LOCUS       sequence-192936-        5624 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Complementary plasmid for aaRS PACE; amber codons in T7 RNAP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5624)
  AUTHORS   Bryson DI, Fan C, Guo LT, Miller C, Soll D, Liu DR
  TITLE     Continuous directed evolution of aminoacyl-tRNA synthetases.
  JOURNAL   Nat Chem Biol. 2017 Oct 16. doi: 10.1038/nchembio.2474.
  PUBMED    29035361
REFERENCE   2  (bases 1 to 5624)
  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. 2017 Oct 16. doi: 10.1038/nchembio.2474."
COMMENT     Sequence Label: pDB023k
FEATURES             Location/Qualifiers
     source          1..5624
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      24..612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     513..532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     766..783
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    798..938
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(864..883)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1024..1046
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1040..1231)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1374..1391)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1544..1630
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1722..1749
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4724..5515)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPMLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(5516..5587)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgctttccag gccaaatagg ccgttgagat cctttttttc tgcgcgtaat ctgctgcttg
       61 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccacct
      121 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
      181 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      241 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      301 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      361 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      421 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      481 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      541 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      601 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      661 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
      721 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
      781 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
      841 caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
      901 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
      961 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     1021 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     1081 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     1141 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     1201 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     1261 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     1321 atgaacaaga ggacatccgg tggctgtttt ggcggatgag agaagatttt cagcctgata
     1381 cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc
     1441 gcggtggtcc cacctgaccc catgccgaac tcagaagtga aactccgtag cgccgatggt
     1501 agtgtggggt ctccccatgc gagagtaggg aactgccagt catcaaataa aacgaaaggc
     1561 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     1621 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
     1681 ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga
     1741 tggccttttt gcgtttctac aaactcgctg aggattctag agcacagctt gatgaaattc
     1801 gccacttgtt agtgtaattc gctaactcat cctggcatgt tgctgttgat tcttcaatca
     1861 gatctttata aatcaaaaag ataaaaaatt ggcacgcaaa ttgtattaac agttcagcag
     1921 gacaatcctg aacgcagaaa tcaagaggac aacattaaga aaagtgaaat gaatagttaa
     1981 ggaggaaaaa aaaatgaaca cgattaacat cgctaagaac gacttctagg acatcgaact
     2041 ggctgctatc ccgttcaaca ctctggctga ccattacggt gagcgtttag ctcgcgaaca
     2101 gttggccctt gagcatgagt cttacgagat gggtgaagca cgcttccgca agatgtttga
     2161 gcgtcaactt aaagctggtg aggttgcgga taacgctgcc gccaagcctc tcatcactac
     2221 cctactccct aagatgattg cacgcatcaa cgactggttt gaggaagtga aagctaagcg
     2281 cggcaagcgc ccgacagcct tccagttcct gcaagaaatc aagccggaag ccgtagcgta
