LOCUS       sequence-447439-        7008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PylRS (AF)/tRNA Pyl orthogonal pair for genetic code expansion that 
            allows site-specific incorporation of noncanonical amino-acids into 
            a POI using the Amber stop codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7008)
  AUTHORS   Milles S, Tyagi S, Banterle N, Koehler C, VanDelinder V, Plass T, 
            Neal AP, Lemke EA
  TITLE     Click strategies for single-molecule protein fluorescence.
  JOURNAL   J Am Chem Soc. 2012 Mar 21;134(11):5187-95. doi: 10.1021/ja210587q. 
            Epub 2012 Mar 5.
  PUBMED    22356317
REFERENCE   2  (bases 1 to 7008)
  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/ja210587q";
            journalName: "J Am Chem Soc"; date: "2012-03-21- 21"; volume: "134";
            issue: "11"; pages: "5187-95"
COMMENT     Sequence Label: pEVOL_PylRS(AF)-tRNA
FEATURES             Location/Qualifiers
     source          1..7008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(480..497)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(614..1159)
                     /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(694..713)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1305..2183)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPSDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        2210..2494
                     /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     2417..2436
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             3902..3919
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3975..3992)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(6334..6993)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6908..6927
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(6994..7008,1..88))
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg
       61 aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg
      121 cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac
      181 aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc aacttactga tttagtgtat
      241 gatggtgttt ttgaggtgct ccagtggctt ctgtttctat cagctgtccc tcctgttcag
      301 ctactgacgg ggtggtgcgt aacggcaaaa gcaccgccgg acatcagcgc tagcggagtg
      361 tatactggct tactatgttg gcactgatga gggtgtcagt gaagtgcttc atgtggcagg
      421 agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat acaggatata ttccgcttcc
      481 tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa
      541 cggggcggag atttcctgga agatgccagg aagatactta acagggaagt gagagggccg
      601 cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct
      661 caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg
      721 gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat
      781 ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag
      841 ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat
      901 cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat
      961 tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag
     1021 ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag
     1081 aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg
     1141 cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc
     1201 agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta
     1261 attctcatgt ttgacagctt atcatcgata agcttggtac ccaattatga caacttgacg
     1321 gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg
     1381 cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg
     1441 gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc
     1501 tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac
     1561 ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga
     1621 tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg
     1681 ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc
     1741 gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc
     1801 ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta
     1861 agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga
     1921 gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcactc
     1981 ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga
     2041 ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg
     2101 gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg
     2161 ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat
     2221 tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc
     2281 aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca
     2341 aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac
     2401 ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga
     2461 cgctttttat cgcaactctc tactgtttct ccatacccgt ttttttgggc taacaggagg
     2521 aattagatct atggacaaaa aaccgctgaa taccctgatc tctgctactg gtctgtggat
     2581 gagtcgtacc ggaaccattc ataaaatcaa acaccacgag gttagccgtt cgaaaatcta
     2641 tattgagatg gcgtgtggcg atcatctggt tgtgaacaat agccgctctt ctcgtacagc
     2701 acgtgcactg cgtcaccaca aatatcgtaa aacctgtaaa cgttgccgtg tgtccgatga
     2761 ggatctgaac aaattcctga caaaagccaa tgaggaccaa acaagcgtga aagtgaaagt
     2821 cgttagcgct cctacccgta ctaaaaaagc aatgccgaaa tccgttgctc gtgcccctaa
