质粒名称: | pDule2-para-aminoPhe |
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出品公司: | Addgene |
目录编号: | 85503 |
存储实验室: | Ryan Mehl |
载体骨架基本信息 |
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载体名称: | pDule2 |
空载体大小: | 5400 bp |
完整质粒大小: | 7003 bp |
载体类型: | 细菌表达 |
载体抗性: | Spectinomycin |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 低拷贝 |
插入基因信息 |
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插入基因名称: | 4-aminoPhenylalanine / pAF / para-aminoPhenylalanine / p-aminoPhe tRNA synthetase and cognate amber suppressing tRNA derived from M. jannaschii Tyrosine synthetase/tRNA system |
物种来源: | Methanocaldococcus jannaschii |
插入大小: | 921 bp |
基因突变: | Y32T, E107T, D158P, I159L, L162A |
启动子: | lpp (constitutive) |
标签/融合蛋白: | 无 |
克隆方法: | 限制性内切酶 |
5’克隆位点: | NcoI(未破坏) |
3’克隆位点: | KpnI (未破坏) |
5’测序引物 | CGTCACTGCGTCTTTTACTG |
文献引用方法 |
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材料和方法部分: | pDule2-para-aminoPhe was a gift from Ryan Mehl (Addgene plasmid # 85503 ; http://n2t.net/addgene:85503 ; RRID:Addgene_85503) |
参考文献部分: | Generation of a bacterium with a 21 amino acid genetic code. Mehl RA, Anderson JC, Santoro SW, Wang L, Martin AB, King DS, Horn DM, Schultz PG. J Am Chem Soc. 2003 Jan 29;125(4):935-9. 10.1021/ja0284153 PubMed 12537491 |
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载体序列
LOCUS pDule2-para-aminoPhe 7003 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid for incorporating the non-canonical amino acid para-aminoPhe with the Mj pAF synthetase and cognate amber suppressing tRNA in Ecoli.. ACCESSION . VERSION . KEYWORDS pDule2-para-aminoPhe SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7003) AUTHORS Mehl RA, Anderson JC, Santoro SW, Wang L, Martin AB, King DS, Horn DM, Schultz PG TITLE Generation of a bacterium with a 21 amino acid genetic code. JOURNAL J Am Chem Soc. 2003 Jan 29;125(4):935-9. PUBMED 12537491 REFERENCE 2 (bases 1 to 7003) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..7003 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1161..1190 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 1206..1223 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1279..1296) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 1449..1535 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind complement(1742..1759) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1876..2421) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(1956..1975) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2572..2663 /gene="bla" /label=AmpR promoter CDS 2664..3455 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" primer_bind complement(3631..3650) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4776..4798 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 4790..4807 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 4791..4807 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 5791..5813 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5969..6847) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /label=araC /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE EKVNDVAVKLS" ORIGIN 1 aactcagaat aagaaatgag gccgctcatg gcgttctgtt gcccgtctca ctggtgaaaa 61 gaaaaacaac cctggcgccg cttctttgag cgaacgatca aaaataagtg gcgccccatc 121 aaaaaaatat tctcaacata aaaaactttg tgtaatactt gtaacgctga gtttacgctt 181 tgaggaatcc cccatggacg aatttgaaat gataaagaga aacacatctg aaattatcag 241 cgaggaagag ttaagagagg ttttaaaaaa agatgaaaaa tctgctacta taggttttga 301 accaagtggt aaaatacatt tagggcatta tctccaaata aaaaagatga ttgatttaca 361 aaatgctgga tttgatataa ttatattgtt ggctgattta cacgcctatt taaaccagaa 421 aggagagttg gatgagatta gaaaaatagg agattataac aaaaaagttt ttgaagcaat 481 ggggttaaag gcaaaatatg tttatggaag tacgttccag cttgataagg attatacact 541 gaatgtctat agattggctt taaaaactac cttaaaaaga gcaagaagga gtatggaact 601 tatagcaaga gaggatgaaa atccaaaggt tgctgaagtt atttatccaa taatgcaggc 661 taatccgttg cattatgctg gcgttgatgt tgcagttgga gggatggagc agagaaaaat 721 acacatgtta gcaagggagc ttttaccaaa aaaggttgtt tgtattcaca accctgtctt 781 aacgggtttg gatggagaag gaaagatgag ttcttcaaaa gggaatttta tagctgttga 841 tgactctcca gaagagatta gggctaagat aaagaaagca tactgcccag ctggagttgt 901 tgaaggaaat ccaataatgg agatagctaa atacttcctt gaatatcctt taaccataaa 961 aaggccagaa aaatttggtg gagatttgac agttaatagc tatgaggagt tagagggttt 1021 atttaaaaat aaggaattgc atccaatgga tttaaaaaat gctgtagctg aagaacttat 1081 aaagatttta gagccaatta gaaagagatt ataactgcag tttcaaacgg gtaccatatg 1141 ggaattcgaa gcttgggccc gaacaaaaac tcatctcaga agaggatctg aatagcgccg 1201 tcgaccatca tcatcatcat cattgagttt aaacggtctc cagcttggct gttttggcgg 1261 atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa 1321 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga 1381 agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg 1441 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt 1501 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagctgtc cctcctgttc 1561 agctactgac ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc gctagcggag 1621 tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct tcatgtggca 1681 ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata tattccgctt 1741 cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg 1801 aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa gtgagagggc 1861 cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg aaatctgacg 1921 ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 1981 cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca ttccgctgtt 2041 atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca gttcgctcca 2101 agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta