LOCUS       sequence-458970-        7008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PylRS (WT)/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(WT)-tRNA
FEATURES             Location/Qualifiers
     source          1..7008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(2112..2771)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2686..2705
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2772..2874)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(3266..3283)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3400..3945)
                     /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(3480..3499)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4091..4969)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPSDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        4996..5280
                     /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     5203..5222
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             6688..6705
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6761..6778)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
ORIGIN
        1 gttagattat caattttaaa aaactaacag ttgtcagcct gtcccgcttt aatatcatac
       61 gccgttatac gttgtttacg ctttgaggaa tcccatatgg acaaaaaacc gctgaatacc
      121 ctgatctctg ctactggtct gtggatgagt cgtaccggaa ccattcataa aatcaaacac
      181 cacgaggtta gccgttcgaa aatctatatt gagatggcgt gtggcgatca tctggttgtg
      241 aacaatagcc gctcttctcg tacagcacgt gcactgcgtc accacaaata tcgtaaaacc
      301 tgtaaacgtt gccgtgtgtc cgatgaggat ctgaacaaat tcctgacaaa agccaatgag
      361 gaccaaacaa gcgtgaaagt gaaagtcgtt agcgctccta cccgtactaa aaaagcaatg
      421 ccgaaatccg ttgctcgtgc ccctaaacca ctggaaaaca ctgaagcagc acaggcacag
      481 ccgtctggaa gcaaattctc tccggccatt cctgtttcta cccaggagtc cgtttctgtt
      541 ccagcaagtg tgagcaccag cattagcagt attagcaccg gtgccaccgc tagcgccctg
      601 gttaaaggca ataccaatcc gattacaagc atgtctgccc cggttcaagc atcagctcca
      661 gcactgacaa aatcccaaac cgatcgtctg gaggttctgc tgaatccgaa agacgaaatc
      721 agcctgaatt ccggcaaacc gtttcgtgaa ctggagagcg aactgctgtc acgtcgtaaa
      781 aaagacctgc aacaaatcta tgccgaagaa cgtgagaact atctggggaa actggaacgt
      841 gaaatcaccc gctttttcgt ggatcgtggc tttctggaga tcaaatcccc gattctgatt
      901 cctctggagt atatcgagcg tatgggcatc gacaatgata ccgaactgag caaacaaatt
      961 ttccgtgtgg ataaaaactt ctgtctgcgc cctatgctgg caccaaatct gtataactat
     1021 ctgcgcaaac tggaccgtgc cctgcctgat cctatcaaaa tcttcgagat cggcccgtgt
     1081 tatcgtaaag agtccgacgg taaagaacat ctggaggagt ttaccatgct gaacttttgc
     1141 caaatgggtt caggttgtac tcgtgagaac ctggaaagca tcatcaccga ttttctgaac
     1201 cacctgggca ttgacttcaa aattgtgggc gacagctgta tggtgtatgg cgacaccctg
     1261 gatgtcatgc acggcgacct ggaactgtct agtgccgttg ttggaccaat tccgctggac
     1321 cgtgagtggg gtatcgacaa accgtggatc ggagcaggat tcggtctgga acgcctgctg
     1381 aaagtgaaac acgacttcaa aaacatcaaa cgtgccgccc gttctgaatc gtattataac
     1441 gggatctcta cgaacctgta actgcagttt caaacgctaa attgcctgat gcgctacgct
     1501 tatcaggcct acatgatctc tgcaatatat tgagtttgcg tgcttttgta ggccggataa
     1561 ggcgttcacg ccgcatccgg caagaaacag caaacaatcc aaaacgccgc gttcagcggc
     1621 gttttttctg cttttcttcg cgaattaatt ccgcttcgca acatgtgagc accggtttat
     1681 tgactaccgg aagcagtgtg accgtgtgct tctcaaatgc ctgaggccag tttgctcagg
     1741 ctctccccgt ggaggtaata attgacgata tgatcagtgc acggctaact aagcggcctg
     1801 ctgactttct cgccgatcaa aaggcatttt gctattaagg gattgacgag ggcgtatctg
     1861 cgcagtaaga tgcgccccgc attggaaacc tgatcatgta gatcgaatgg actctaaatc
     1921 cgttcagccg ggttagattc ccggggtttc cgccaaattc gaaaagcctg ctcaacgagc
     1981 aggctttttt gcatgctcga gcagctcagg gtcgaatttg ctttcgaatt tctgccattc
     2041 atccgcttat tatcacttat tcaggcgtag caaccaggcg tttaagggca ccaataactg
     2101 ccttaaaaaa attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc
     2161 attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc
     2221 agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg
     2281 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg
     2341 aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc
     2401 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc
     2461 gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat
     2521 atcaccagct caccgtcttt cattgccata cggaattccg gatgagcatt catcaggcgg
     2581 gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa
     2641 aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat
     2701 gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt
     2761 tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc
     2821 ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaacgtct
     2881 cattttcgcc aaaagttggc ccagggcttc ccggtatcaa cagggacacc aggatttatt
     2941 tattctgcga agtgatcttc cgtcacaggt atttattcgg cgcaaagtgc gtcgggtgat
