LOCUS       sequence-458349-        6057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mutant E. coli TyrRS from a derepressed lac promoter for 
            proteome-wide incorporation of tyrosine in E. coli/K. pneumoniae..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6057)
  AUTHORS   Loynd C, Singha Roy SJ, Canarelli SE, Bak DW, Sundaresh B, Babbitz 
            Z, van Opijnen T, Weerapana E, Chatterjee A
  TITLE     Cell-selective multiplexed bioorthogonal noncanonical amino acid 
            tagging for nascent proteomics.
  JOURNAL   Nat Chem Biol. 2025 Oct 2. doi: 10.1038/s41589-025-02039-3.
  PUBMED    41039161
REFERENCE   2  (bases 1 to 6057)
  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"; journalName: "Nat Chem 
            Biol. 2025 Oct 2. doi: 10.1038/s41589-025-02039-3."
COMMENT     Sequence Label: pBBR1-EcTyrRS(VSMA,D165G)
FEATURES             Location/Qualifiers
     source          1..6057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(928..950)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    939..955
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(963..969,970..987,988..993))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1008..1029
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1434..2228)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1545..1564)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2155..2174
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      3708..4479
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             4480..5142
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
ORIGIN
        1 cctgctccgc cagtacatac tgggcgcgcg gtgctttacc gctgttttta tcttcttctt
       61 ccagggcgtt gatctcttca atgctcataa aggtgaagaa cttcaggaag cggtaaacgt
      121 cggcacgcgc agtgttgatc cagaactggt agaatttgta cgggctggtt ttcttcggat
      181 ctaaccagac tgcgccgcct tcagttttac caaatttggt gccatctgct ttagtgatca
      241 gcggaacggt caggccaaac acctgattct gatgcagacg acgggtcagg tcgataccag
      301 aagtgatgtt accccactgg tcagaaccac caatttgcag caccacaccg tactgtttgt
      361 tcgcacaggc catactataa ccctgcaaca ggttgtagga aaactcagtg aacgaaatcc
      421 cctgaccttc acggttgaga cgctgcttaa ccgcttcttt gttgatcatc tggttaacgg
      481 agaagtgttt gccaatatcg cgcaggaagg tcagcacatt catattgccg aaccagtcat
      541 agttgttcgc cgcgatagca gagttttctc cacagtcgaa atcgaggaac ggggcaacct
      601 gcttacggat tttgtccacc cactcctgaa cagtttcttc ggtgttcagc ttacgctcgg
      661 cagctttgaa gctcgggtcg ccaatcagac ccgtcgcgcc gcctaccagc gcaaccggct
      721 tgtggcccgc ctgctggaag cgtttcaggc ataacaatgg aacaagatgc cccaaatgca
      781 agctgtcagc ggtaggatcg aagccgcaca cgagcgcgat cgggccttgc gccagtcgct
      841 ctgctaacgc ttcctcgtcc gtcacctggg ctaccagccc ccgctcttgc aattgtttaa
      901 tcaagttact gcttgccata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
      961 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1021 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1081 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgcatgcat
     1141 aaaaactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga
     1201 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg
     1261 ggggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc
     1321 ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg
     1381 tgatggcagg ttgggcgtcg cttggtcggt catttcgaac cccagagtcc cgctcagaag
     1441 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa
     1501 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc
     1561 aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa
     1621 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga
     1681 tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc
     1741 tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct
     1801 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc
     1861 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac
     1921 aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca
     1981 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc
     2041 tcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc
     2101 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag
     2161 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt
     2221 tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag atcttgatcc cctgcgccat
     2281 cagatccttg gcggcaagaa agccatccag tttactttgc agggcttccc aaccttacca
     2341 gagggcgccc cagctggcaa ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc
     2401 tatcgccatg taagcccact gcaagctacc tgctttctct ttgcgcttgc gttttccctt
