LOCUS pEVOL_ABK_Addgen 6801 bp ds-DNA linear 11-APR-2019 DEFINITION tRNA synthetase/tRNA pair for the in vivo incorporation of a photocrosslinker, p-benzoyl-l-phenylalanine into proteins in E coli in response to the amber codon, TAG. ACCESSION . VERSION . KEYWORDS pEVOL-ABK_Addgene SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6801) AUTHORS Chin JW, Martin AB, King DS, Wang L, Schultz PG TITLE Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli. JOURNAL Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11020-4. Epub 2002 Aug 1. PUBMED 12154230 REFERENCE 2 (bases 1 to 6801) AUTHORS . TITLE Direct Submission JOURNAL Exported 2019年4月11日 from SnapGene 2.8.3 http://www.snapgene.com COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers promoter 22..306 /gene="araBAD" /note="araBAD promoter" /note="promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction (Guzman et al., 1995)" /label=araBAD /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 CDS 343..1602 /codon_start=1 /note="AbK-RS, mbPylRS-L274M/C313A/T349F(Ai et al., 2011)" /translation="MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGD H LVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKV K KAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDR V EALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVD R GFLEIKSPILIPAEYVERMGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYMRKLDRI L PGPIKIFEVGPCYRKESDGKEHLEEFTMVNFAQMGSGCTRENLEALIKEFLDYLEIDF E IVGDSCMVFGDTLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHG F KNIKRASRSESYYNGISTNL" /label=AbK-RS, mbPylRS-L274M/C313A/T349F(Ai et al., 2011) /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 CDS 1612..1629 /codon_start=1 /product="6xHis affinity tag" /note="6xHis" /translation="HHHHHH" /label=6xHis affinity tag /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 promoter 1937..1995 /note="E.coli glnS promoter" /label=E.coli glnS promoter /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 CDS 2029..3288 /codon_start=1 /note="AbK-RS, mbPylRS-L274M/C313A/T349F(Ai et al., 2011)" /translation="MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGD H LVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKV K KAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDR V EALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVD R GFLEIKSPILIPAEYVERMGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYMRKLDRI L PGPIKIFEVGPCYRKESDGKEHLEEFTMVNFAQMGSGCTRENLEALIKEFLDYLEIDF E IVGDSCMVFGDTLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHG F KNIKRASRSESYYNGISTNL" /label=AbK-RS, mbPylRS-L274M/C313A/T349F(Ai et al., 2011)(1) /ApEinfo_label=AbK-RS, mbPylRS-L274M/C313A/T349F(Ai et al., 2011) /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 misc_feature 3418..3462 /note="glnS terminator" /label=glnS terminator /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 promoter 3649..3710 /note="E. coli proK promoter" /label=E. coli proK promoter /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 misc_feature 3711..3782 /note="Pyrrolysyl tRNA (TAG), Chatterjee et al., 2013" /label=Pyrrolysyl tRNA (TAG), Chatterjee et al., 2013 /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 misc_feature 3783..3817 /note="E. coli proK terminator" /label=E. coli proK terminator /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 CDS complement(3939..4598) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /note="CmR" /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF L KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFS S LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVAN M DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" /label=chloramphenicol acetyltransferase /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 promoter complement(4599..4701) /note="cat promoter" /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" /label=cat promoter /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 rep_origin complement(5227..5772) /direction=LEFT /note="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." /label=p15A ori /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 CDS