质粒名称: | pPL5071_TEF1*-Cre_URA3 |
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出品公司: | Addgene |
目录编号: | 60930 |
存储实验室: | Robert Piper |
载体骨架基本信息 |
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载体名称: | pSH47 |
完整质粒大小: | 6906 bp |
载体类型: | Yeast Expression |
筛选标记: | URA3 |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | Top10 |
拷贝数: | 低拷贝 |
插入基因信息 |
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插入基因名称: | Cre / Cre-recombinase |
物种来源: | E. coli |
插入大小: | 1032 bp |
启动子: | mutant TEF1 |
标签/融合蛋白: | Tag / Fusion Protein |
克隆方法: | 未知 |
5’测序引物 | cgataccgtcgaggggcagagcc |
3’测序引物 | catattttccgtcgatgttgaaatcc |
文献引用方法 |
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材料和方法部分: | pPL5071_TEF1*-Cre_URA3 was a gift from Robert Piper (Addgene plasmid # 60930 ; http://n2t.net/addgene:60930 ; RRID:Addgene_60930) |
参考文献部分: | Puromycin and Methotrexate Resistance Cassettes and Optimized cre-recombinase Expression Plasmids for use in Yeast. MacDonald C, Piper RC. Yeast. 2015 Feb 16. doi: 10.1002/yea.3069. 10.1002/yea.3069 PubMed 25688547 |
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载体序列
LOCUS pPL5071_TEF1*-Cre_URA3 6906 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Constitutive expression vector for Cre-recombinase containing URA3 selection marker.. ACCESSION . VERSION . KEYWORDS pPL5071_TEF1*-Cre_URA3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6906) AUTHORS MacDonald C, Piper RC TITLE Puromycin and Methotrexate Resistance Cassettes and Optimized cre-recombinase Expression Plasmids for use in Yeast. JOURNAL Yeast. 2015 Feb 16. doi: 10.1002/yea.3069. PUBMED 25688547 REFERENCE 2 (bases 1 to 6906) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..6906 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..19 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" CDS 553..1584 /codon_start=1 /product="site-specific recombinase" /label=Cre /note="Cre is a site-specific recombinase from bacteriophage P1. Recombination occurs at loxP sequences." /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE DGD" terminator 1902..2149 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(1918..1936) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" primer_bind complement(2167..2186) /label=T7 /note="T7 promoter, forward primer" promoter complement(2168..2186) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2196..2213) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2196..2212) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2205..2227) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2353..2808 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2440..2459) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2650..2671 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2939..3742) /codon_start=1 /gene="S. cerevisiae URA3" /product="orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis" /label=URA3 /note="yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)" /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN" promoter complement(3743..3958) /gene="S. cerevisiae URA3" /label=URA3 promoter primer_bind complement(3982..4001) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4101..4123 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4161..4179) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" misc_feature 4220..4723 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" promoter 4760..4864 /gene="bla" /label=AmpR promoter CDS 4865..5725 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(5083..5102) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5896..6484 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6385..6404 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6638..6655 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6772..6793 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6808..6838 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6846..6862 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 6851..6873 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6870..6886 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6870..6886 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6905..19 /label=T3 /note="T3 promoter, forward primer" ORIGIN 1 aattaaccct cactaaaggg aacaaaagct ggatagcttc aaaatgtctc tactcctttt 61 ttactcttcc agattttctc ggactccgcg caccgccgta ccacttcaaa acacccaagc 121 acagcatact aaattccccc tcctccttcc tctagggtgc cgttaattac ccgtactaaa 181 ggtttggaaa aggaaaaaga gaccgcctcg tccctttttc ttcgtcggag aaggcaataa 241 aaatttttat cacgtttctt tctcttgaaa actttttttt cgattttgtt ctctttcgac 301 gacctcccat tgatatttga gttaacaaac ggtcttcaat ttctcaagtt tcagcttcat 361 ttttcctgtt ctattacaac tttttttact tcttgctcat tggaaagaaa gcatagcaat 421 ctaatctaag ttttaattac aaaaactagt ggatcccccg ggctgcagga attcgatatc 481 aagcttatcg ataccgtcga ggggcagagc cgatcctgta cactttactt aaaaccatta 541 tctgagtgtt aaatgtccaa tttactgacc gtacaccaaa atttgcctgc attaccggtc 601 gatgcaacga gtgatgaggt tcgcaagaac ctgatggaca tgttcaggga tcgccaggcg 661 ttttctgagc atacctggaa aatgcttctg tccgtttgcc ggtcgtgggc ggcatggtgc 721 aagttgaata accggaaatg gtttcccgca gaacctgaag atgttcgcga ttatcttcta 781 tatcttcagg cgcgcggtct ggcagtaaaa actatccagc aacatttggg ccagctaaac 841 atgcttcatc gtcggtccgg gctgccacga ccaagtgaca gcaatgctgt ttcactggtt 901 atgcggcgga tccgaaaaga aaacgttgat gccggtgaac gtgcaaaaca ggctctagcg 961 ttcgaacgca ctgatttcga ccaggttcgt tcactcatgg aaaatagcga tcgctgccag 1021 gatatacgta atctggcatt tctggggatt gcttataaca ccctgttacg tatagccgaa 1081 attgccagga tcagggttaa agatatctca cgtactgacg gtgggagaat gttaatccat 1141 attggcagaa cgaaaacgct ggttagcacc gcaggtgtag agaaggcact tagcctgggg 1201 gtaactaaac tggtcgagcg atggatttcc gtctctggtg tagctgatga tccgaataac 1261 tacctgtttt gccgggtcag aaaaaatggt gttgccgcgc catctgccac cagccagcta 1321 tcaactcgcg ccctggaagg gatttttgaa gcaactcatc gattgattta cggcgctaag 1381 gatgactctg gtcagagata cctggcctgg tctggacaca gtgcccgtgt cggagccgcg 1441 cgagatatgg cccgcgctgg