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT
 4201 TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA
 4261 GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT CTGTCTATTT CGTTCATCCA
 4321 TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC
 4381 CCAGTGCTGC AATGATACCG CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA
 4441 ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC GCCTCCATCC
 4501 AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA
 4561 ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC GTCGTTTGGT ATGGCTTCAT
 4621 TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG
 4681 CGGTTAGCTC CTTCGGTCCT CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC
 4741 TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA AGATGCTTTT
 4801 CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA GTGTATGCGG CGACCGAGTT
 4861 GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA TAGCAGAACT TTAAAAGTGC
 4921 TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG GATCTTACCG CTGTTGAGAT
 4981 CCAGTTCGAT GTAACCCACT CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA
 5041 GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA
 5101 CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA TTATTGAAGC ATTTATCAGG
 5161 GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA GAAAAATAAA CAAATAGGGG
 5221 TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CTGACGTC