| 出品公司: | Invitrogen |
|---|---|
| 载体名称: | pcDNA4/HisMax C |
| 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: | 5257 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 C 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 |
买家导航
pcDNA4/HisMax C载体质粒基本信息
pcDNA4/HisMax C质粒图谱载体图谱和pcDNA4/HisMax C载体序列质粒序列多克隆位点信息
pcDNA4/HisMax C质粒载体简介
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. For antibody to the Xpress epitope, please see the next page.
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.
pcDNA4/HisMax C质粒序列载体序列
LOCUS pcDNA™4/HisMax C 5257 bp DNA SYN
DEFINITION pcDNA™4/HisMax C
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
FEATURES Location/Qualifiers
source 1..5257
/organism="pcDNA™4/HisMax C"
/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(1264..1281)
/label="BGH_rev_primer"
terminator 1267..1494
/label="bGH_PA_terminator"
rep_origin 1557..1863
/label="f1_origin"
misc_feature complement(1977..1997)
/label="pBABE_3_primer"
misc_feature complement(1983..2199)
/label="SV40_enhancer"
promoter 1995..2264
/label="SV40_promoter"
rep_origin 2163..2240
/label="SV40_origin"
misc_feature 2225..2244
/label="SV40pro_F_primer"
promoter 2358..2425
/label="EM7_promoter"
gene 2426..2797
/label="bleo"
/gene="bleo"
misc_feature 2426..2800
/label="sh_ble"
terminator 2933..3052
/label="SV40_PA_terminator"
misc_feature 3021..3040
/label="EBV_rev_primer"
promoter complement(3096..3114)
/label="M13_reverse_primer"
misc_feature complement(3113..3135)
/label="M13_pUC_rev_primer"
promoter complement(3149..3178)
/label="lac_promoter"
rep_origin complement(3487..4106)
/label="pBR322_origin"
gene complement(4261..5121)
/label="Ampicillin"
/gene="Ampicillin"
CDS complement(4261..5121)
/label="ORF frame 2"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
promoter complement(5163..5191)
/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 AGGTACCAGG ATCCAGTGTG GTGGAATTCT
1201 GCAGATATCC AGCACAGTGG CGGCCGCTCG AGTCTAGAGG GCCCGTTTAA ACCCGCTGAT
1261 CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC CATCTGTTGT TTGCCCCTCC CCCGTGCCTT
1321 CCTTGACCCT GGAAGGTGCC ACTCCCACTG TCCTTTCCTA ATAAAATGAG GAAATTGCAT
1381 CGCATTGTCT GAGTAGGTGT CATTCTATTC TGGGGGGTGG GGTGGGGCAG GACAGCAAGG
1441 GGGAGGATTG GGAAGACAAT AGCAGGCATG CTGGGGATGC GGTGGGCTCT ATGGCTTCTG
1501 AGGCGGAAAG AACCAGCTGG GGCTCTAGGG GGTATCCCCA CGCGCCCTGT AGCGGCGCAT
1561 TAAGCGCGGC GGGTGTGGTG GTTACGCGCA GCGTGACCGC TACACTTGCC AGCGCCCTAG
1621 CGCCCGCTCC TTTCGCTTTC TTCCCTTCCT TTCTCGCCAC GTTCGCCGGC TTTCCCCGTC
1681 AAGCTCTAAA TCGGGGCATC CCTTTAGGGT TCCGATTTAG TGCTTTACGG CACCTCGACC
1741 CCAAAAAACT TGATTAGGGT GATGGTTCAC GTAGTGGGCC ATCGCCCTGA TAGACGGTTT
1801 TTCGCCCTTT GACGTTGGAG TCCACGTTCT TTAATAGTGG ACTCTTGTTC CAAACTGGAA
1861 CAACACTCAA CCCTATCTCG GTCTATTCTT TTGATTTATA AGGGATTTTG GGGATTTCGG
1921 CCTATTGGTT AAAAAATGAG CTGATTTAAC AAAAATTTAA CGCGAATTAA TTCTGTGGAA
1981 TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GGCAGGCAGA AGTATGCAAA
2041 GCATGCATCT CAATTAGTCA GCAACCAGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA
2101 GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAT AGTCCCGCCC CTAACTCCGC
2161 CCATCCCGCC CCTAACTCCG CCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT
2221 TTTTTATTTA TGCAGAGGCC GAGGCCGCCT CTGCCTCTGA GCTATTCCAG AAGTAGTGAG
2281 GAGGCTTTTT TGGAGGCCTA GGCTTTTGCA AAAAGCTCCC GGGAGCTTGT ATATCCATTT
2341 TCGGATCTGA TCAGCACGTG TTGACAATTA ATCATCGGCA TAGTATATCG GCATAGTATA
2401 ATACGACAAG GTGAGGAACT AAACCATGGC CAAGTTGACC AGTGCCGTTC CGGTGCTCAC
