LOCUS       sequence-502867-        4044 bp    DNA     circular SYN 12-MAR-2026
DEFINITION  Machinery plasmid expressing Methanomethylophilus alvus aaRS/tRNA 
            for TAG suppression and incorporation of trifluoromethyl-tryptophan 
            (tfm-Trp) into sfGFP150TAG in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4044)
  AUTHORS   Augustin G, Bhinderwala F, Alexander ND, Hernandez I, Stanisheuski 
            S, Monnie CM, Eddins AJ, Gangarde YM, Soloshonok VA, Oiarbide M, 
            Landa A, Cooley RB, Gronenborn AM, Mehl RA
  TITLE     Endogenous Site-Specific Encoding of Trifluoromethyl-Bearing 
            Phenylalanine and Tryptophan for in-Cell (19)F NMR.
  JOURNAL   J Am Chem Soc. 2026 Feb 12. doi: 10.1021/jacs.5c18349.
  PUBMED    41684198
REFERENCE   2  (bases 1 to 4044)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 12, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Am Chem 
            Soc. 2026 Feb 12. doi: 10.1021/jacs.5c18349."
COMMENT     Sequence Label: pAJE90-Ma-tfmW-RSA2
FEATURES             Location/Qualifiers
     source          1..4044
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      427..436
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1784..1801)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1784..1800)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1793..1815)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2145..2189
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2227..2245)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2241..2288
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2459..3250)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(3251..3342)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3457..4044
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3945..3964
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt ttctagattt
       61 cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt
      121 aattctcatg tttgacagct tatcatcgat gcgaccgagt gagctagcta tggatcctcg
      181 ggagttgtca gcctgtcccg cttataagat catacgccgt tatacgttgt ttacgctttg
      241 aggaatccca tatgacagtg aaatacacag atgcccagat ccagcgcctg cgggagtatg
      301 gcaacggcac ctatgagcag aaggtgtttg aagatctggc ctctagagat gcagccttct
      361 ccaaggagat gtccgtggct tccacagaca acgagaaaaa gatcaagggc atgattgcca
      421 accccagccg ccatgggctg acccagctga tgaatgacat cgccgacgcc ctggtggccg
      481 agggcttcat cgaggtcagg acccccatct tcatttctaa ggacgcgctg gctcgcatga
      541 ccatcaccga ggacaagccc ctgttcaagc aggtgttctg gatcgatgag aagagggctc
      601 tgaggcccat gctcgccccc aacgtttact ccgtgatgcg ggacctgcgc gaccacacgg
      661 acggccctgt gaaaattttc gaaatgggct cctgctttag gaaagaaagc cacagcggaa
      721 tgcacctgga ggagttcacc atgctggggc tgattgacat ggggccaaga ggagatgcca
      781 cagaagtgct gaagaactac atctcagtgg tcatgaaggc tgctggactg cccgactatg
      841 atttggtgca ggaagagagc gatgtctaca aagaaaccat tgatgtggag atcaatggcc
      901 aggaggtgtg ctctgctagt gtgggccccc actacctgga cgccgcccac gacgtgcatg
      961 aaccctggag tggagcgggc tttggcctgg agaggctgct gaccataaga gaaaagtaca
     1021 gcactgtgaa gaaaggcggc gcctccatct cctacttgaa tggagccaag atcaacagcg
     1081 gctgaagcct gcagtttcaa acgctaaatt gcctgatgcg ctacgcttat caggcctaca
     1141 tgatctctgc aatatattga gtttgcgtgc ttttgtaggc cggataaggc gttcacgccg
     1201 catccggcaa gaaacagcaa acaatccaaa acgccgcgtt cggcggtcga cacagatgta
     1261 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     1321 ggcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgct
     1381 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     1441 gtcatgcccc gcgcccaccg gaaggagcta ccggcagcgg tgcggactgt tgtaactcag
     1501 aataagaaat gaggccgctc atggcgttct gttgcccgtc tcactggtga aaagaaaaac
     1561 aaccctggcg ccgcttcttt gagcgaacga tcaaaaataa gtggcgcccc atcaaaaaaa
     1621 tattctcaac ataaaaaact ttgtgtaata cttgtaacgc tagatctggg ggacggtccg
     1681 gcgaccagcg ggtctctaaa acctagcata gcggggttcg acaccccggt ctctcgccaa
     1741 ctagtatcct tagcgaaagc taaggatttt ttttaagctt ggcactggcc gtcgttttac
     1801 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     1861 ctttcgccag acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     1921 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagccc gagatgcgcc
     1981 gcgtgcggct gctggagatg gcggacgcga tggatatgtt ctggcggccg cataatgctt
     2041 aagtcgaaca gaaagtaatc gtattgtaca cggccgcata atcgaaatta aattggatat
     2101 cggcaggcca cgcgatcgct gacgtcggta ccctcgagtc tggtaaagaa accgctgctg
     2161 cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg
     2221 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     2281 gttttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt ccggtagtca
     2341 ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga
     2401 cgaccgggtc atcgtggccg gatcttgcgg cccctcggct tgaacgaatt gttagacatt
     2461 atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct tccaactgat
     2521 ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta gcttcaagta
     2581 tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg acatccttcg
     2641 gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc actacatttc
     2701 gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca tttagcgcct
     2761 caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga cctaccaagg
     2821 caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg atcgtggctg
     2881 gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc agttcgcgct
     2941 tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact tctacagcgc
     3001 ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg atcaaagctc
     3061 gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata tcactgtgtg
     3121 gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac gtcggttcga
     3181 gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg gcgatcaccg
     3241 cttccctcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     3301 tgagcggata catatttgaa tgtatttaga aaaataaaca aatagctagc gggtggtgcg
     3361 taacggcaaa agcaccgccg gacatcagcg ctagcggagt gtatactttt cgttccactg
     3421 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3481 aatctgctgc ttgcaaacaa aaaaaccacc gctttgacgg tggtttgttt gccggatcaa
     3541 gagcggctaa ctctttttcc gaaggtaact ggctgacgca gagcgcagat accaaatact
     3601 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3661 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3721 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3781 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3841 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3901 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3961 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4021 tcaggggggc ggagcctatg gaaa
//