agtttcaata ccggagatca tgcaagctgg tggctggacc 1501 aatgtaaata ttgtcatgaa ctatatccgt aacctggata gtgaaacagg ggcaatggtg 1561 cgcctgctgg aagatggcga ttagccatta acgcgtaaat gattgctata attatttgat 1621 atttatggtg acatatgaga aaggatttca acatcgacgg aaaatatgta gtgctgtctg 1681 taagcactaa tattcagtcg ccagccgtca ttgtcactgt aaagctgagc gatagaatgc 1741 ctgatattga ctcaatatcc gttgcgtttc ctgtcaaaag tatgcgtagt gctgaacatt 1801 tcgtgatgaa tgccaccgag gaagaagcac ggcgcggttt tgctaaagtg atgtctgagt 1861 ttggcgaact cttgggtaag gttggaattg tcgacctcga gtcatgtaat tagttatgtc 1921 acgcttacat tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca 1981 acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt 2041 tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact 2101 gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgcg gccggtaccc 2161 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt 2221 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 2281 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 2341 aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 2401 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 2461 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 2521 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 2581 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 2641 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 2701 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 2761 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta 2821 ttttctcctt acgcatctgt gcggtatttc acaccgcata gggtaataac tgatataatt 2881 aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat acagtttttt 2941 agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg taacgttcac 3001 cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata ataatgtcag 3061 atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg tctcccttgt 3121 catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct cttccaccca 3181 tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca atgtcaacag 3241 tacccttagt atattctcca gtagataggg agcccttgca tgacaattct gctaacatca 3301 aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg ctaacaatac 3361 ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt ctgtatacac 3421 ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg tcttcgaaga 3481 gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc tccatggaaa 3541 aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct aatgcttcaa 3601 ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag tttgtttgct 3661 tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta gcagcacgtt 3721 ccttatatgt agctttcgac atgatttatc ttcgtttcct gcaggttttt gttctgtgca 3781 gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg cgtatatata 3841 ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt accgaatcaa 3901 aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat gaattgaaaa 3961 ggtggtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg 4021 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta 4081 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc 4141 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat 4201 aataatggtt tcttagacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa 4261 tgatggaata atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat 4321 tttagaaagt aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa 4381 ggtttaaaaa atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc 4441 agattaaata gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg 4501 tgtgtggtct tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa 4561 gagcaagata aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa 4621 aacaaaaact attttttctt taatttcttt ttttactttc tatttttaat ttatatattt 4681 atattaaaaa atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg 4741 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 4801 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 4861 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 4921 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 4981 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 5041 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 5101 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 5161 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 5221 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 5281 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 5341 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 5401 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 5461 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 5521 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 5581 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 5641 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 5701 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 5761 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 5821 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 5881 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 5941 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 6001 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 6061 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 6121 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 6181 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 6241 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 6301 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 6361 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 6421 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 6481 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 6541 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 6601 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 6661 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 6721 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 6781 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 6841 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa 6901 gcgcgc //
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