pDual-GC

价格:2000元

联系方式:I47-825O-882O

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载体基本信息

出品公司: Agilent
载体名称: pDual-GC Dual expression vector; pDual-GC
质粒类型: 真核原核双系统表达载体;克隆载体
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: 原核启动子lac;真核启动子CMV
载体大小: 6640 bp
5' 测序引物及序列: --
3' 测序引物及序列: --
载体标签: His tag ;CPB tag
载体抗性: 氨苄青霉素
筛选标记: 新霉素Neomycin 和卡那霉素 Kanamycin
克隆宿主: XL1-Blue MRF'
表达宿主: E.coli  BL21(DE3) 
产品目录号: #214503
稳定性: --
组成型/诱导型: 真核细胞组成型表达;原核细胞IPTG诱导型表达
病毒/非病毒: 非病毒

载体质粒图谱和多克隆位点信息

pDual-GC载体图谱



pDual-GC 载体特征

载体简介

pDual-GC载体介绍

The pDual-GC vector, which is based on Agilent’s pDual expression vector, is designed for high-level protein expression in mammalian and bacterial cells (see Figure 1). The vector contains the promoter and enhancer region of the human cytomegalovirus (CMV) immediate early gene‡ for constitutive expression of the clones in either transiently or stably transfected mammalian cells. Inducible gene expression in prokaryotes is directed from the hybrid T7/lacO promoter; the vector carries a copy of the lac repressor gene (lacIq), which mediates tight repression in the absence of isopropyl-β-D-thio-galactopyranoside (IPTG). Expression is therefore regulated using IPTG in bacteria that contain the T7 RNA polymerase, for example, BL21(DE3) bacterial cells. A tandem arrangement of the bacterial Shine-Dalgarno1 and mammalian Kozak2 ribosomal binding sites (RBS)allows for efficient expression of the ORF in both bacterial and mammalian systems.
The unique cloning region of the pDual GC expression vector is characterized by the presence of two Eam1104 I recognition sequences (CTCTTC) directed in opposite orientations and separated by a spacer region encoding the β-lactamase gene with a prokaryotic promoter.Digesting the vector with the Eam1104 I restriction enzyme creates a 3-nucleotide 5´ overhang that is complementary to the translation initiation codon (ATG) of the DNA insert.
Inserts must be generated by PCR amplification with primers that contain Eam1104 I recognition sites and a minimal flanking sequence at their 5´ termini. The ability of Eam1104 I to cleave several bases downstream of its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA insert. The elimination of extraneous nucleotides and the generation of unique, nonpalindromic sticky ends permit the formation of directional seamless junctions during the subsequent ligation to the pDual GC expression vector.

3 In both bacterial and mammalian cells, the dominant selectable marker is the neomycin phosphotransferase gene, which is under the control of the β-lactamase promoter in bacterial cells and the SV40 promoter in mammalian cells. Expression of the neomycin phosphotransferase gene in mammalian cells allows stable clone selection with G418, whereas in bacteria the gene confers resistance to kanamycin selection. All pDual GC clones express a fusion protein consisting of the cDNA, a thrombin cleavage site, three copies of the c-myc epitope tag, and a single copy of the 6×His purification tag. The c-myc epitope is derived from the human c-myc gene and contains 10 amino acid residues (EQKLISEEDL).

4 This allows for convenient and sensitive detection of expressed proteins with anti–c-myc antibody. The 6×His purification tag consists of six histidine residues and allows for quick and easy purification of the fusion protein from bacterial cells.

5 A thrombin cleavage site between the protein encoded by the cDNA and the c-myc and 6×His tags allows the removal of both tags when desired, for example, following protein purification. 


