LOCUS Exported 5301 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Double mutant of wild type mmPylRS designed for incorporation of non-canonical amino acids with mono-substituted phenylalanine derivatives and tyrosinyl ethers. ACCESSION . VERSION . KEYWORDS pEVOL-pylT-N346A/C348A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5301) AUTHORS Wang YS, Fang X, Wallace AL, Wu B, Liu WR TITLE A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum. JOURNAL J Am Chem Soc. 2012 Feb 15;134(6):2950-3. doi: 10.1021/ja211972x. Epub 2012 Feb 6. PUBMED 22289053 REFERENCE 2 (bases 1 to 5301) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5301 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1408..1425 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1481..1498) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" CDS complement(2133..2792) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 2707..2726 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(2793..2895) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" primer_bind complement(3287..3304) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3421..3966) /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(3501..3520) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4112..4990) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /label=araC /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPSDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE EKVNDVAVKLS" promoter 5017..5301 /gene="araBAD" /label=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)" primer_bind 5224..5243 /label=pBAD Forward /note="For vectors with E. coli araBAD promoter, forward primer" ORIGIN 1 acccgttttt ttgggctaac aggaggaatt actagtatgg ataaaaaacc actaaacact 61 ctgatatctg caaccgggct ctggatgtcc aggaccggaa caattcataa aataaaacac 121 cacgaagtct ctcgaagcaa aatctatatt gaaatggcat gcggtgacca ccttgttgta 181 aacaactcca ggagcagcag gactgcaaga gcgctcaggc accacaaata caggaagacc 241 tgcaaacgct gcagggtttc ggatgaggat ctcaataagt tcctcacaaa ggcaaacgaa 301 gaccagacaa gcgtaaaagt caaggtcgtt tctgccccta ccagaacgaa aaaggcaatg 361 ccaaaatccg ttgcgagagc cccgaaacct cttgagaata cagaagcggc acaggctcaa 421 ccttctggat ctaaattttc acctgcgata ccggtttcca cccaagagtc agtttctgtc 481 ccggcatctg tttcaacatc aatatcaagc atttctacag gagcaactgc atccgcactg 541 gtaaaaggga atacgaaccc cattacatcc atgtctgccc ctgttcaggc aagtgccccc 601 gcacttacga agagccagac tgacaggctt gaagtcctgt taaacccaaa agatgagatt 661 tccctgaatt ccggcaagcc tttcagggag cttgagtccg aattgctctc tcgcagaaaa 721 aaagacctgc agcagatcta cgcggaagaa agggagaatt atctggggaa actcgagcgt 781 gaaattacca ggttctttgt ggacaggggt tttctggaaa taaaatcccc gatcctgatc 841 cctcttgagt atatcgaaag gatgggcatt gataatgata ccgaactttc aaaacagatc 901 ttcagggttg acaagaactt ctgcctgaga cccatgcttg ctccaaacct ttacaactac 961 ctgcgcaagc ttgacagggc cctgcctgat ccaataaaaa tttttgaaat aggcccatgc 1021 tacagaaaag agtccgacgg caaagaacac ctcgaagagt ttaccatgct ggcgttcgcg 1081 cagatgggat cgggatgcac acgggaaaat cttgaaagca taattacgga cttcctgaac 1141 cacctgggaa ttgatttcaa gatcgtaggc gattcctgca tggtctatgg ggataccctt 1201 gatgtaatgc acggagacct ggaactttcc tctgcagtag tcggacccat accgcttgac 1261 cgggaatggg gtattgataa accctggata ggggcaggtt tcgggctcga acgccttcta 1321 aaggttaaac acgactttaa aaatatcaag agagctgcaa ggtccgagtc ttactataac 1381 gggatttcta ccaacctgta agtcgaccat catcatcatc atcattgagt ttaaacggtc 1441 tccagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag 1501 aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac 1561 ctgaccccat gccgaactca gaagtgaaac gccgcatccg gcaagaaaca gcaaacaatc 1621 caaaacgccg cgttcagcgg cgttttttct gcttttcttc gcgaattaat tccgcttcgc 1681 aacatgtgag caccggttta ttgactaccg gaagcagtgt gaccgtgtgc ttctcaaatg 1741 cctgaggcca gtttgctcag gctctccccg tggaggtaat aattgacgat atgatcagtg 1801 cacggctaac taagcggcct gctgactttc tcgccgatca aaaggcattt tgctattaag 1861 ggattgacga gggcgtatct gcgcagtaag atgcgccccg cattggaaac ctgatcatgt 1921 agatcgaatg gactctaaat ccgttcagcc gggttagatt cccggggttt ccgccaaatt 1981 cgaaaagcct gctcaacgag caggcttttt tgcatgctcg agcagctcag ggtcgaattt 2041 gctttcgaat ttctgccatt catccgctta ttatcactta ttcaggcgta gcaaccaggc 2101 gtttaagggc accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag 2161 tactgttgta attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga 2221 acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtg 2281 aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc 2341 acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc 2401 aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg 2461 tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa 2521 caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat acggaattcc 2581 ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta 2641 tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta 2701 cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca 2761 acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc 2821 gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct 2881 cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg cccagggctt cccggtatca 2941 acagggacac caggatttat ttattctgcg aagtgatctt ccgtcacagg tatttattcg 3001 gcgcaaagtg cgtcgggtga tgctgccaac ttactgattt agtgtatgat ggtgtttttg 3061 aggtgctcca gtggcttctg tttctatcag ctgtccctcc tgttcagcta ctgacggggt 3121 ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctag cggagtgtat actggcttac 3181 tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg 3241 caccggtgcg tcagcagaat atgtgataca ggatatattc cgcttcctcg ctcactgact 3301 cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt 3361 tcctggaaga tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt 3421 tttccatagg ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg 3481 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggcggct ccctcgtgcg 3541 ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc 3601 tcattccacg cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc 3661 acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 3721 acccggaaag acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag 3781 ttagtcttga agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc 3841 gctcctccaa gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa 3901 ccgccctgca aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga 3961 tctcaagaag atcatcttat taatcagata aaatatttct agatttcagt gcaatttatc 4021 tcttcaaatg tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgtttg 4081 acagcttatc atcgataagc ttggtaccca attatgacaa cttgacggct acatcattca 4141 ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat tttttaaata 4201 cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg gcgataggca 4261 tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg cgccagctta 4321 agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa 4381 catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg ctgatgtact 4441 gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta atcgcttcca 4501 tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa tagcgccctt 4561 ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt 4621 catccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc cattcatgcc 4681 agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc tccggatgac 4741 gaccgtagtg atgaatctct cctggcggga acagcaaaat atcactcggt cggcaaacaa 4801 attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg agaatataac 4861 ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc tcaatcggcg 4921 ttaaacccgc caccagatgg gcattaaacg agtatcccgg cagcagggga tcattttgcg 4981 cttcagccat acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca 5041 tcagacattg ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc 5101 cgcttattaa aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca 5161 aaagtgtcta taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt 5221 gctatgccat agcattttta tccataagat tagcggatcc tacctgacgc tttttatcgc 5281 aactctctac tgtttctcca t //