LOCUS       sequence-250214-        5301 bp    DNA     circular SYN 15-DEC-2025
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    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1021/ja211972x";
            journalName: "J Am Chem Soc"; date: "2012-02-15- 15"; volume: "134";
            issue: "6"; pages: "2950-3"
COMMENT     Sequence Label: pEVOL-pylT-N346A/C348A
FEATURES             Location/Qualifiers
     source          1..5301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
//