     3001 gctgccaact tactgattta gtgtatgatg gtgtttttga ggtgctccag tggcttctgt
     3061 ttctatcagc tgtccctcct gttcagctac tgacggggtg gtgcgtaacg gcaaaagcac
     3121 cgccggacat cagcgctagc ggagtgtata ctggcttact atgttggcac tgatgagggt
     3181 gtcagtgaag tgcttcatgt ggcaggagaa aaaaggctgc accggtgcgt cagcagaata
     3241 tgtgatacag gatatattcc gcttcctcgc tcactgactc gctacgctcg gtcgttcgac
     3301 tgcggcgagc ggaaatggct tacgaacggg gcggagattt cctggaagat gccaggaaga
     3361 tacttaacag ggaagtgaga gggccgcggc aaagccgttt ttccataggc tccgcccccc
     3421 tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga caggactata
     3481 aagataccag gcgtttcccc ctggcggctc cctcgtgcgc tctcctgttc ctgcctttcg
     3541 gtttaccggt gtcattccgc tgttatggcc gcgtttgtct cattccacgc ctgacactca
     3601 gttccgggta ggcagttcgc tccaagctgg actgtatgca cgaacccccc gttcagtccg
     3661 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggaaaga catgcaaaag
     3721 caccactggc agcagccact ggtaattgat ttagaggagt tagtcttgaa gtcatgcgcc
     3781 ggttaaggct aaactgaaag gacaagtttt ggtgactgcg ctcctccaag ccagttacct
     3841 cggttcaaag agttggtagc tcagagaacc ttcgaaaaac cgccctgcaa ggcggttttt
     3901 tcgttttcag agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga tcatcttatt
     3961 aatcagataa aatatttcta gatttcagtg caatttatct cttcaaatgt agcacctgaa
     4021 gtcagcccca tacgatataa gttgtaattc tcatgtttga cagcttatca tcgataagct
     4081 tggtacccaa ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac
     4141 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat
     4201 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca
     4261 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct
     4321 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga
     4381 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat
     4441 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct
     4501 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga
     4561 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg
     4621 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt
     4681 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc
     4741 ctggcgggaa cagcaaaata tcactcggtc ggcaaacaaa ttctcgtccc tgatttttca
     4801 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt
     4861 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg
     4921 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac
     4981 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg
     5041 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt
     5101 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca
     5161 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat
     5221 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat
     5281 acccgttttt ttgggctaac aggaggaatt agatctatgg acaaaaaacc gctgaatacc
     5341 ctgatctctg ctactggtct gtggatgagt cgtaccggaa ccattcataa aatcaaacac
     5401 cacgaggtta gccgttcgaa aatctatatt gagatggcgt gtggcgatca tctggttgtg
     5461 aacaatagcc gctcttctcg tacagcacgt gcactgcgtc accacaaata tcgtaaaacc
     5521 tgtaaacgtt gccgtgtgtc cgatgaggat ctgaacaaat tcctgacaaa agccaatgag
     5581 gaccaaacaa gcgtgaaagt gaaagtcgtt agcgctccta cccgtactaa aaaagcaatg
     5641 ccgaaatccg ttgctcgtgc ccctaaacca ctggaaaaca ctgaagcagc acaggcacag
     5701 ccgtctggaa gcaaattctc tccggccatt cctgtttcta cccaggagtc cgtttctgtt
     5761 ccagcaagtg tgagcaccag cattagcagt attagcaccg gtgccaccgc tagcgccctg
     5821 gttaaaggca ataccaatcc gattacaagc atgtctgccc cggttcaagc atcagctcca
     5881 gcactgacaa aatcccaaac cgatcgtctg gaggttctgc tgaatccgaa agacgaaatc
     5941 agcctgaatt ccggcaaacc gtttcgtgaa ctggagagcg aactgctgtc acgtcgtaaa
     6001 aaagacctgc aacaaatcta tgccgaagaa cgtgagaact atctggggaa actggaacgt
     6061 gaaatcaccc gctttttcgt ggatcgtggc tttctggaga tcaaatcccc gattctgatt
     6121 cctctggagt atatcgagcg tatgggcatc gacaatgata ccgaactgag caaacaaatt
     6181 ttccgtgtgg ataaaaactt ctgtctgcgc cctatgctgg caccaaatct gtataactat
     6241 ctgcgcaaac tggaccgtgc cctgcctgat cctatcaaaa tcttcgagat cggcccgtgt
     6301 tatcgtaaag agtccgacgg taaagaacat ctggaggagt ttaccatgct gaacttttgc
     6361 caaatgggtt caggttgtac tcgtgagaac ctggaaagca tcatcaccga ttttctgaac
     6421 cacctgggca ttgacttcaa aattgtgggc gacagctgta tggtgtatgg cgacaccctg
     6481 gatgtcatgc acggcgacct ggaactgtct agtgccgttg ttggaccaat tccgctggac
     6541 cgtgagtggg gtatcgacaa accgtggatc ggagcaggat tcggtctgga acgcctgctg
     6601 aaagtgaaac acgacttcaa aaacatcaaa cgtgccgccc gttctgaatc gtattataac
     6661 gggatctcta cgaacctgta agtcgaccat catcatcatc atcattgagt ttaaacggtc
     6721 tccagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag
     6781 aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
     6841 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
     6901 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
     6961 tgggccttgt ttgtgagctc ccggtcatca atcatcccca taatcctt
//