     2881 accactggaa aacactgaag cagcacaggc acagccgtct ggaagcaaat tctctccggc
     2941 cattcctgtt tctacccagg agtccgtttc tgttccagca agtgtgagca ccagcattag
     3001 cagtattagc accggtgcca ccgctagcgc cctggttaaa ggcaatacca atccgattac
     3061 aagcatgtct gccccggttc aagcatcagc tccagcactg acaaaatccc aaaccgatcg
     3121 tctggaggtt ctgctgaatc cgaaagacga aatcagcctg aattccggca aaccgtttcg
     3181 tgaactggag agcgaactgc tgtcacgtcg taaaaaagac ctgcaacaaa tctatgccga
     3241 agaacgtgag aactatctgg ggaaactgga acgtgaaatc acccgctttt tcgtggatcg
     3301 tggctttctg gagatcaaat ccccgattct gattcctctg gagtatatcg agcgtatggg
     3361 catcgacaat gataccgaac tgagcaaaca aattttccgt gtggataaaa acttctgtct
     3421 gcgccctatg ctagcaccaa atctggctaa ctatctgcgc aaactggacc gtgccctgcc
     3481 tgatcctatc aaaatcttcg agatcggccc gtgttatcgt aaagagtccg acggtaaaga
     3541 acatctggag gagtttacca tgctgaactt ttgccaaatg ggttcaggtt gtactcgtga
     3601 gaacctggaa agcatcatca ccgattttct gaaccacctg ggcattgact tcaaaattgt
     3661 gggcgacagc tgtatggtgt ttggcgacac cctggatgtc atgcacggcg acctggaact
     3721 gtctagtgcc gttgttggac caattccgct ggaccgtgag tggggtatcg acaaaccgtg
     3781 gatcggagca ggattcggtc tggaacgcct gctgaaagtg aaacacgact tcaaaaacat
     3841 caaacgtgcc gcccgttctg aatcgtatta taacgggatc tctacgaacc tgtaagtcga
     3901 ccatcatcat catcatcatt gagtttaaac ggtctccagc ttggctgttt tggcggatga
     3961 gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag
     4021 aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg
     4081 aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag
     4141 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc ttgtttgtga gctcccggtc
     4201 atcaatcatc cccataatcc ttgttagatt atcaatttta aaaaactaac agttgtcagc
     4261 ctgtcccgct ttaatatcat acgccgttat acgttgttta cgctttgagg aatcccatat
     4321 ggacaaaaaa ccgctgaata ccctgatctc tgctactggt ctgtggatga gtcgtaccgg
     4381 aaccattcat aaaatcaaac accacgaggt tagccgttcg aaaatctata ttgagatggc
     4441 gtgtggcgat catctggttg tgaacaatag ccgctcttct cgtacagcac gtgcactgcg
     4501 tcaccacaaa tatcgtaaaa cctgtaaacg ttgccgtgtg tccgatgagg atctgaacaa
     4561 attcctgaca aaagccaatg aggaccaaac aagcgtgaaa gtgaaagtcg ttagcgctcc
     4621 tacccgtact aaaaaagcaa tgccgaaatc cgttgctcgt gcccctaaac cactggaaaa
     4681 cactgaagca gcacaggcac agccgtctgg aagcaaattc tctccggcca ttcctgtttc
     4741 tacccaggag tccgtttctg ttccagcaag tgtgagcacc agcattagca gtattagcac
     4801 cggtgccacc gctagcgccc tggttaaagg caataccaat ccgattacaa gcatgtctgc
     4861 cccggttcaa gcatcagctc cagcactgac aaaatcccaa accgatcgtc tggaggttct
     4921 gctgaatccg aaagacgaaa tcagcctgaa ttccggcaaa ccgtttcgtg aactggagag
     4981 cgaactgctg tcacgtcgta aaaaagacct gcaacaaatc tatgccgaag aacgtgagaa
     5041 ctatctgggg aaactggaac gtgaaatcac ccgctttttc gtggatcgtg gctttctgga
     5101 gatcaaatcc ccgattctga ttcctctgga gtatatcgag cgtatgggca tcgacaatga
     5161 taccgaactg agcaaacaaa ttttccgtgt ggataaaaac ttctgtctgc gccctatgct
     5221 agcaccaaat ctggctaact atctgcgcaa actggaccgt gccctgcctg atcctatcaa
     5281 aatcttcgag atcggcccgt gttatcgtaa agagtccgac ggtaaagaac atctggagga
     5341 gtttaccatg ctgaactttt gccaaatggg ttcaggttgt actcgtgaga acctggaaag
     5401 catcatcacc gattttctga accacctggg cattgacttc aaaattgtgg gcgacagctg
     5461 tatggtgttt ggcgacaccc tggatgtcat gcacggcgac ctggaactgt ctagtgccgt
     5521 tgttggacca attccgctgg accgtgagtg gggtatcgac aaaccgtgga tcggagcagg
     5581 attcggtctg gaacgcctgc tgaaagtgaa acacgacttc aaaaacatca aacgtgccgc
     5641 ccgttctgaa tcgtattata acgggatctc tacgaacctg taactgcagt ttcaaacgct
     5701 aaattgcctg atgcgctacg cttatcaggc ctacatgatc tctgcaatat attgagtttg
     5761 cgtgcttttg taggccggat aaggcgttca cgccgcatcc ggcaagaaac agcaaacaat
     5821 ccaaaacgcc gcgttcagcg gcgttttttc tgcttttctt cgcgaattaa ttccgcttcg
     5881 caacatgtga gcaccggttt attgactacc ggaagcagtg tgaccgtgtg cttctcaaat
     5941 gcctgaggcc agtttgctca ggctctcccc gtggaggtaa taattgacga tatgatcagt
     6001 gcacggctaa ctaagcggcc tgctgacttt ctcgccgatc aaaaggcatt ttgctattaa
     6061 gggattgacg agggcgtatc tgcgcagtaa gatgcgcccc gcattggaaa cctgatcatg
     6121 tagatcgaat ggactctaaa tccgttcagc cgggttagat tcccggggtt tccgccaaat
     6181 tcgaaaagcc tgctcaacga gcaggctttt ttgcatgctc gagcagctca gggtcgaatt
     6241 tgctttcgaa tttctgccat tcatccgctt attatcactt attcaggcgt agcaaccagg
     6301 cgtttaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc actcatcgca
     6361 gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg
     6421 aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatggt
     6481 gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact
     6541 cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag ggaaataggc
     6601 caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc
     6661 gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta
     6721 acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca tacggaattc
     6781 cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt
     6841 atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttataggt
     6901 acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt gggatatatc
     6961 aacggtggta tatccagtga tttttttctc cattttagct tccttagc
//