complement(5918..6796) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /note="araC" /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKG Y ILNLTIRGQGVVKNQGREFVCRPSDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAY W HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLR R MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGI S VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGC E EKVNDVAVKLS" /label=L-arabinose regulatory protein /ApEinfo_fwdcolor=pink /ApEinfo_revcolor=pink /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0 ORIGIN 1 tcatactccc gccattcaga gaagaaacca attgtccata ttgcatcaga cattgccgtc 61 actgcgtctt ttactggctc ttctcgctaa ccaaaccggt aaccccgctt attaaaagca 121 ttctgtaaca aagcgggacc aaagccatga caaaaacgcg taacaaaagt gtctataatc 181 acggcagaaa agtccacatt gattatttgc acggcgtcac actttgctat gccatagcat 241 ttttatccat aagattagcg gatcctacct gacgcttttt atcgcaactc tctactgttt 301 ctccataccc gtttttttgg gctaacagga ggaattagat ccatggataa aaaaccatta 361 gatgttttaa tatctgcgac cgggctctgg atgtccagga ctggcacgct ccacaaaatc 421 aagcaccatg aggtctcaag aagtaaaata tacattgaaa tggcgtgtgg agaccatctt 481 gttgtgaata attccaggag ttgtagaaca gccagagcat tcagacatca taagtacaga 541 aaaacctgca aacgatgtag ggtttcggac gaggatatca ataattttct cacaagatca 601 accgaaagca aaaacagtgt gaaagttagg gtagtttctg ctccaaaggt caaaaaagct 661 atgccgaaat cagtttcaag ggctccgaag cctctggaaa attctgtttc tgcaaaggca 721 tcaacgaaca catccagatc tgtaccttcg cctgcaaaat caactccaaa ttcgtctgtt 781 cccgcatcgg ctcctgctcc ttcacttaca agaagccagc ttgatagggt tgaggctctc 841 ttaagtccag aggataaaat ttctctaaat atggcaaagc ctttcaggga acttgagcct 901 gaacttgtga caagaagaaa aaacgatttt cagcggctct ataccaatga tagagaagac 961 tacctcggta aactcgaacg tgatattacg aaatttttcg tagaccgggg ttttctggag 1021 ataaagtctc ctatccttat tccggcggaa tacgtggaga gaatgggtat taataatgat 1081 actgaacttt caaaacagat cttccgggtg gataaaaatc tctgcttgag gccaatgctt 1141 gccccgactc tgtataacta tatgcgaaaa ctcgatagga ttttaccagg cccaataaaa 1201 attttcgaag tcggaccttg ttaccggaaa gagtctgacg gcaaagagca cctggaagaa 1261 tttactatgg tgaacttcgc tcagatgggt tcgggatgta ctcgggaaaa tcttgaagct 1321 ctcatcaaag agtttctgga ctatctggaa atcgacttcg aaatcgtagg agattcctgt 1381 atggtctttg gggatactct tgatataatg cacggggacc tggagctttc ttcggcagtc 1441 gtcgggccag tttctcttga tagagaatgg ggtattgaca aaccatggat aggtgcaggt 1501 tttggtcttg aacgcttgct caaggttatg cacggcttta aaaacattaa gagggcatca 1561 aggtccgaat cttactataa tgggatttca accaatctat aataagtcga ccatcatcat 1621 catcatcatt gagtttaaac ggtctccagc ttggctgttt tggcggatga gagaagattt 1681 tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg 1741 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta 1801 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata 1861 aaacgaaagg ctcagtcgaa agactgggcc ttgtttgtga gctcccggtc atcaatcatc 1921 cccataatcc ttgttagatt atcaatttta aaaaactaac agttgtcagc ctgtcccgct 1981 ttaatatcat acgccgttat acgttgttta cgctttgagg aatcccatat ggataaaaaa 2041 ccattagatg ttttaatatc tgcgaccggg ctctggatgt ccaggactgg cacgctccac 2101 aaaatcaagc accatgaggt ctcaagaagt aaaatataca ttgaaatggc gtgtggagac 2161 catcttgttg tgaataattc caggagttgt agaacagcca gagcattcag acatcataag 2221 tacagaaaaa cctgcaaacg atgtagggtt tcggacgagg atatcaataa ttttctcaca 2281 agatcaaccg aaagcaaaaa cagtgtgaaa gttagggtag tttctgctcc aaaggtcaaa 2341 aaagctatgc cgaaatcagt ttcaagggct ccgaagcctc tggaaaattc tgtttctgca 2401 aaggcatcaa cgaacacatc cagatctgta ccttcgcctg caaaatcaac tccaaattcg 2461 tctgttcccg catcggctcc tgctccttca cttacaagaa gccagcttga tagggttgag 2521 gctctcttaa gtccagagga taaaatttct ctaaatatgg caaagccttt cagggaactt 2581 gagcctgaac ttgtgacaag aagaaaaaac gattttcagc ggctctatac caatgataga 2641 gaagactacc tcggtaaact cgaacgtgat attacgaaat ttttcgtaga ccggggtttt 2701 ctggagataa agtctcctat ccttattccg gcggaatacg tggagagaat gggtattaat 2761 aatgatactg aactttcaaa acagatcttc cgggtggata aaaatctctg cttgaggcca 2821 atgcttgccc cgactctgta taactatatg cgaaaactcg ataggatttt accaggccca 2881 ataaaaattt tcgaagtcgg accttgttac cggaaagagt ctgacggcaa agagcacctg 2941 gaagaattta ctatggtgaa cttcgctcag atgggttcgg gatgtactcg ggaaaatctt 3001 gaagctctca tcaaagagtt tctggactat ctggaaatcg acttcgaaat cgtaggagat 3061 tcctgtatgg tctttgggga tactcttgat ataatgcacg gggacctgga