pDual Expression Vectors - Details & Specifications

The pDual expression vector directs expression of heterologous genes in both mammalian and prokaryotic systems. For constitutive expression in mammalian cells, the pDual expression vector contains a mutagenized version of the promoter/enhancer of the human cytomegalovirus (CMV) immediate early gene. Inducible gene expression in prokaryotes is directed from the hybrid T7/lacO promoter; the pDual expression vector carries a copy of the lac repressor gene (lacIq), which mediates tight repression in the absence of isopropyl- ß -D-thio-galactopyranoside (IPTG). 
Efficient translation of mRNA generated in either the mammalian or prokaryotic system is achieved by a tandemly arranged Shine-Dalgarno/Kozak consensus sequence. In both bacterial and mammalian cells, the dominant selectable marker is the neomycin phosphotransferase gene which is under the control of the ß-lactamase promoter in bacterial cells and the SV40 promoter inmammalian cells (Figure 1). Expression of the neomycin phosphotransferase gene in mammaian cells allows stable clone selection with G418, whereas in bacteria the gene confers resistance to kanamycin selection.
Figure 1. Western Blot Analysis of Transfected Mammalian Lysates From CHO cell lysates, 1 mg of protein was electrophoresed on a 4-20% tris-glycine-SDS gel, then transferred to a nitrocellulose membrane, and reacted with antiluciferase (A) or anti-c-myc (B) antibodies. Antibody binding was detected using chemiluminescence methods. The difference in molecular weights of the different detected proteins is due to the use of epitope and purification tags. Estimated molecular weights: pDual GC + Luciferase is 68 kDa, pCMV-Tag5 + Luciferase is 63 kDa, and luciferase protein is 61 kDa. The difference in chemiluminescent signal between pDual GC + Luciferase and pCMV-Tag5 + Luciferase (B) is probably due to the presence of three copies of the c-myc epitope in the pDUAL GC vector and only one copy of the c-myc epitope in the pCMV-Tag5 vector. The lower molecular weight band that is detected at about 40 kDa with the anti-c-myc antibody (B) was also detected in control cells that had notThe unique cloning region of the pDual expression vector is characterized by the presence of two Eam1104 I recognition sequences (CTCTTC) directed in opposite orientations and separated by a spacer region encoding two EcoR I sites. Digesting the vector with the Eam1104 I restriction enzyme creates a 3-nucleotide 5´ overhang that is complementary to the translation initiation codon (ATG) of the DNA insert. 
Inserts must be generated by PCR amplification with primers that contain Eam1104 I recognition sites and a minimal flanking sequence at their 5´ termini. The ability of Eam1104 I to cleave several bases downstream of its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA insert. The elimination of extraneous nucleotides and the generation of unique, nonpalindromic sticky ends permit the formation of directional seamless junctions during the subsequent ligation to the pDual expression vector.
The pDual vector contains the Calmodulin Binding Peptide (CBP) affinity tag, located 3´ to the cloning site, for optional fusion of the affinity tag to the carboxy terminus of the protein-coding sequence of interest. The CBP-affinity tag is preceded by a thrombin cleavage site which allows the removal of the fusion tag from the protein of interest.


载体序列

pDual GC, 6640 bp                              version 027003

NOTE: The following sequence has been verified for accuracy
at the junctions. The remainder of the sequence has been 
obtained from existing data. 


