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pRetro-Lib
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出品公司: | Ybscience |
---|---|
载体名称: | pRetro-Lib |
质粒类型: | 逆转录病毒载体;文库克隆与表达载体 |
高拷贝/低拷贝: | 低拷贝 |
克隆方法: | 限制性内切酶,多克隆位点 |
启动子: | -- |
载体大小: | 4891 bp |
5' 测序引物及序列: | 5'-AGCCCTCACTCCTTCTCTAG-3' |
3' 测序引物及序列: | 5'-ACCTACAGGTGGGGTCTTTCATTCCC-3' |
载体标签: | -- |
载体抗性: | 氨苄青霉素 |
筛选标记: | -- |
克隆菌株: | DH5α 等 |
宿主细胞(系): | 常规细胞系(293、CV-1、CHO等) |
备注: | -- |
稳定性: | 稳表达 或 瞬表达 |
组成型/诱导型: | 组成型 |
病毒/非病毒: | 逆转录病毒 |
pRetro-Lib载体描述pRetro-Lib, derived from Moloney murine leukemia virus (MoMuLV), is designed for delivery and expression of retroviral libraries or genes (1–3). Upon transfection into a packaging cell line, pRetro-Lib can transiently express, or integrate and stably express, a transcript containing the gene of interest. The 5' viral LTR in this vector contains a viral promoter that controls expression of genes cloned into the MCS. pRetro-Lib also contains the Col E1 origin of replication and E. coli Ampr gene for propagation and antibiotic selection in bacteria. pRetro-Lib can be used as an alternative vector in the construction of a cDNA library using the SMART? cDNA Library Construction Kit (Cat. No. 634901). Retroviral expression libraries can be constructed by directionally cloning the SfiI(dT)30 primed cDNA into the SfiIA/B sites of pRetro-Lib. Restriction digestion with SfiI will remove the Stuffer fragment, prior to cloning.pRetro-Lib facilitates expression cloning in combination with the benefits of retroviral gene delivery. Upon transfection into a high-titer packaging cell line, RNA from the vector is packaged into infectious, replication-incompetent retroviral particles. That is, these retroviral particles can infect target cells and transmit the gene of interest (which is cloned between the viral LTR sequences), but cannot replicate within these cells since the cells lack the viral structural genes. pRetro-Lib does not contain the structural genes necessary for particle formation and replication, gag, pol, and env; these genes are stably integrated into the packaging line. Subsequent introduction of pRetro-Lib, containing Ψ+, transcription and processing elements, and the gene of interest produces high-titer, replication-incompetent infectious virus.Propagation in E. coli: Suitable host strains: DH5α, and other general purpose strains. Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) to E. coli hosts. E. coli replication origin: Col E1 Copy number: low Plasmid incompatibility group: pBR322NOTE: The viral supernatants produced by this retroviral vector could, depending on your cloned insert, containpotentially hazardous recombinant virus. Due caution must be exercised in the production and handling ofrecombinant retrovirus. Appropriate NIH, regional, and institutional guidelines apply.References:1. Coffin, J. M. & Varmus, H. E., Eds. (1996) Retroviruses (Cold Spring Harbor Laboratory Press, NY).2. Ausubel, F. M., et al. (1994) Current Protocols in Molecular Biology (Greene Publishing Associates, Inc. & John Wiley & Sons, Inc.).3. Miller, A. D. & Rosman, G. J. (1989) BioTechniques 7:980–990.
LOCUS pRetro-Lib 4891 bp DNA circular 20-NOV-2009COMMENT Created by Clontech Laboratories Inc. http://www.clontech.comCOMMENT Cat. No. 635002FEATURES Location/Qualifiers misc_feature 1..589 /label=5'\LTR misc_feature 659..1468 /label=Extended\packaging\signal misc_feature 1470..1502 /label=MCS-A misc_feature 1503..1694 /label=Stuffer\Fragment misc_feature 1695..1723 /label=MCS-B misc_feature 1763..2356 /label=3'\LTR misc_feature 2892..2892 /label=pUC\origin misc_feature complement(3652..4512) /label=Amp(R)BASE COUNT 1184 a 1325 c 1187 g 1195 t ORIGIN 1 tttgaaagac cccacccgta ggtggcaagc tagcttaagt aacgccactt tgcaaggcat 61 ggaaaaatac ataactgaga atagaaaagt tcagatcaag gtcaggaaca aagaaacagc 121 tgaataccaa acaggatatc tgtggtaagc ggttcctgcc ccggctcagg gccaagaaca 181 gatgagacag ctgagtgatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg 241 ctcggggcca agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagtgaa 301 tcatcagatg tttccagggt gccccaagga cctgaaaatg accctgtacc ttatttgaac 361 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc cgctctccga gctcaataaa 421 agagcccaca acccctcact cggcgcgcca gtcttccgat agactgcgtc gcccgggtac 481 ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggtctcg ctgttccttg 541 ggagggtctc ctctgagtga ttgactaccc acgacggggg tctttcattt gggggctcgt 601 ccgggatttg gagacccctg cccagggacc accgacccac caccgggagg taagctggcc 661 agcaacttat ctgtgtctgt ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg 721 tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt 781 ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg 841 cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt 901 aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa 961 ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct 1021 gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt 1081 gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa 1141 gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc 1201 gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc 1261 tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt 1321 tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc 1381 atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta 1441 tccagccctc actccttctc taggcgccgg aattcgttag gccattacgg cctgcaggat 1501 ccgaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt 1561 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 1621 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 1681 atcttatcat gtctggatcc ggccgcctcg gccaaacatc gataaaataa aagattttat 1741 ttagtctcca gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt 1801 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagag aagttcagat 1861 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 1921 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 1981 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 2041 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 2101 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 2161 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 2221 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 2281 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 2341 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc 2401 gacccaccac cgggaggtaa gctggctgcc tcgcgcgttt cggtgatgac ggtgaaaacc 2461 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca 2521 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc 2581 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt 2641 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg 2701 catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 2761 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 2821 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 2881 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 2941 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 3001 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 3061 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 3121 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 3181 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 3241 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 3301 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 3361 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 3421 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 3481 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 3541 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 3601 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 3661 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 3721 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 3781 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 3841 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 3901 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 3961 cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 4021 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 4081 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 4141 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 4201 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 4261 ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 4321 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 4381 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 4441 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 4501 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 4561 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 4621 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 4681 taaaaatagg cgtatcacga ggccctttcg tcttcaagaa ttgctagcaa ttgctagcaa 4741 ttgctagcaa ttcataccag atcaccgaaa actgtcctcc aaatgtgtcc ccctcacact 4801 cccaaattcg cgggcttctg cctcttagac cactctaccc tattccccac actcaccgga 4861 gccaaagccg cggcccttcc gtttctttgc t
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