LOCUS       sequence-136017-        6124 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA synthetase/tRNA pair for the in vivo incorporation of several 
            non-standard amino acids, into proteins in E coli in response to the
            amber codon, TAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6124)
  AUTHORS   Amiram M, Haimovich AD, Fan C, Wang YS, Aerni HR, Ntai I, Moonan DW,
            Ma NJ, Rovner AJ, Hong SH, Kelleher NL, Goodman AL, Jewett MC, Soll 
            D, Rinehart J, Isaacs FJ
  TITLE     Evolution of translation machinery in recoded bacteria enables 
            multi-site incorporation of nonstandard amino acids.
  JOURNAL   Nat Biotechnol. 2015 Dec;33(12):1272-1279. doi: 10.1038/nbt.3372. 
            Epub 2015 Nov 16.
  PUBMED    26571098
REFERENCE   2  (bases 1 to 6124)
  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: "10.1038/nbt.3372"; 
            journalName: "Nat Biotechnol"; date: "2015-12"; volume: "33"; issue:
            "12"; pages: "1272-1279"
COMMENT     Sequence Label: pEvol-pAcFRS.1.t1
FEATURES             Location/Qualifiers
     source          1..6124
                     /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             3458..3475
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3531..3548)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(5450..6109)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6024..6043
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(6110..6124,1..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 atggacgaat ttgaaatgat aaagagaaac acatctgaaa ttatcagcga
     2581 ggaagagtta agagaggttt taaaaaaaga tgaaaaatct gctctgatag gttttgaacc
     2641 aagtggtaaa atacatttag ggcattatct ccaaataaaa aagatgattg atttacaaaa
     2701 tgctggattt gatataatta tattgttggc ggatttacac gcctatttaa accagaaagg
     2761 agagttggat gagattagaa aaataggaga ttataacaaa aaagtttttg aagcaatggg
     2821 gttaaaggca aaatatgttt atggaagtga attccagctt gataaggatt atacactgaa
     2881 tgtctataga ttggctttaa aaactacctt aaaaagagca agaaggagta tggaacttat
     2941 agcaagagag gatgaaaatc caaaggttgc tgaagttatc tatccaataa tgcaggttaa
     3001 tggttgtcat tataggggcg ttgatgttga tgttggaggg atggagcaga gaaaaataca
     3061 catgttagca agggagcttt taccaaaaaa ggttgtttgt attcacaacc ctgtcttaac
     3121 gggtttggat ggagaaggaa agatgagttc ttcaaaaggg aattttatag ctgttgatga
     3181 ctctccagaa gagattaggg ctaagataaa gaaagcatac tgcccagctg gagttgttga
     3241 aggaaatcca ataatggaga tagctaaata cttccttgaa tatcctttaa ccataaaagg
     3301 gccagaaaaa tttggtggag atttgacagt taatagctat gaggagttag agagtttatt
     3361 taaaaataag gaattgcatc caatgcgctt aaaaaatgct gtagctgaag aacttataaa
     3421 gattttagag ccaattagaa agagattata agtcgaccat catcatcatc atcattgagt
     3481 ttaaacggtc tccagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag
     3541 attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg
     3601 gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt
     3661 gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca
     3721 gtcgaaagac tgggccttgt ttgtgagctc ccggtcatca atcatcccca taatccttgt
     3781 tagattatca attttaaaaa actaacagtt gtcagcctgt cccgctttaa tatcatacgc
     3841 cgttatacgt tgtttacgct ttgaggaatc ccatatggac gaatttgaaa tgataaagag
     3901 aaacacatct gaaattatca gcgaggaaga gttaagagag gttttaaaaa aagatgaaaa
     3961 atctgctctg ataggttttg aaccaagtgg taaaatacat ttagggcatt atctccaaat
     4021 aaaaaagatg attgatttac aaaatgctgg atttgatata attatattgt tggcggattt
     4081 acacgcctat ttaaaccaga aaggagagtt ggatgagatt agaaaaatag gagattataa
     4141 caaaaaagtt tttgaagcaa tggggttaaa ggcaaaatat gtttatggaa gtgaattcca
     4201 gcttgataag gattatacac tgaatgtcta tagattggct ttaaaaacta ccttaaaaag
     4261 agcaagaagg agtatggaac ttatagcaag agaggatgaa aatccaaagg ttgctgaagt
     4321 tatctatcca ataatgcagg ttaatggttg tcattatagg ggcgttgatg ttgatgttgg
     4381 agggatggag cagagaaaaa tacacatgtt agcaagggag cttttaccaa aaaaggttgt
     4441 ttgtattcac aaccctgtct taacgggttt ggatggagaa ggaaagatga gttcttcaaa
     4501 agggaatttt atagctgttg atgactctcc agaagagatt agggctaaga taaagaaagc
     4561 atactgccca gctggagttg ttgaaggaaa tccaataatg gagatagcta aatacttcct
     4621 tgaatatcct ttaaccataa aagggccaga aaaatttggt ggagatttga cagttaatag
     4681 ctatgaggag ttagagagtt tatttaaaaa taaggaattg catccaatgc gcttaaaaaa
     4741 tgctgtagct gaagaactta taaagatttt agagccaatt agaaagagat tataactgca
     4801 gtttcaaacg ctaaattgcc tgatgcgcta cgcttatcag gcctacatga tctctgcaat
     4861 atattgagtt tgcgtgcttt tgtaggccgg ataaggcgtt cacgccgcat ccggcaagaa
     4921 acagcaaaca atccaaaacg ccgcgttcag cggcgttttt tctgcttttc ttcgcgaatt
     4981 aattccgctt cgcaacatgt gagcaccggt ttattgacta ccggaagcag tgtgaccgtg
     5041 tgcttctcaa atgcctgagg ccagtttgct caggctctcc ccgtggaggt aataattgac
     5101 gatatgatca gtgcacggct aactaagcgg cctgctgact ttctcgccga tcaaaaggca
     5161 ttttgctatt aagggattga cgagggcgta tctgcgcagt aagatgcgcc ccgcattccg
     5221 gcggtagttc agcagggcag aacggcggac tctaaatccg catggcaggg gttcaaatcc
     5281 cctccgccgg accaaattcg aaaagcctgc tcaacgagca ggcttttttg catgctcgag
     5341 cagctcaggg tcgaatttgc tttcgaattt ctgccattca tccgcttatt atcacttatt
     5401 caggcgtagc accaggcgtt taagggcacc aataactgcc ttaaaaaaat tacgccccgc
     5461 cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat
     5521 cacagacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat
     5581 aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat
     5641 caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc
     5701 ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta
     5761 gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct
     5821 catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca
     5881 ttgccatacg gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg
     5941 gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa
     6001 cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat
     6061 gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct
     6121 tagc
//
