| 出品公司: | Clontech |
|---|---|
| 载体名称: | pIRESpuro3 |
| 质粒类型: | 哺乳动物细胞表达载体 |
| 启动子: | CMV |
| 表达水平: | 高 |
| 克隆方法: | 多克隆位点，限制性内切酶 |
| 载体大小: | 5157 bp |
| 5' 测序引物: | CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3' |
| 3' 测序引物: | pIRES-R:5'-GCCCTAGATGCATGCTCG-3' |
| 载体标签: | 无 |
| 载体抗性: | 氨苄青霉素（Ampicillin） |
| 筛选标记: | 嘌呤霉素（Puromycin） |
| 克隆菌株: | DH5α, HB101 |
| 宿主细胞（系）: | 常规细胞系，293、CV-1、CHO等 |
| 备注: |
pIRESpuro3载体含有IRES元件，目的基因和抗性基因以双顺反子的形式表达。
|
| 产品目录号: | 6986-1 |
| 稳定性: | 稳表达 或 稳表达 |
| 组成型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
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
载体序列
LOCUS pIRESpuro3 5171 bp DNA circular 16-NOV-2009 COMMENT Created by Clontech Laboratories Inc. http://www.biofeng.com COMMENT Cat. No. 631619 FEATURES Location/Qualifiers misc_feature 309..810 /label=CMV\IE\Promoter/Enhancer misc_feature 863..879 /label=T7\RNA\Polymerase\Promoter misc_feature 912..1015 /label=MCS misc_feature 1015..1310 /label=IVS misc_feature 1336..1926 /label=IRES misc_feature 1959..2555 /label=Puro(R) misc_feature 2822..2827 /label=SV40\polyA\signal misc_feature 2851..2856 /label=SV40\polyA\signal misc_feature 3411..4010 /label=pUC\origin misc_feature complement(4172..5032) /label=Amp(R) BASE COUNT 1207 a 1429 c 1303 g 1232 t ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 ctctcccccc cccccctctc cctccccccc ccctaacgtt 1381 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc 1441 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc 1501 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag 1561 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg 1621 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat 1681 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga 1741 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc 1801 cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg 1861 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga 1921 tgataagctt gccacaaccc acaaggagac gaccttccat gaccgagtac aagcccacgg 1981 tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg caccctcgcc gccgcgttcg 2041 ccgactaccc cgccacgcgc cacaccgtcg acccggaccg ccacatcgag cgggtcaccg 2101 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg 2161 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt 2221 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac 2281 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg 2341 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag 2401 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc 2461 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac 2521 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc 2581 gcccgaccga aaggagcgca cgaccccatg gctccgaccg aagccacccg gggcggcccc 2641 gccgaccccg cacccgcccc cgaggcccac cgactctaga taactgatca taatcagcca 2701 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct 2761 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta 2821 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 2881 ttgtggtttg tccaaactca tcaatgtatc ttaacgctcg agtgcattct agttgtggtt 2941 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct 3001 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 3061 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 3121 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 3181 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 3241 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 3301 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 3361 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 3421 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 3481 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 3541 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 3601 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 3661 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 3721 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 3781 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 3841 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 3901 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 3961 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 4021 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 4081 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 4141 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 4201 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 4261 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 4321 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 4381 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 4441 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 4501 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 4561 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 4621 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 4681 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 4741 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 4801 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 4861 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 4921 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 4981 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 5041 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 5101 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 5161 cacctgacgt c //
