质粒名称: | pCMV-MfPylRS (human-opti) |
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出品公司: | addgene |
目录编号: | 164081 |
存储实验室: | Tao Liu |
载体骨架基本信息 |
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载体名称: | pCMV |
空载体大小: | 5988 bp |
完整质粒大小: | 7235bp |
载体类型: | Mammalian Expression, Synthetic Biology |
筛选标记: | Hygromycin |
载体抗性: | 氨苄西林 |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | MfPylRS(human-opti) |
物种来源: | Methanosarcina flavescens |
插入大小: | 1251bp |
启动子: | CMV |
克隆方法: | Gibson Cloning |
5’测序引物 | CMV-forward |
3’测序引物 | BGHR |
文献引用方法 |
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材料和方法部分: | pCMV-MfPylRS (human-opti) was a gift from Tao Liu (Addgene plasmid # 164081 ; http://n2t.net/addgene:164081 ; RRID:Addgene_164081) |
参考文献部分: | Thermophilic Pyrrolysyl-tRNA Synthetase Mutants for Enhanced Mammalian Genetic Code Expansion. Hu L, Qin X, Huang Y, Cao W, Wang C, Wang Y, Ling X, Chen H, Wu D, Lin Y, Liu T. ACS Synth Biol. 2020 Oct 16;9(10):2723-2736. doi: 10.1021/acssynbio.0c00257. Epub 2020 Sep 23. 10.1021/acssynbio.0c00257 PubMed 32931698 |
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LOCUS pCMV-MfPylRS (human-opti) 7235 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION To express a Methanosarcina flavescens derived PylRS in mammalian cells for genetic code expansion. ACCESSION . VERSION . KEYWORDS pCMV-MfPylRS (human-opti) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7235) AUTHORS Hu L, Qin X, Huang Y, Cao W, Wang C, Wang Y, Ling X, Chen H, Wu D, Lin Y, Liu T TITLE Thermophilic Pyrrolysyl-tRNA Synthetase Mutants for Enhanced Mammalian Genetic Code Expansion. JOURNAL ACS Synth Biol. 2020 Oct 16;9(10):2723-2736. doi: 10.1021/acssynbio.0c00257. Epub 2020 Sep 23. PUBMED 32931698 REFERENCE 2 (bases 1 to 7235) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..7235 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 581..605 /label=LNCX /note="Human CMV promoter, forward primer" promoter 815..833 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 815..832 /label=SP6 /note="SP6 promoter, forward primer" primer_bind 846..865 /label=pMT2-F /note="Synthetic intron, forward primer" primer_bind complement(2174..2191) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2180..2404 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2450..2878 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2537..2556) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2747..2768 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2887..2907) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2892..3221 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3072..3207 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3134..3153 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3270..4295 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" polyA_signal 4425..4546 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4462..4481) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4516..4535 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4595..4611) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4595..4611) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4608..4630) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4619..4635 /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)." promoter complement(4643..4673) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4688..4709 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4826..4843) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4997..5585) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5077..5096) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5756..6616) /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 6379..6398 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6617..6721) /gene="bla" /label=AmpR promoter promoter complement(6819..7059) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(6869..6888) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(7039..7059) /label=hU6-F /note="Human U6 promoter, forward primer" ORIGIN 1 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat 661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg 721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca 781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat 841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacg gaagcttgcc 901 atggacaaga agcccctgga tgtgctgatc tctgccaccg gactgtggat gagcaggacc 961 ggcacactgc acaagatcaa gcaccacgag gtgtctaagc ggaagatcta catcgagatg 1021 gcctgcggcg acagactggt ggtgaacaat agcaggaact gtcgcaccgc ccgggccttc 1081 agacaccaca agtaccgcaa gacatgcaag tattgtaagg tgtccgacga ggatatctct 1141 aattttctga ccaagagcac agagaacaat agctccgtga aggtgaaggt ggtgtccgcc 1201 cccaaggtga agaaggccat gcctaagtct gtgagcaggg caccaaagcc tctggagacc 1261 cctgtgagcg cccagacatc cgcctctcgc agcggatccg tgcacgcaaa gtctgccagc 1321 aactctagct cctctacctc cgtgccagca cccgccctga caaggtctca gctggacagg 1381 gtggaggccc tgctgagccc agaggacaag gtgtccctgg atgcagcaaa gcccttccgg 1441 gagctggagt ccgagctgct ggtgcggaga aagggcgatc tgcagcggat ctacgccaac 1501 gagagagaga attatctggg caagctggag agggacatca ccaagttctt tgtggatcgc 1561 ggctttctgg agatcaagag cccaatcctg atccccgccg agtacgtgga gcggatgggc 1621 atcgactccg attctgagct gtccaagcag gtgttcaggg tggacaagaa tttttgcctg 1681 cgccctatgc tggccccaaa cctgtacaat tatctgcgga agctggacag agtgctgcct 1741 gatccagtga agatcttcga gatcggccct tgttatagaa aggagtccga gggcaaggag 1801 cacctggagg agttcaccat gctgaacttt tgccagatgg gctctggctg tacacgggag 1861 aatctggagg ccctgatcag agagttcctg gactacctgg