2461 CGCGCGCGAC GTCGCCGGAG CGGTCGAGTT CTGGACCGAC CGGCTCGGGT TCTCCCGGGA
2521 CTTCGTGGAG GACGACTTCG CCGGTGTGGT CCGGGACGAC GTGACCCTGT TCATCAGCGC
2581 GGTCCAGGAC CAGGTGGTGC CGGACAACAC CCTGGCCTGG GTGTGGGTGC GCGGCCTGGA
2641 CGAGCTGTAC GCCGAGTGGT CGGAGGTCGT GTCCACGAAC TTCCGGGACG CCTCCGGGCC
2701 GGCCATGACC GAGATCGGCG AGCAGCCGTG GGGGCGGGAG TTCGCCCTGC GCGACCCGGC
2761 CGGCAACTGC GTGCACTTCG TGGCCGAGGA GCAGGACTGA CACGTGCTAC GAGATTTCGA
2821 TTCCACCGCC GCCTTCTATG AAAGGTTGGG CTTCGGAATC GTTTTCCGGG ACGCCGGCTG
2881 GATGATCCTC CAGCGCGGGG ATCTCATGCT GGAGTTCTTC GCCCACCCCA ACTTGTTTAT
2941 TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA ATAAAGCATT
3001 TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC AATGTATCTT ATCATGTCTG
3061 TATACCGTCG ACCTCTAGCT AGAGCTTGGC GTAATCATGG TCATAGCTGT TTCCTGTGTG
3121 AAATTGTTAT CCGCTCACAA TTCCACACAA CATACGAGCC GGAAGCATAA AGTGTAAAGC
3181 CTGGGGTGCC TAATGAGTGA GCTAACTCAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT
3241 CCAGTCGGGA AACCTGTCGT GCCAGCTGCA TTAATGAATC GGCCAACGCG CGGGGAGAGG
3301 CGGTTTGCGT ATTGGGCGCT CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT
3361 TCGGCTGCGG CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT CCACAGAATC
3421 AGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA
3481 AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA
3541 TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC
3601 CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC
3661 CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCAATGC TCACGCTGTA GGTATCTCAG
3721 TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA
3781 CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC
3841 GCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG GCGGTGCTAC
3901 AGAGTTCTTG AAGTGGTGGC CTAACTACGG CTACACTAGA AGGACAGTAT TTGGTATCTG
3961 CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA
4021 AACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC GCAGAAAAAA
4081 AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT GGAACGAAAA
4141 CTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT AGATCCTTTT
4201 AAATTAAAAA TGAAGTTTTA AATCAATCTA AAGTATATAT GAGTAAACTT GGTCTGACAG
4261 TTACCAATGC TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC GTTCATCCAT
4321 AGTTGCCTGA CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC CATCTGGCCC
4381 CAGTGCTGCA ATGATACCGC GAGACCCACG CTCACCGGCT CCAGATTTAT CAGCAATAAA
4441 CCAGCCAGCC GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA
4501 GTCTATTAAT TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA
4561 CGTTGTTGCC ATTGCTACAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA TGGCTTCATT
4621 CAGCTCCGGT TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC
4681 GGTTAGCTCC TTCGGTCCTC CGATCGTTGT CAGAAGTAAG TTGGCCGCAG TGTTATCACT
4741 CATGGTTATG GCAGCACTGC ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC
4801 TGTGACTGGT GAGTACTCAA CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG
4861 CTCTTGCCCG GCGTCAATAC GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCT
4921 CATCATTGGA AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC TGTTGAGATC
4981 CAGTTCGATG TAACCCACTC GTGCACCCAA CTGATCTTCA GCATCTTTTA CTTTCACCAG
5041 CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA AAATGCCGCA AAAAAGGGAA TAAGGGCGAC
5101 ACGGAAATGT TGAATACTCA TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG
5161 TTATTGTCTC ATGAGCGGAT ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT
5221 TCCGCGCACA TTTCCCCGAA AAGTGCCACC TGACGTC
//
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