    1  GCACTTTTCG GGGAAATGTG CGCGGAACCC CTATTTGTTT ATTTTTCTAA
   51  ATACATTCAA ATATGTATCC GCTCATGAGA CAATAACCCT GATAAATGCT
  101  TCAATAATAT TGAAAAAGGA AGAATCCTGA GGCGGAAAGA ACCAGCTGTG
  151  GAATGTGTGT CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA
  201  GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG
  251  TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA
  301  GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG CCCCTAACTC
  351  CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT TTTTTTTATT
  401  TATGCAGAGG CCGAGGCCGC CTCGGCCTCT GAGCTATTCC AGAAGTAGTG
  451  AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG CAAAGATCGA TCAAGAGACA
  501  GGATGAGGAT CGTTTCGCAT GATTGAACAA GATGGATTGC ACGCAGGTTC
  551  TCCGGCCGCT TGGGTGGAGA GGCTATTCGG CTATGACTGG GCACAACAGA
  601  CAATCGGCTG CTCTGATGCC GCCGTGTTCC GGCTGTCAGC GCAGGGGCGC
  651  CCGGTTCTTT TTGTCAAGAC CGACCTGTCC GGTGCCCTGA ATGAACTGCA
  701  AGACGAGGCA GCGCGGCTAT CGTGGCTGGC CACGACGGGC GTTCCTTGCG
  751  CAGCTGTGCT CGACGTTGTC ACTGAAGCGG GAAGGGACTG GCTGCTATTG
  801  GGCGAAGTGC CGGGGCAGGA TCTCCTGTCA TCTCACCTTG CTCCTGCCGA
  851  GAAAGTATCC ATCATGGCTG ATGCAATGCG GCGGCTGCAT ACGCTTGATC
  901  CGGCTACCTG CCCATTCGAC CACCAAGCGA AACATCGCAT CGAGCGAGCA
  951  CGTACTCGGA TGGAAGCCGG TCTTGTCGAT CAGGATGATC TGGACGAAGA
 1001  ACATCAGGGG CTCGCGCCAG CCGAACTGTT CGCCAGGCTC AAGGCGAGCA
 1051  TGCCCGACGG CGAGGATCTC GTCGTGACCC ATGGCGATGC CTGCTTGCCG
 1101  AATATCATGG TGGAAAATGG CCGCTTTTCT GGATTCATCG ACTGTGGCCG
 1151  GCTGGGTGTG GCGGACCGCT ATCAGGACAT AGCGTTGGCT ACCCGTGATA
 1201  TTGCTGAAGA ACTTGGCGGC GAATGGGCTG ACCGCTTCCT CGTGCTTTAC
 1251  GGTATCGCCG CTCCCGATTC GCAGCGCATC GCCTTCTATC GCCTTCTTGA
 1301  CGAGTTCTTC TGAGCGGGAC TCTGGGGTTC GAAATGACCG ACCAAGCGAC
 1351  GCCCAACCTG CCATCACGAG ATTTCGATTC CACCGCCGCC TTCTATGAAA
 1401  GGTTGGGCTT CGGAATCGTT TTCCGGGACG CCGGCTGGAT GATCCTCCAG
 1451  CGCGGGGATC TCATGCTGGA GTTCTTCGCC CACCCTAGGG GGAGGCTAAC
 1501  TGAAACACGG AAGGAGACAA TACCGGAAGG AACCCGCGCT ATGACGGCAA
 1551  TAAAAAGACA GAATAAAACG CACGGTGTTG GGTCGTTTGT TCATAAACGC
 1601  GGGGTTCGGT CCCAGGGCTG GCACTCTGTC GATACCCCAC CGAGACCCCA
 1651  TTGGGGCCAA TACGCCCGCG TTTCTTCCTT TTCCCCACCC CACCCCCCAA
 1701  GTTCGGGTGA AGGCCCAGGG CTCGCAGCCA ACGTCGGGGC GGCAGGCCCT
 1751  GCCATAGCCT CAGGTTACTC ATATATACTT TAGATTGATT TAAAACTTCA
 1801  TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT AATCTCATGA
 1851  CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA
 1901  GAAAAGATCA AAGGATCTTC TTGAGATCCT TTTTTTCTGC GCGTAATCTG
 1951  CTGCTTGCAA ACAAAAAAAC CACCGCTACC AGCGGTGGTT TGTTTGCCGG
 2001  ATCAAGAGCT ACCAACTCTT TTTCCGAAGG TAACTGGCTT CAGCAGAGCG
 2051  CAGATACCAA ATACTGTCCT TCTAGTGTAG CCGTAGTTAG GCCACCACTT
 2101  CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA ATCCTGTTAC
 2151  CAGTGGCTGC TGCCAGTGGC GATAAGTCGT GTCTTACCGG GTTGGACTCA