     2461 gtccagatag cccagtagct gacattcatc ccaggtggca cttttcgggg aaatgtgcgc
     2521 gcccgcgttc ctgctggcgc tgggcctgtt tctggcgctg gacttcccgc tgttccgtca
     2581 gcagcttttc gcccacggcc ttgatgatcg cggcggcctt ggcctgcata tcccgattca
     2641 acggccccag ggcgtccaga acgggcttca ggcgctcccg aaggtctcgg gccgtctctt
     2701 gggcttgatc ggccttcttg cgcatctcac gcgctcctgc ggcggcctgt agggcaggct
     2761 catacccctg ccgaaccgct tttgtcagcc ggtcggccac ggcttccggc gtctcaacgc
     2821 gctttgagat tcccagcttt tcggccaatc cctgcggtgc ataggcgcgt ggctcgaccg
     2881 cttgcgggct gatggtgacg tggcccactg gtggccgctc cagggcctcg tagaacgcct
     2941 gaatgcgcgt gtgacgtgcc ttgctgccct cgatgccccg ttgcagccct agatcggcca
     3001 cagcggccgc aaacgtggtc tggtcgcggg tcatctgcgc tttgttgccg atgaactcct
     3061 tggccgacag cctgccgtcc tgcgtcagcg gcaccacgaa cgcggtcatg tgcgggctgg
     3121 tttcgtcacg gtggatgctg gccgtcacga tgcgatccgc cccgtacttg tccgccagcc
     3181 acttgtgcgc cttctcgaag aacgccgcct gctgttcttg gctggccgac ttccaccatt
     3241 ccgggctggc cgtcatgacg tactcgaccg ccaacacagc gtccttgcgc cgcttctctg
     3301 gcagcaactc gcgcagtcgg cccatcgctt catcggtgct gctggccgcc cagtgctcgt
     3361 tctctggcgt cctgctggcg tcagcgttgg gcgtctcgcg ctcgcggtag gcgtgcttga
     3421 gactggccgc cacgttgccc attttcgcca gcttcttgca tcgcatgatc gcgtatgccg
     3481 ccatgcctgc ccctcccttt tggtgtccaa ccggctcgac gggggcagcg caaggcggtg
     3541 cctccggcgg gccactcaat gcttgagtat actcactaga ctttgcttcg caaagtcgtg
     3601 accgcctacg gcggctgcgg cgccctacgg gcttgctctc cgggcttcgc cctgcgcggt
     3661 cgctgcgctc ccttgccagc ccgtggatat gtggacgatg gccgcgagcg gccaccggct
     3721 ggctcgcttc gctcggcccg tggacaaccc tgctggacaa gctgatggac aggctgcgcc
     3781 tgcccacgag cttgaccaca gggattgccc accggctacc cagccttcga ccacataccc
     3841 accggctcca actgcgcggc ctgcggcctt gccccatcaa tttttttaat tttctctggg
     3901 gaaaagcctc cggcctgcgg cctgcgcgct tcgcttgccg gttggacacc aagtggaagg
     3961 cgggtcaagg ctcgcgcagc gaccgcgcag cggcttggcc ttgacgcgcc tggaacgacc
     4021 caagcctatg cgagtggggg cagtcgaagg gcgaagcccg cccgcctgcc ccccgagcct
     4081 cacggcggcg agtgcggggg ttccaagggg gcagcgccac cttgggcaag gccgaaggcc
     4141 gcgcagtcga tcaacaagcc ccggaggggc cactttttgc cggaggggga gccgcgccga
     4201 aggcgtgggg gaaccccgca ggggtgccct tctttgggca ccaaagaact agatataggg
     4261 cgaaatgcga aagacttaaa aatcaacaac ttaaaaaagg ggggtacgca acagctcatt
     4321 gcggcacccc ccgcaatagc tcattgcgta ggttaaagaa aatctgtaat tgactgccac
     4381 ttttacgcaa cgcataattg ttgtcgcgct gccgaaaagt tgcagctgat tgcgcatggt
     4441 gccgcaaccg tgcggcaccc ctaccgcatg gagataagca tggccacgca gtccagagaa
     4501 atcggcattc aagccaagaa caagcccggt cactgggtgc aaacggaacg caaagcgcat
     4561 gaggcgtggg ccgggcttat tgcgaggaaa cccacggcgg caatgctgct gcatcacctc
     4621 gtggcgcaga tgggccacca gaacgccgtg gtggtcagcc agaagacact ttccaagctc
     4681 atcggacgtt ctttgcggac ggtccaatac gcagtcaagg acttggtggc cgagcgctgg
     4741 atctccgtcg tgaagctcaa cggccccggc accgtgtcgg cctacgtggt caatgaccgc
     4801 gtggcgtggg gccagccccg cgaccagttg cgcctgtcgg tgttcagtgc cgccgtggtg
     4861 gttgatcacg acgaccagga cgaatcgctg ttggggcatg gcgacctgcg ccgcatcccg
     4921 accctgtatc cgggcgagca gcaactaccg accggccccg gcgaggagcc gcccagccag
     4981 cccggcattc cgggcatgga accagacctg ccagccttga ccgaaacgga ggaatgggaa
     5041 cggcgcgggc agcagcgcct gccgatgccc gatgagccgt gttttctgga cgatggcgag
     5101 ccgttggagc cgccgacacg ggtcacgctg ccgcgccggt agcacttggg ttgcgcagca
     5161 acccgtaagt gcgctgttcc agactatcgg ctgtagccgc ctcgccgccc tataccttgt
     5221 ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct gaatggaagc
     5281 cggcggcacc tcgctaacgg attcaccgtt tttatcaggc tctgggaggc agaataaatg
     5341 atcatatcgt caattattac ctccacgggg agagcctgag caaactggcc tcaggcattt
     5401 gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca
     5461 taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat
     5521 ttctgccatt catccgctta ttatcactta ttcaggcgta gcaaccaggc gtttaagggc
     5581 accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tacggcctat
     5641 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5701 cttatttcca gcaaatcaga cagtaattct ttttaccgcg acgcagtaag gtaaaacgac
     5761 caaacagacg atcttcttct ttaaagaagt attcaggatc ggactgtttt tcaccgttaa
     5821 tggtgatggc attggaggcg atagttttac gtgcctgacc acgggaaggt tgcagttcag
     5881 aatcgaccag tgcctgcatc aggtctgcgc ccttttccat ctcaaccatc ggtacgccgt
     5941 cctgcgccag ctgttcgaag tccgcttcac tcagcgcact caaagaaccg ctgaacaggc
     6001 attcggtaat acgttttgcc gcctgtaaac cttcttcacc gtgaaccaga cgagtca
//
