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載體基本信息
| Clontech |
| pIRESpuro3 |
| 哺乳動物細胞表達載體 |
| CMV |
| 高 |
| 多克隆位點，限制性內切酶 |
| 5157 bp |
| CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3' |
| pIRES-R:5'-GCCCTAGATGCATGCTCG-3' |
| / |
| Ampicillin |
| Puromycin |
Express 2 genes on one transcript with IRES. |
| 6986-1 |
| 穩表達 Stable |
| 組成型 Constitutive |
| 非病毒 |
載體質粒圖譜和多克隆位點信息
載體簡介
Description
pIRESpuro3 contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA (1–3). After selection with puromycin, nearly all surviving colonies will stably express the gene of interest, thus decreasing the need to screen large numbers of colonies to find functional clones. To select for cells that express high levels of the gene of interest, the selective pressure for antibiotic resistance was increased by shifting the puromycin resistance gene downstream to a less optimal position for translation as directed by the IRES sequence (3). By decreasing the level of expression of the antibiotic resistance marker, the selective pressure on the entire expression cassette is increased, resulting in selection for cells that express the entire transcript, including the gene of interest, at high levels. The expression cassette of pIRESpuro3 contains the human cytomegalovirus (CMV) major immediate early promoter/enhancer followed by a multiple cloning site (MCS) that precedes stop codons in all three reading frames, a synthetic intron known to enhance the stability of the mRNA (4), the ECMV IRES followed by the gene encoding puromycin resistance (puromycin-N-acetyltransferase; 5), and the polyadenylation signal from SV40. Ribosomes can enter the bicistronic mRNA at the 5' end to translate the gene of interest and at the ECMV IRES to translate the antibiotic resistance marker.
Use
When using the pIRESpuro3 Vector, the antibiotic exerts selective pressure on the whole expression cassette; thus, a high dose of antibiotic will select for cells expressing a high level of the gene of interest. This selective pressure also ensures that the expression of the gene of interest will be stable
載體序列
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 gcttggtacc
901 gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc ggtcgtacgt
961 ctccggattc gaattcggat ccgcggccgc atagataact gatccagtgt gctggaatta
1021 attcgctgtc tgcgagggcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga
1081 cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg
1141 cggtgatgcc tttgagggtg gccgcgtcca tctggtcaga aaagacaatc tttttgttgt
1201 caagcttgag gtgtggcagg cttgagatct ggccatacac ttgagtgaca atgacatcca
1261 ctttgccttt ctctccacag gtgtccactc ccaggtccaa ctgcaggtcg agcatgcatc
1321 tagggcggcc aattccgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc
1381 ttggaataag gccggtgtgc gtttgtctat atgtgatttt ccaccatatt gccgtctttt
1441 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
1501 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
1561 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
1621 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
1681 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
1741 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
1801 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
1861 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata agcttgccac
1921 aacccacaag gagacgacct tccatgaccg agtacaagcc cacggtgcgc ctcgccaccc
1981 gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
2041 cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg caagaactct
2101 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
2161 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
2221 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
2281 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
2341 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
2401 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
2461 ggctcggctt caccgtcacc gccgacgtcg agtgcccgaa ggaccgcgcg acctggtgca
2521 tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga
2581 gcgcacgacc ccatggctcc gaccgaagcc gacccgggcg gccccgccga ccccgcaccc
2641 gcccccgagg cccaccgact ctagataact gatcataatc agccatacca catttgtaga
2701 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
2761 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
2821 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
2881 actcatcaat gtatcttaac gcgtcgagtg cattctagtt gtggtttgtc caaactcatc
2941 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
3001 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
3061 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
3121 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
3181 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
3241 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
3301 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
3361 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
3421 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
3481 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
3541 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc
3601 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
3661 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
3721 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
3781 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
3841 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
3901 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
3961 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
4021 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
4081 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
4141 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
4201 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
4261 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
4321 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
4381 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
4441 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
4501 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
4561 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
4621 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
4681 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
4741 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
4801 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
4861 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
4921 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
4981 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
5041 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
5101 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
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