LOCUS       sequence-364441-        6128 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 6128)
  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 6128)
  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-pAzFRS.2.t1
FEATURES             Location/Qualifiers
     source          1..6128
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(556..840)
                     /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     complement(614..633)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             867..1745
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPSDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     rep_origin      1891..2436
                     /direction=RIGHT
                     /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     2337..2356
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2553..2570
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2962..3064
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3065..3724
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3131..3150)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     5627..5644
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(5700..5717)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 ctgctccatc cctccaacag caacatcaac gcccctataa tgacaaccat taacctgcat
       61 tattggatag ataacttcag caacctttgg attttcatcc tctcttgcta taagttccat
      121 actccttctt gctcttttta aggtagtttt taaagccaat ctatagacat tcagtgtata
      181 atccttatca agcatataag tacttccata aacatatttt gcctttaacc ccattgcttc
      241 aaaaactttt ttgttataat ctcctatttt tctaatctca tccaactctc ctttctggtt
      301 taaataggcg tgtaaatcag ccaacaatat aattatatca aatccagcat tttgtaaatc
      361 aatcatcttt tttatttgga gataatgccc taaatgtatt ttaccacttg gttcaaaacc
      421 tatcagagca gatttttcat ctttttttaa aacctctctt aactcttcct cgctgataat
      481 ttcagatgtg tttctcttta tcatttcaaa ttcgtccata gatctaattc ctcctgttag
      541 cccaaaaaaa cgggtatgga gaaacagtag agagttgcga taaaaagcgt caggtaggat
      601 ccgctaatct tatggataaa aatgctatgg catagcaaag tgtgacgccg tgcaaataat
      661 caatgtggac ttttctgccg tgattataga cacttttgtt acgcgttttt gtcatggctt
      721 tggtcccgct ttgttacaga atgcttttaa taagcggggt taccggtttg gttagcgaga
      781 agagccagta aaagacgcag tgacggcaat gtctgatgca atatggacaa ttggtttctt
      841 ctctgaatgg cgggagtatg aaaagtatgg ctgaagcgca aaatgatccc ctgctgccgg
      901 gatactcgtt taatgcccat ctggtggcgg gtttaacgcc gattgaggcc aacggttatc
      961 tcgatttttt tatcgaccga ccgctgggaa tgaaaggtta tattctcaat ctcaccattc
     1021 gcggtcaggg ggtggtgaaa aatcagggac gagaatttgt ttgccgaccg agtgatattt
     1081 tgctgttccc gccaggagag attcatcact acggtcgtca tccggaggct cgcgaatggt
     1141 atcaccagtg ggtttacttt cgtccgcgcg cctactggca tgaatggctt aactggccgt
     1201 caatatttgc caatacgggg ttctttcgcc cggatgaagc gcaccagccg catttcagcg
     1261 acctgtttgg gcaaatcatt aacgccgggc aaggggaagg gcgctattcg gagctgctgg
     1321 cgataaatct gcttgagcaa ttgttactgc ggcgcatgga agcgattaac gagtcgctcc
     1381 atccaccgat ggataatcgg gtacgcgagg cttgtcagta catcagcgat cacctggcag
     1441 acagcaattt tgatatcgcc agcgtcgcac agcatgtttg cttgtcgccg tcgcgtctgt
     1501 cacatctttt ccgccagcag ttagggatta gcgtcttaag ctggcgcgag gaccaacgta
     1561 tcagccaggc gaagctgctt ttgagcacca cccggatgcc tatcgccacc gtcggtcgca
     1621 atgttggttt tgacgatcaa ctctatttct cgcgggtatt taaaaaatgc accggggcca
     1681 gcccgagcga gttccgtgcc ggttgtgaag aaaaagtgaa tgatgtagcc gtcaagttgt
     1741 cataattggg taccaagctt atcgatgata agctgtcaaa catgagaatt acaacttata
     1801 tcgtatgggg ctgacttcag gtgctacatt tgaagagata aattgcactg aaatctagaa
     1861 atattttatc tgattaataa gatgatcttc ttgagatcgt tttggtctgc gcgtaatctc
     1921 ttgctctgaa aacgaaaaaa ccgccttgca gggcggtttt tcgaaggttc tctgagctac
     1981 caactctttg aaccgaggta actggcttgg aggagcgcag tcaccaaaac ttgtcctttc
     2041 agtttagcct taaccggcgc atgacttcaa gactaactcc tctaaatcaa ttaccagtgg
     2101 ctgctgccag tggtgctttt gcatgtcttt ccgggttgga ctcaagacga tagttaccgg
     2161 ataaggcgca gcggtcggac tgaacggggg gttcgtgcat acagtccagc ttggagcgaa
     2221 ctgcctaccc ggaactgagt gtcaggcgtg gaatgagaca aacgcggcca taacagcgga
     2281 atgacaccgg taaaccgaaa ggcaggaaca ggagagcgca cgagggagcc gccaggggga
     2341 aacgcctggt atctttatag tcctgtcggg tttcgccacc actgatttga gcgtcagatt
     2401 tcgtgatgct tgtcaggggg gcggagccta tggaaaaacg gctttgccgc ggccctctca
     2461 cttccctgtt aagtatcttc ctggcatctt ccaggaaatc tccgccccgt tcgtaagcca
