LOCUS       sequence-192398-        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: pDB023f
FEATURES             Location/Qualifiers
     source          1..5624
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      18..606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     507..526
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     760..777
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    792..932
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(858..877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1018..1040
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1034..1225)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1368..1385)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1538..1624
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1716..1743
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4718..5509)
                     /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(5510..5581)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccaggccaaa taggccgttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
       61 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc acctcttttt
      121 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
      181 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
      241 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
      301 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
      361 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
      421 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      481 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      541 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      601 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      661 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
      721 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
      781 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
      841 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      901 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      961 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     1021 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     1081 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     1141 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     1201 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     1261 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     1321 aagaggacat ccggtggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt
     1381 aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg
     1441 gtcccacctg accccatgcc gaactcagaa gtgaaactcc gtagcgccga tggtagtgtg
     1501 gggtctcccc atgcgagagt agggaactgc cagtcatcaa ataaaacgaa aggctcagtc
     1561 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     1621 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
     1681 acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct
     1741 ttttgcgttt ctacaaactc gctgaggatt ctagagcaca gcttgatgaa attcgccact
     1801 tgttagtgta attcgctaac tcatcctggc atgttgctgt tgattcttca atcagatctt
     1861 tataaatcaa aaagataaaa aattggcacg caaattgtat taacagttca gcaggacaat
     1921 cctgaacgca gaaatcaaga ggacaacatt aagaaaagtg aaatgaatag ttaaggagga
     1981 aaaaaaaatg aacacgatta acatcgctaa gaacgacttc taggacatcg aactggctgc
     2041 tatcccgttc aacactctgg ctgaccatta cggtgagcgt ttagctcgcg aacagttggc
     2101 ccttgagcat gagtcttacg agatgggtga agcacgcttc cgcaagatgt ttgagcgtca
     2161 acttaaagct ggtgaggttg cggataacgc tgccgccaag cctctcatca ctaccctact
     2221 ccctaagatg attgcacgca tcaacgactg gtttgaggaa gtgaaagcta agcgcggcaa
     2281 gcgcccgaca gccttccagt tcctgcaaga aatcaagccg gaagccgtag cgtacatcac
     2341 cattaagacc actctggctt gcctaaccag tgctgacaat acaaccgttc aggctgtagc
     2401 aagcgcaatc ggtcgggcca ttgaggacga ggctcgcttc ggtcgtatcc gtgaccttga
     2461 agctaagcac ttcaagaaaa acgttgagga acaactcaac aagcgcgtag ggcacgtcta