gctttcttcg 3121 gcagtcgtcg ggccagtttc tcttgataga gaatggggta ttgacaaacc atggataggt 3181 gcaggttttg gtcttgaacg cttgctcaag gttatgcacg gctttaaaaa cattaagagg 3241 gcatcaaggt ccgaatctta ctataatggg atttcaacca atctataact gcagtttcaa 3301 acgctaaatt gcctgatgcg ctacgcttat caggcctaca tgatctctgc aatatattga 3361 gtttgcgtgc ttttgtaggc cggataaggc gttcacgccg catccggcaa gaaacagcaa 3421 acaatccaaa acgccgcgtt cagcggcgtt ttttctgctt ttcttcgcga attaattccg 3481 cttcgcaaca tgtgagcacc ggtttattga ctaccggaag cagtgtgacc gtgtgcttct 3541 caaatgcctg aggccagttt gctcaggctc tccccgtgga ggtaataatt gacgatatga 3601 tcagtgcacg gctaactaag cggcctgctg actttctcgc cgatcaaaag gcattttgct 3661 attaagggat tgacgagggc gtatctgcgc agtaagatgc gccccgcatt ggaaacctga 3721 tcatgtagat cgaacggact ctaaatccgt tcagccgggt tagattcccg gggtttccgc 3781 caaattcgaa aagcctgctc aacgagcagg cttttttgca tgctcgagca gctcagggtc 3841 gaatttgctt tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa 3901 ccaggcgttt aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca 3961 tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca 4021 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc 4081 atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg 4141 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa 4201 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg 4261 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg 4321 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg 4381 aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg 4441 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta 4501 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat 4561 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa 4621 aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg 4681 gaacctctta cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg 4741 gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt 4801 tattcggcgc aaagtgcgtc gggtgatgct gccaacttac tgatttagtg tatgatggtg 4861 tttttgaggt gctccagtgg cttctgtttc tatcagctgt ccctcctgtt cagctactga 4921 cggggtggtg cgtaacggca aaagcaccgc cggacatcag cgctagcgga gtgtatactg 4981 gcttactatg ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa 5041 aggctgcacc ggtgcgtcag cagaatatgt gatacaggat atattccgct tcctcgctca 5101 ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg 5161 gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa 5221 gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca 5281 gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct 5341 cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg 5401 tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact 5461 gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg 5521 agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta 5581 gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt 5641 gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc 5701 gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca 5761 aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat ttcagtgcaa 5821 tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca 5881 tgtttgacag cttatcatcg ataagcttgg tacccaatta tgacaacttg acggctacat 5941 cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg gtgcattttt 6001 taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg acggtggcga 6061 taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg tcctcgcgcc 6121 agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc gacggcgaca 6181 agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg tgatcgctga 6241 tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac tcgttaatcg 6301 cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc tccgaatagc 6361 gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa tgcggctggt 6421 gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag ttaagccatt 6481 catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg cgagcctccg 6541 gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca ctcggtcggc 6601 aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg agattgagaa 6661 tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg ttggcctcaa 6721 tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc aggggatcat 6781 tttgcgcttc agccatactt t //