     2521 tttccgctcg ccgcagtcga acgaccgagc gtagcgagtc agtgagcgag gaagcggaat
     2581 atatcctgta tcacatattc tgctgacgca ccggtgcagc cttttttctc ctgccacatg
     2641 aagcacttca ctgacaccct catcagtgcc aacatagtaa gccagtatac actccgctag
     2701 cgctgatgtc cggcggtgct tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag
     2761 ggacagctga tagaaacaga agccactgga gcacctcaaa aacaccatca tacactaaat
     2821 cagtaagttg gcagcatcac ccgacgcact ttgcgccgaa taaatacctg tgacggaaga
     2881 tcacttcgca gaataaataa atcctggtgt ccctgttgat accgggaagc cctgggccaa
     2941 cttttggcga aaatgagacg ttgatcggca cgtaagaggt tccaactttc accataatga
     3001 aataagatca ctaccgggcg tattttttga gttatcgaga ttttcaggag ctaaggaagc
     3061 taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa
     3121 agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct
     3181 ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt
     3241 tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga
     3301 cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac
     3361 tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat
     3421 atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat
     3481 tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa
     3541 cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca
     3601 aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt
     3661 ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc
     3721 gtaatttttt taaggcagtt attggtgccc ttaaacgcct ggttgctacg cctgaataag
     3781 tgataataag cggatgaatg gcagaaattc gaaagcaaat tcgaccctga gctgctcgag
     3841 catgcaaaaa agcctgctcg ttgagcaggc ttttcgaatt tggtccggcg gaggggattt
     3901 gaacccctgc catgcggatt tagagtccgc cgttctgccc tgctgaacta ccgccggaat
     3961 gcggggcgca tcttactgcg cagatacgcc ctcgtcaatc ccttaatagc aaaatgcctt
     4021 ttgatcggcg agaaagtcag caggccgctt agttagccgt gcactgatca tatcgtcaat
     4081 tattacctcc acggggagag cctgagcaaa ctggcctcag gcatttgaga agcacacggt
     4141 cacactgctt ccggtagtca ataaaccggt gctcacatgt tgcgaagcgg aattaattcg
     4201 cgaagaaaag cagaaaaaac gccgctgaac gcggcgtttt ggattgtttg ctgtttcttg
     4261 ccggatgcgg cgtgaacgcc ttatccggcc tacaaaagca cgcaaactca atatattgca
     4321 gagatcatgt aggcctgata agcgtagcgc atcaggcaat ttagcgtttg aaactgcagt
     4381 tataatctct ttctaattgg ctctaaaatc tttataagtt cttcagctac agcatttttt
     4441 aagcgcattg gatgcaattc cttattttta aataaactct ctaactcctc atagctatta
     4501 actgtcaaat ctccaccaaa tttttctggc ccttttatgg ttaaaggata ttcaaggaag
     4561 tatttagcta tctccattat tggatttcct tcaacaactc cagctgggca gtatgctttc
     4621 tttatcttag ccctaatctc ttctggagag tcatcaacag ctataaaatt cccttttgaa
     4681 gaactcatct ttccttctcc atccaaaccc gttaagacag ggttgtgaat acaaacaacc
     4741 ttttttggta aaagctccct tgctaacatg tgtatttttc tctgctccat ccctccaaca
     4801 gcaacatcaa cgcccctata atgacaacca ttaacctgca ttattggata gataacttca
     4861 gcaacctttg gattttcatc ctctcttgct ataagttcca tactccttct tgcccttttt
     4921 aaggtagttt ttaaagccaa tctatagaca ttcagtgtat aatccttatc aagcatataa
     4981 gtacttccat aaacatattt tgcctttaac cccattgctt caaaaacttt tttgttataa
     5041 tctcctattt ttctaatctc atccaactct cctttctggt ttaaataggc gtgtaaatca
     5101 gccaacaata taattatatc aaatccagca ttttgtaaat caatcatctt ttttatttgg
     5161 agataatgcc ctaaatgtat tttaccactt ggttcaaaac ctatcagagc agatttttca
     5221 tcctttttta aaacctctct taactcttcc tcgctgataa tttcagatgt gtttctcttt
     5281 atcatttcaa attcgtccat atgggattcc tcaaagcgta aacaacgtat aacggcgtat
     5341 gatattaaag cgggacaggc tgacaactgt tagtttttta aaattgataa tctaacaagg
     5401 attatgggga tgattgatga ccgggagctc acaaacaagg cccagtcttt cgactgagcc
     5461 tttcgtttta tttgatgcct ggcagttccc tactctcgca tggggagacc ccacactacc
     5521 atcggcgcta cggcgtttca cttctgagtt cggcatgggg tcaggtggga ccaccgcgct
     5581 actgccgcca ggcaaattct gttttatcag accgcttctg cgttctgatt taatctgtat
     5641 caggctgaaa atcttctctc atccgccaaa acagccaagc tggagaccgt ttaaactcaa
     5701 tgatgatgat gatgatggtc gacttattat aatctctttc taattggctc taaaatcttt
     5761 ataagttctt cagctacagc attttttaag cgcattggat gcaattcctt atttttaaat
     5821 aaactctcta actcctcata gctattaact gtcaaatctc caccaaattt ttctggccct
     5881 tttatggtta aaggatattc aaggaagtat ttagctatct ccattattgg atttccttca
     5941 acaactccag ctgggcagta tgctttcttt atcttagccc taatctcttc tggagagtca
     6001 tcaacagcta taaaattccc ttttgaagaa ctcatctttc cttctccatc caaacccgtt
     6061 aagacagggt tgtgaataca aacaaccttt tttggtaaaa gctcccttgc taacatgtgt
     6121 atttttct
//
