| 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 |
买家导航
pDual-GC载体质粒基本信息
pDual-GC质粒图谱载体图谱和pDual-GC载体序列质粒序列多克隆位点信息
pDual-GC质粒载体简介
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.
pDual-GC质粒序列载体序列
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