gcatcgattt tgagatcgtg 1921 ggcgacagct gcatggtgta tggcgacacc ctggatgtga tgcacggcaa cctggagctg 1981 agctccgccg tgatcggacc aatccctctg gatagggagt ggggcatcaa taagccatgg 2041 atcggagcag gattcggact ggagcggctg ctgaaagtga tgcacggctt taagaacatc 2101 aagagggccg cccgcagcga gtcctactat aatggcatct ctacaagcct gtgaggatcc 2161 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 2221 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 2281 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 2341 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct 2401 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt 2461 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 2521 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 2581 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 2641 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 2701 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 2761 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg 2821 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa 2881 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 2941 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 3001 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 3061 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 3121 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga 3181 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta 3241 tatccatttt cggatctgat cagcacgtga tgaaaaagcc tgaactcacc gcgacgtctg 3301 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg 3361 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa 3421 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg 3481 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca 3541 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg 3601 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga 3661 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca 3721 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca 3781 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag 3841 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca 3901 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca 3961 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc 4021 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc 4081 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg 4141 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc 4201 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc 4261 gacgccccag cactcgtccg agggcaaagg aatagcacgt gctacgagat ttcgattcca 4321 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga 4381 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag 4441 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 4501 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac 4561 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 4621 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 4681 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 4741 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 4801 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 4861 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 4921 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 4981 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 5041 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 5101 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 5161 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 5221 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 5281 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 5341 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 5401 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc 5461 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 5521 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 5581 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 5641 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 5701 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 5761 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 5821 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 5881 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 5941 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 6001 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 6061 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 6121 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 6181 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 6241 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 6301 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 6361 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 6421 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 6481 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 6541 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 6601 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 6661 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 6721 gcacatttcc ccgaaaaatg gcggaaaccc cgggaatcta acccggctga acggatttag 6781 agtccattcg atctacatga tcaggtttcc ggtgtttcgt cctttccaca agatatataa 6841 agccaagaaa tcgaaatact ttcaagttac ggtaagcata tgatagtcca ttttaaaaca 6901 taattttaaa actgcaaact acccaagaaa ttattacttt ctacgtcacg tattttgtac 6961 taatatcttt gtgtttacag tcaaattaat tctaattatc tctctaacag ccttgtatcg 7021 tatatgcaaa tatgaaggaa tcatgggaaa taggccctct tcctgcccga ccttcgcgat 7081 gtacgggcca gatatacgcc aatattggcc attagccata ttattcattg gttatatagc 7141 ataaatcaat attggctatt ggccattgca tacgttgtat ctatatcata atatgtacat 7201 ttatattggc tcatgtccaa tatgaccgcc atgtt //
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