     2521 caagaaagca tttatgcaag ttgtcgaggc tgacatgctc tctaagggtc tactcggtgg
     2581 cgaggcgtgg tcttagtggc ataaggaaga ctctattcat gtaggagtac gctgcatcga
     2641 gatgctcatt gagtcaaccg gaatggttag cttacaccgc caaaatgctg gcgtagtagg
     2701 tcaagactct gagactatcg aactcgcacc tgaatacgct gaggctatcg caacccgtgc
     2761 aggtgcgctg gctggcatct ctccgatgtt ccaaccttgc gtagttcctc ctaagccgtg
     2821 gactggcatt actggtggtg gctattgggc taacggtcgt cgtcctctgg cgctggtgcg
     2881 tactcacagt aagaaagcac tgatgcgcta cgaagacgtt tacatgcctg aggtgtacaa
     2941 agcgattaac attgcgcaaa acaccgcatg gaaaatcaac aagaaagtcc tagcggtcgc
     3001 caacgtaatc accaagtgga agcattgtcc ggtcgaggac atccctgcga ttgagcgtga
     3061 agaactcccg atgaaaccgg aagacatcga catgaatcct gaggctctca ccgcgtggaa
     3121 acgtgctgcc gctgctgtgt accgcaagga caaggctcgc aagtctcgcc gtatcagcct
     3181 tgagttcatg cttgagcaag ccaataagtt tgctaaccat aaggccatct ggttccctta
     3241 caacatggac tggcgcggtc gtgtttacgc tgtgtcaatg ttcaacccgc aaggtaacga
     3301 tatgaccaaa ggactgctta cgctggcgaa aggtaaacca atcggtaagg aaggttacta
     3361 ctggctgaaa atccacggtg caaactgtgc gggtgtcgat aaggttccgt tccctgagcg
     3421 catcaagttc attgaggaaa accacgagaa catcatggct tgcgctaagt ctccactgga
     3481 gaacacttgg tgggctgagc aagattctcc gttctgcttc cttgcgttct gctttgagta
     3541 cgctggggta cagcaccacg gcctgagcta taactgctcc cttccgctgg cgtttgacgg
     3601 gtcttgctct ggcatccagc acttctccgc gatgctccga gatgaggtag gtggtcgcgc
     3661 ggttaacttg cttcctagtg aaaccgttca ggacatctac gggattgttg ctaagaaagt
     3721 caacgagatt ctacaagcag acgcaatcaa tgggaccgat aacgaagtag ttaccgtgac
     3781 cgatgagaac actggtgaaa tctctgagaa agtcaagctg ggcactaagg cactggctgg
     3841 tcaatggctg gcttacggtg ttactcgcag tgtgactaag cgttcagtca tgacgctggc
     3901 ttacgggtcc aaagagttcg gcttccgtca acaagtgctg gaagatacca ttcagccagc
     3961 tattgattcc ggcaagggtc tgatgttcac tcagccgaat caggctgctg gatacatggc
     4021 taagctgatt tgggaatctg tgagcgtgac ggtggtagct gcggttgaag caatgaactg
     4081 gcttaagtct gctgctaagc tgctggctgc tgaggtcaaa gataagaaga ctggagagat
     4141 tcttcgcaag cgttgcgctg tgcattgggt aactcctgat ggtttccctg tgtggcagga
     4201 atacaagaag cctattcaga cgcgcttgaa cctgatgttc ctcggtcagt tccgcttaca
     4261 gcctaccatt aacaccaaca aagatagcga gattgatgca cacaaacagg agtctggtat
     4321 cgctcctaac tttgtacaca gccaagacgg tagccacctt cgtaagactg tagtgtgggc
     4381 acacgagaag tacggaatcg aatcttttgc actgattcac gactccttcg gtaccattcc
     4441 ggctgacgct gcgaacctgt tcaaagcagt gcgcgaaact atggttgaca catatgagtc
     4501 ttgtgatgta ctggctgatt tctacgacca gttcgctgac cagttgcacg agtctcaatt
     4561 ggacaaaatg ccagcacttc cggctaaagg taacttgaac ctccgtgaca tcttagagtc
     4621 ggacttcgcg ttcgcgtaac ttaattaacg gcactcctca gcaaatataa tgaccctctt
     4681 gataacccaa gagggcattt tttaatgccc atggcgttta tttgccgact accttggtga
     4741 tctcgccttt cacgtagtgg acaaattctt ccaactgatc tgcgcgcgag gccaagcgat
     4801 cttcttcttg tccaagataa gcctgtctag cttcaagtat gacgggctga tactgggccg
     4861 gcaggcgctc cattgcccag tcggcagcga catccttcgg cgcgattttg ccggttactg
     4921 cgctgtacca aatgcgggac aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg
     4981 gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa
     5041 ccggatcaaa gagttcctcc gccgctggac ctaccaaggc aacgctatgt tctcttgctt
     5101 ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa
     5161 tgtcattgcg ctgccattct ccaaattgca gttcgcgctt agctggataa cgccacggaa
     5221 tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg gagaatctcg ctctctccag
     5281 gggaagccga agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc
     5341 ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg ccatccactg
     5401 cggagccgta caaatgtacg gccagcaacg tcggttcgag atggcgctcg atgacgccaa
     5461 ctacctctga tagttgagtc gatacttcgg cgatcaccgc ttccctcata ctcttccttt
     5521 ttcaatatta ttgaagcatt tatcagggtt attgtttcat gagcgtatac atatttgaat
     5581 gtatttagaa aaatattaat tgcgttgcgc tcactgcccg cttt
//