 2201  AGACGATAGT TACCGGATAA GGCGCAGCGG TCGGGCTGAA CGGGGGGTTC
 2251  GTGCACACAG CCCAGCTTGG AGCGAACGAC CTACACCGAA CTGAGATACC
 2301  TACAGCGTGA GCTATGAGAA AGCGCCACGC TTCCCGAAGG GAGAAAGGCG
 2351  GACAGGTATC CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC GCACGAGGGA
 2401  GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC GGGTTTCGCC
 2451  ACCTCTGACT TGAGCGTCGA TTTTTGTGAT GCTCGTCAGG GGGGCGGAGC
 2501  CTATGGAAAA ACGCCAGCAA CGCGGCCTTT TTACGGTTCC TGGCCTTTTG
 2551  CTGGCCTTTT GCTCACATGT TCTTTCCTGC GTTATCCCCT GATTCTGTGG
 2601  ATAACCGTAT TACCGTAATG AGTGAGCTAA CTTACATTAA TTGCGTTGCG
 2651  CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT
 2701  GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCCAGGGT
 2751  GGTTTTTCTT TTCACCAGTG AGACGGGCAA CAGCTGATTG CCCTTCACCG
 2801  CCTGGCCCTG AGAGAGTTGC AGCAAGCGGT CCACGCTGGT TTGCCCCAGC
 2851  AGGCGAAAAT CCTGTTTGAT GGTGGTTAAC GGCGGGATAT AACATGAGCT
 2901  GTCTTCGGTA TCGTCGTATC CCACTACCGA GATATCCGCA CCAACGCGCA
 2951  GCCCGGACTC GGTAATGGCG CGCATTGCGC CCAGCGCCAT CTGATCGTTG
 3001  GCAACCAGCA TCGCAGTGGG AACGATGCCC TCATTCAGCA TTTGCATGGT
 3051  TTGTTGAAAA CCGGACATGG CACTCCAGTC GCCTTCCCGT TCCGCTATCG
 3101  GCTGAATTTG ATTGCGAGTG AGATATTTAT GCCAGCCAGC CAGACGCAGA
 3151  CGCGCCGAGA CAGAACTTAA TGGGCCCGCT AACAGCGCGA TTTGCTGGTG
 3201  ACCCAATGCG ACCAGATGCT CCACGCCCAG TCGCGTACCG TCTTCATGGG
 3251  AGAAAATAAT ACTGTTGATG GGTGTCTGGT CAGAGACATC AAGAAATAAC
 3301  GCCGGAACAT TAGTGCAGGC AGCTTCCACA GCAATGGCAT CCTGGTCATC
 3351  CAGCGGATAG TTAATGATCA GCCCACTGAC GCGTTGCGCG AGAAGATTGT
 3401  GCACCGCCGC TTTACAGGCT TCGACGCCGC TTCGTTCTAC CATCGACACC
 3451  ACCACGCTGG CACCCAGTTG ATCGGCGCGA GATTTAATCG CCGCGACAAT
 3501  TTGCGACGGC GCGTGCAGGG CCAGACTGGA GGTGGCAACG CCAATCAGCA
 3551  ACGACTGTTT GCCCGCCAGT TGTTGTGCCA CGCGGTTGGG AATGTAATTC
 3601  AGCTCCGCCA TCGCCGCTTC CACTTTTTCC CGCGTTTTCG CAGAAACGTG
 3651  GCTGGCCTGG TTCACCACGC GGGAAACGGT CTGATAAGAG ACACCGGCAT
 3701  ACTCTGCGAC ATCGTATAAC GTTACTGGTT TCACATTCAC CACCCTGAAT
 3751  TGACTCTCTT TCGGGCGCTA TCATGCCATA CCGCGAAAGG TTTTGCGCCA
 3801  TTCGATTATT AATAGTAATC AATTACGGGG TCATTAGTTC ATAGCCCATA
 3851  TATGGAGTTC CGCGTTACAT AACTTACGGT AAATGGCCCG CCTGGCTGAC
 3901  CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA
 3951  GTAACGCCAA TAGGGACTTT CCATTGACGT CAATGGGTGG AGTATTTACG
 4001  GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTACGC
 4051  CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG
 4101  TACATGACCT TATGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT
 4151  CATCGCTATT ACCATGGTGA TGCGGTTTTG GCAGTACATC AATGGGCGTG
 4201  GATAGCGGTT TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC
 4251  AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG
 4301  TAACAACTCC GCCCCATTGA CGCAAATGGG CGGTAGGCGT GCCTAATGGG
 4351  AGGTCTATAT AAGCAGAGCT GGTTTAGTGA ACCGTCAGAT CCGCTAGCTA
