LOCUS       sequence-182107-        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"
     primer_bind     489..508
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     742..759
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    774..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1350..1367)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1520..1606
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1698..1725
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4700..5491)
                     /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(5492..5563)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(5624,1..588)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
       61 gcggtggttt gtttgccgga tcaagagcta ccacctcttt ttccgaaggt aactggcttc
      121 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
      181 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      241 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      301 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      361 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      421 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      481 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      541 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      601 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
      661 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
      721 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
      781 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acaagaggac atccggtggc
     1321 tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga acgcagaagc
     1381 ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg
     1441 ccgaactcag aagtgaaact ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga
     1501 gtagggaact gccagtcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1561 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     1621 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     1681 caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac
     1741 tcgctgagga ttctagagca cagcttgatg aaattcgcca cttgttagtg taattcgcta
     1801 actcatcctg gcatgttgct gttgattctt caatcagatc tttataaatc aaaaagataa
     1861 aaaattggca cgcaaattgt attaacagtt cagcaggaca atcctgaacg cagaaatcaa
     1921 gaggacaaca ttaagaaaag tgaaatgaat agttaaggag gaaaaaaaaa tgaacacgat
     1981 taacatcgct aagaacgact tctaggacat cgaactggct gctatcccgt tcaacactct
     2041 ggctgaccat tacggtgagc gtttagctcg cgaacagttg gcccttgagc atgagtctta
     2101 cgagatgggt gaagcacgct tccgcaagat gtttgagcgt caacttaaag ctggtgaggt
     2161 tgcggataac gctgccgcca agcctctcat cactacccta ctccctaaga tgattgcacg
     2221 catcaacgac tggtttgagg aagtgaaagc taagcgcggc aagcgcccga cagccttcca
     2281 gttcctgcaa gaaatcaagc cggaagccgt agcgtacatc accattaaga ccactctggc
     2341 ttgcctaacc agtgctgaca atacaaccgt tcaggctgta gcaagcgcaa tcggtcgggc
     2401 cattgaggac gaggctcgct tcggtcgtat ccgtgacctt gaagctaagc acttcaagaa
     2461 aaacgttgag gaacaactca acaagcgcgt agggcacgtc tacaagaaag catttatgca
     2521 agttgtcgag gctgacatgc tctctaaggg tctactcggt ggcgaggcgt ggtcttagtg
     2581 gcataaggaa gactctattc atgtaggagt acgctgcatc gagatgctca ttgagtcaac
     2641 cggaatggtt agcttacacc gccaaaatgc tggcgtagta ggtcaagact ctgagactat
     2701 cgaactcgca cctgaatacg ctgaggctat cgcaacccgt gcaggtgcgc tggctggcat
     2761 ctctccgatg ttccaacctt gcgtagttcc tcctaagccg tggactggca ttactggtgg
     2821 tggctattgg gctaacggtc gtcgtcctct ggcgctggtg cgtactcaca gtaagaaagc
     2881 actgatgcgc tacgaagacg tttacatgcc tgaggtgtac aaagcgatta acattgcgca
     2941 aaacaccgca tggaaaatca acaagaaagt cctagcggtc gccaacgtaa tcaccaagtg
     3001 gaagcattgt ccggtcgagg acatccctgc gattgagcgt gaagaactcc cgatgaaacc
     3061 ggaagacatc gacatgaatc ctgaggctct caccgcgtgg aaacgtgctg ccgctgctgt
     3121 gtaccgcaag gacaaggctc gcaagtctcg ccgtatcagc cttgagttca tgcttgagca
     3181 agccaataag tttgctaacc ataaggccat ctggttccct tacaacatgg actggcgcgg
     3241 tcgtgtttac gctgtgtcaa tgttcaaccc gcaaggtaac gatatgacca aaggactgct
     3301 tacgctggcg aaaggtaaac caatcggtaa ggaaggttac tactggctga aaatccacgg
     3361 tgcaaactgt gcgggtgtcg ataaggttcc gttccctgag cgcatcaagt tcattgagga
     3421 aaaccacgag aacatcatgg cttgcgctaa gtctccactg gagaacactt ggtgggctga
     3481 gcaagattct ccgttctgct tccttgcgtt ctgctttgag tacgctgggg tacagcacca
     3541 cggcctgagc tataactgct cccttccgct ggcgtttgac gggtcttgct ctggcatcca
     3601 gcacttctcc gcgatgctcc gagatgaggt aggtggtcgc gcggttaact tgcttcctag
     3661 tgaaaccgtt caggacatct acgggattgt tgctaagaaa gtcaacgaga ttctacaagc
     3721 agacgcaatc aatgggaccg ataacgaagt agttaccgtg accgatgaga acactggtga
     3781 aatctctgag aaagtcaagc tgggcactaa ggcactggct ggtcaatggc tggcttacgg
     3841 tgttactcgc agtgtgacta agcgttcagt catgacgctg gcttacgggt ccaaagagtt
     3901 cggcttccgt caacaagtgc tggaagatac cattcagcca gctattgatt ccggcaaggg
     3961 tctgatgttc actcagccga atcaggctgc tggatacatg gctaagctga tttgggaatc
     4021 tgtgagcgtg acggtggtag ctgcggttga agcaatgaac tggcttaagt ctgctgctaa
     4081 gctgctggct gctgaggtca aagataagaa gactggagag attcttcgca agcgttgcgc
     4141 tgtgcattgg gtaactcctg atggtttccc tgtgtggcag gaatacaaga agcctattca
     4201 gacgcgcttg aacctgatgt tcctcggtca gttccgctta cagcctacca ttaacaccaa
     4261 caaagatagc gagattgatg cacacaaaca ggagtctggt atcgctccta actttgtaca
     4321 cagccaagac ggtagccacc ttcgtaagac tgtagtgtgg gcacacgaga agtacggaat
     4381 cgaatctttt gcactgattc acgactcctt cggtaccatt ccggctgacg ctgcgaacct
     4441 gttcaaagca gtgcgcgaaa ctatggttga cacatatgag tcttgtgatg tactggctga
     4501 tttctacgac cagttcgctg accagttgca cgagtctcaa ttggacaaaa tgccagcact
     4561 tccggctaaa ggtaacttga acctccgtga catcttagag tcggacttcg cgttcgcgta
     4621 acttaattaa cggcactcct cagcaaatat aatgaccctc ttgataaccc aagagggcat
     4681 tttttaatgc ccatggcgtt tatttgccga ctaccttggt gatctcgcct ttcacgtagt
     4741 ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct tgtccaagat
     4801 aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc tccattgccc
     4861 agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac caaatgcggg
     4921 acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag ttccatagcg
     4981 ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca aagagttcct
     5041 ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc aagatagcca
     5101 gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg cgctgccatt
     5161 ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa
     5221 caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc gaagtttcca
     5281 aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc accgtaacca
     5341 gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg tacaaatgta
     5401 cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct gatagttgag
     5461 tcgatacttc ggcgatcacc gcttccctca tactcttcct ttttcaatat tattgaagca
     5521 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaatatta
     5581 attgcgttgc gctcactgcc cgctttccag gccaaatagg ccgt
//