     2341 catcaccatt aagaccactc tggcttgcct aaccagtgct gacaatacaa ccgttcaggc
     2401 tgtagcaagc gcaatcggtc gggccattga ggacgaggct cgcttcggtc gtatccgtga
     2461 ccttgaagct aagcacttca agaaaaacgt tgaggaacaa ctcaacaagc gcgtagggca
     2521 cgtctacaag aaagcattta tgcaagttgt cgaggctgac atgctctcta agggtctact
     2581 cggtggcgag gcgtggtctt agtggcataa ggaagactct attcatgtag gagtacgctg
     2641 catcgagatg ctcattgagt caaccggaat ggttagctta caccgccaaa atgctggcgt
     2701 agtaggtcaa gactctgaga ctatcgaact cgcacctgaa tacgctgagg ctatcgcaac
     2761 ccgtgcaggt gcgctggctg gcatctctcc gatgttccaa ccttgcgtag ttcctcctaa
     2821 gccgtggact ggcattactg gtggtggcta ttgggctaac ggtcgtcgtc ctctggcgct
     2881 ggtgcgtact cacagtaaga aagcactgat gcgctacgaa gacgtttaca tgcctgaggt
     2941 gtacaaagcg attaacattg cgcaaaacac cgcatggaaa atcaacaaga aagtcctagc
     3001 ggtcgccaac gtaatcacca agtggaagca ttgtccggtc gaggacatcc ctgcgattga
     3061 gcgtgaagaa ctcccgatga aaccggaaga catcgacatg aatcctgagg ctctcaccgc
     3121 gtggaaacgt gctgccgctg ctgtgtaccg caaggacaag gctcgcaagt ctcgccgtat
     3181 cagccttgag ttcatgcttg agcaagccaa taagtttgct aaccataagg ccatctggtt
     3241 cccttacaac atggactggc gcggtcgtgt ttacgctgtg tcaatgttca acccgcaagg
     3301 taacgatatg accaaaggac tgcttacgct ggcgaaaggt aaaccaatcg gtaaggaagg
     3361 ttactactgg ctgaaaatcc acggtgcaaa ctgtgcgggt gtcgataagg ttccgttccc
     3421 tgagcgcatc aagttcattg aggaaaacca cgagaacatc atggcttgcg ctaagtctcc
     3481 actggagaac acttggtggg ctgagcaaga ttctccgttc tgcttccttg cgttctgctt
     3541 tgagtacgct ggggtacagc accacggcct gtagtataac tgctcccttc cgctggcgtt
     3601 tgacgggtct tgctctggca tccagcactt ctccgcgatg ctccgagatg aggtaggtgg
     3661 tcgcgcggtt aacttgcttc ctagtgaaac cgttcaggac atctacggga ttgttgctaa
     3721 gaaagtcaac gagattctac aagcagacgc aatcaatggg accgataacg aagtagttac
     3781 cgtgaccgat gagaacactg gtgaaatctc tgagaaagtc aagctgggca ctaaggcact
     3841 ggctggtcaa tggctggctt acggtgttac tcgcagtgtg actaagcgtt cagtcatgac
     3901 gctggcttac gggtccaaag agttcggctt ccgtcaacaa gtgctggaag ataccattca
     3961 gccagctatt gattccggca agggtctgat gttcactcag ccgaatcagg ctgctggata
     4021 catggctaag ctgatttggg aatctgtgag cgtgacggtg gtagctgcgg ttgaagcaat
     4081 gaactggctt aagtctgctg ctaagctgct ggctgctgag gtcaaagata agaagactgg
     4141 agagattctt cgcaagcgtt gcgctgtgca ttgggtaact cctgatggtt tccctgtgtg
     4201 gcaggaatac aagaagccta ttcagacgcg cttgaacctg atgttcctcg gtcagttccg
     4261 cttacagcct accattaaca ccaacaaaga tagcgagatt gatgcacaca aacaggagtc
     4321 tggtatcgct cctaactttg tacacagcca agacggtagc caccttcgta agactgtagt
     4381 gtgggcacac gagaagtacg gaatcgaatc ttttgcactg attcacgact ccttcggtac
     4441 cattccggct gacgctgcga acctgttcaa agcagtgcgc gaaactatgg ttgacacata
     4501 tgagtcttgt gatgtactgg ctgatttcta cgaccagttc gctgaccagt tgcacgagtc
     4561 tcaattggac aaaatgccag cacttccggc taaaggtaac ttgaacctcc gtgacatctt
     4621 agagtcggac ttcgcgttcg cgtaacttaa ttaacggcac tcctcagcaa atataatgac
     4681 cctcttgata acccaagagg gcatttttta atgcccatgg cgtttatttg ccgactacct
     4741 tggtgatctc gcctttcacg tagtggacaa attcttccaa ctgatctgcg cgcgaggcca
     4801 agcgatcttc ttcttgtcca agataagcct gtctagcttc aagtatgacg ggctgatact
     4861 gggccggcag gcgctccatt gcccagtcgg cagcgacatc cttcggcgcg attttgccgg
     4921 ttactgcgct gtaccaaatg cgggacaacg taagcactac atttcgctca tcgccagccc
     4981 agtcgggcgg cgagttccat agcgttaagg tttcatttag cgcctcaaat agatcctgtt
     5041 caggaaccgg atcaaagagt tcctccgccg ctggacctac caaggcaacg ctatgttctc
     5101 ttgcttttgt cagcaagata gccagatcaa tgtcgatcgt ggctggctcg aagatacctg
     5161 caagaatgtc attgcgctgc cattctccaa attgcagttc gcgcttagct ggataacgcc
     5221 acggaatgat gtcgtcgtgc acaacaatgg tgacttctac agcgcggaga atctcgctct
     5281 ctccagggga agccgaagtt tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat
     5341 caagccttac ggtcaccgta accagcaaat caatatcact gtgtggcttc aggccgccat
     5401 ccactgcgga gccgtacaaa tgtacggcca gcaacatcgg ttcgagatgg cgctcgatga
     5461 cgccaactac ctctgatagt tgagtcgata cttcggcgat caccgcttcc ctcatactct
     5521 tcctttttca atattattga agcatttatc agggttattg tttcatgagc gtatacatat
     5581 ttgaatgtat ttagaaaaat attaattgcg ttgcgctcac tgcc
//