tccggtaact 2161 atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca gccactggta 2221 attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca 2281 agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt ggtagctcag 2341 agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca agagattacg 2401 cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata tttctagatt 2461 tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg 2521 taattctcat gtttgacagc ttatcatcga tgcgaccgag tgtgctagct atttgtttat 2581 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 2641 aataatattg aaaaaggaag agtatgaggg aagcggtgat cgccgaagta tcgactcaac 2701 tatcagaggt agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt 2761 tgtacggctc cgcagtggat ggcggcctga agccacacag tgatattgat ttgctggtta 2821 cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa 2881 cttcggcttc ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc 2941 acgacgacat cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc 3001 agcgcaatga cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta 3061 tcttgctgac aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac 3121 tctttgatcc ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat 3181 ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca 3241 tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa 3301 tggagcgcct gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg 3361 gacaagaaga agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccactacg 3421 tgaaaggcga gatcaccaag gtagtcggca aataatgtct aacaattcgt tcaagccgag 3481 gggccgcaag atccggccac gatgacccgg tcgtcggttc agggcagggt cgttaaatag 3541 ccgcttatgt ctattgctgg tttaccggga tcctctacgc cggacgcatc gtggccggca 3601 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag 3661 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc 3721 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg 3781 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag 3841 agcgtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg 3901 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg 3961 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga 4021 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg 4081 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg 4141 cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga 4201 ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg 4261 tagatgacga ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt 4321 cgatcattgg accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg 4381 ggttggcatg gattgtaggc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg 4441 gtgcatggag ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt 4501 caccactcca agaattggag ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa 4561 cccttggcag aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc 4621 gggctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact 4681 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt ctggcgaaag ggggatgtgc 4741 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 4801 ggccagtgcc aagcttaaaa aaaatcctta gctttcgcta aggatctgca gtggtccggc 4861 gggccggatt tgaaccagcg ccatgcggat ttagagtccg ccgttctgcc ctgctgaact 4921 accgccggga attcagcgtt acaagtatta cacaaagttt tttatgttga gaatattttt 4981 ttgatggggc gccacttatt tttgatcgtt cgctcaaaga agcggcgcca gggttgtttt 5041 tcttttcacc agtgagacgg gcaacagaac gccatgagcg gcctcatttc ttattctgag 5101 ttacaacagt ccgcaccgct gccggtagct ccttccggtg ggcgcggggc atgactatcg 5161 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc 5221 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgccctgca 5281 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat 5341 ctgtattaac gaagcgctaa ccgtttttat catgctctgg gaggcagaat aaatgatcat 5401 atcgtcaatt attacctcca cggggagagc ctgagcaaac tggcctcagg catttgagaa 5461 gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc 5521 ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg aatttgcttt cgaatttctg 5581 ccattcatcc gcttattatc acttattcag gcgtagcaac caggcgttta agggcaccaa 5641 taactgcctt aaaaaaatta cgccccgccc tgccactcat cgcagttgac tgggtcatgg 5701 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 5761 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 5821 cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg 5881 cataatgtgc ctgtcaaatg gacgaagcag ggattctgca aaccctatgc tactccgtca 5941 agccgtcaat tgtctgattc gttaccaatt atgacaactt gacggctaca tcattcactt 6001 tttcttcaca accggcacgg aactcgctcg ggctggcccc ggtgcatttt ttaaataccc 6061 gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc 6121 gggtggtgct caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga 6181 cgctaatccc taactgctgg cggaaaagat gtgacagacg cgacggcgac aagcaaacat 6241 gctgtgcgac gctggcgata tcaaaattgc tgtctgccag gtgatcgctg atgtactgac 6301 aagcctcgcg tacccgatta tccatcggtg gatggagcga ctcgttaatc gcttccatgc 6361 gccgcagtaa caattgctca agcagattta tcgccagcag ctccgaatag cgcccttccc 6421 cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat 6481 ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca gttaagccat tcatgccagt 6541 aggcgcgcgg acgaaagtaa acccactggt gataccattc gcgagcctcc ggatgacgac 6601 cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt 6661 ctcgtccctg atttttcacc accccctgac cgcgaatggt gagattgaga atataacctt 6721 tcattcccag cggtcggtcg ataaaaaaat cgagataacc gttggcctca atcggcgtta 6781 aacccgccac cagatgggca ttaaacgagt atcccggcag caggggatca ttttgcgctt 6841 cagccatact tttcatactc ccgccattca gagaagaaac caattgtcca tattgcatca 6901 gacattgccg tcactgcgtc ttttactggc tcttctcgct aaccaaaccg gtaacccggg 6961 accaagttta ctcatatata cggacagcgg tgcggactgt tgt //
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