pDual-GC

pDual-GC

pDual-GC载体基本信息

pDual-GC载体质粒图谱和多克隆位点信息

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. 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.

pDual-GC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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