 4401  ATACGACTCA CTATAGGGGA ATTGTGAGCG GATAACAATT CCCCTTGTTT
 4451  AAACTTTAAG AGGAGGGCCA CCATGGGAAG AGAATTCGTG CGCGGAACCC
 4501  CTATTTGTTT ATTTTTCTAA ATACATTCAA ATATGTATCC GCTCATGAGA
 4551  CAATAACCCT GATAAATGCT TCAATAATAT TGAAAAAGGA GGAGTATGAG
 4601  TATTCAACAT TTCCGTGTCG CCCTTATTCC CTTTTTTGCG GCATTTTGCC
 4651  TTCCTGTTTT TGCTCACCCA GAAACGCTGG TGAAAGTAAA AGATGCTGAA
 4701  GATCAGTTGG GTGCACGAGT GGGTTACATC GAACTGGATC TCAACAGCGG
 4751  TAAGATCCTT GAGAGTTTTC GCCCCGAAGA ACGTTTTCCA ATGATGAGCA
 4801  CTTTTAAAGT TCTGCTATGT GGCGCGGTAT TATCCCGTAT TGACGCCGGG
 4851  CAAGAGCAAC TCGGTCGCCG CATACACTAT TCTCAGAATG ACTTGGTTGA
 4901  GTACTCACCA GTCACAGAAA AGCATCTTAC GGATGGCATG ACAGTAAGAG
 4951  AATTATGCAG TGCTGCCATA ACCATGAGTG ATAACACTGC GGCCAACTTA
 5001  CTTCTGACAA CGATCGGAGG ACCGAAGGAG CTAACCGCTT TTTTGCACAA
 5051  CATGGGGGAT CATGTAACTC GCCTTGATCG TTGGGAACCG GAGCTGAATG
 5101  AAGCCATACC AAACGACGAG CGTGACACCA CGATGCCTGT AGCAATGGCA
 5151  ACAACGTTGC GCAAACTATT AACTGGCGAA CTACTTACTC TAGCTTCCCG
 5201  GCAACAATTA ATAGACTGGA TGGAGGCGGA TAAAGTTGCA GGACCACTTC
 5251  TGCGCTCGGC CCTTCCGGCT GGCTGGTTTA TTGCTGATAA ATCTGGAGCC
 5301  GGTGAGCGTG GGTCTCGCGG TATCATTGCA GCACTGGGGC CAGATGGTAA
 5351  GCCCTCCCGT ATCGTAGTTA TCTACACGAC GGGGAGTCAG GCAACTATGG
 5401  ATGAACGAAA TAGACAGATC GCTGAGATAG GTGCCTCACT GATTAAGCAT
 5451  TGGTAACTGT CAGACCAAGT TTACTCATAT ATAGAATTCA AGCTTTCCTC
 5501  TTCACTTAGT TTAAACACTG CGGCCGCGCT GCTGGTTCCG CGTGGTTCTA
 5551  CTAGTGAGCA GAAACTCATC TCTGAAGAAG ATCTGGAACA AAAGTTGATT
 5601  TCAGAAGAAG ATCTGGAACA GAAGCTCATC TCTGAGGAAG ATCTGGGTAC
 5651  CGCTGGCTCC GCTGCTGGTT CTAGACATCA CCATCACCAT CACTAATGAT
 5701  AATCCGCGTG GTTTCCAATA CCCAGCTTTG ACTTGACTTG ATAACAGGTA
 5751  AGTGTACCCG GATCCGGGGA ATTGTGAGCG GATAACTTCC CCGATCCGCC
 5801  CTTCCCAACA GTTGCGCAGC CTGAATGGCG AATGGAGATC CAATTTTTAA
 5851  GTGTATAATG TGCTAAACTA CTGATTCTAA TTGTTTGTGT ATTTTAGATT
 5901  CCAGACATGA TAAGATACAT TGATGAGTTT GGACAAACCA CAACTAGAAT
 5951  GCAGTGAAAA AAATGCTTTA TTTGTGAAAT TTGTGATGCT ATTGCTTTAT
 6001  TTGTAACCAT TATAAGCTGC AATAAACAAG TTAACAACAA CAATTGCATT
 6051  CATTTTATGT TTCAGGTTCA GGGGGAGATG TGGGAGGTTT TTTAAAGCAA
 6101  GTAAAACCTC TACAAATGTG GTAACGCGTA TAACCCCTTG GGGCCTCTAA
 6151  ACGGGTCTTG AGGGGTTTTT TGACGCGTAA ATTGTAAGCG TTAATATTTT
 6201  GTTAAAATTC GCGTTAAATT TTTGTTAAAT CAGCTCATTT TTTAACCAAT
 6251  AGGCCGAAAT CGGCAAAATC CCTTATAAAT CAAAAGAATA GACCGAGATA
 6301  GGGTTGAGTG TTGTTCCAGT TTGGAACAAG AGTCCACTAT TAAAGAACGT
 6351  GGACTCCAAC GTCAAAGGGC GAAAAACCGT CTATCAGGGC GATGGCCCAC
 6401  TACGTGAACC ATCACCCTAA TCAAGTTTTT TGGGGTCGAG GTGCCGTAAA
 6451  GCACTAAATC GGAACCCTAA AGGGAGCCCC CGATTTAGAG CTTGACGGGG
 6501  AAAGCCGGCG AACGTGGCGA GAAAGGAAGG GAAGAAAGCG AAAGGAGCGG
 6551  GCGCTAGGGC GCTGGCAAGT GTAGCGGTCA CGCTGCGCGT AACCACCACA
 6601  CCCGCCGCGC TTAATGCGCC GCTACAGGGC GCGTCAGGTG


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