LOCUS       sequence-182109-        5620 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 5620)
  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 5620)
  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: pDB023f1
FEATURES             Location/Qualifiers
     source          1..5620
                     /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(4696..5487)
                     /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(5488..5559)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(5620,1..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 gaaaaatgaa cacgattaac
     1981 atcgctaaga acgacttcta ggacatcgaa ctggctgcta tcccgttcaa cactctggct
     2041 gaccattacg gtgagcgttt agctcgcgaa cagttggccc ttgagcatga gtcttacgag
     2101 atgggtgaag cacgcttccg caagatgttt gagcgtcaac ttaaagctgg tgaggttgcg
     2161 gataacgctg ccgccaagcc tctcatcact accctactcc ctaagatgat tgcacgcatc
     2221 aacgactggt ttgaggaagt gaaagctaag cgcggcaagc gcccgacagc cttccagttc
     2281 ctgcaagaaa tcaagccgga agccgtagcg tacatcacca ttaagaccac tctggcttgc
     2341 ctaaccagtg ctgacaatac aaccgttcag gctgtagcaa gcgcaatcgg tcgggccatt
     2401 gaggacgagg ctcgcttcgg tcgtatccgt gaccttgaag ctaagcactt caagaaaaac
     2461 gttgaggaac aactcaacaa gcgcgtaggg cacgtctaca agaaagcatt tatgcaagtt
     2521 gtcgaggctg acatgctctc taagggtcta ctcggtggcg aggcgtggtc ttagtggcat
     2581 aaggaagact ctattcatgt aggagtacgc tgcatcgaga tgctcattga gtcaaccgga
     2641 atggttagct tacaccgcca aaatgctggc gtagtaggtc aagactctga gactatcgaa
     2701 ctcgcacctg aatacgctga ggctatcgca acccgtgcag gtgcgctggc tggcatctct
     2761 ccgatgttcc aaccttgcgt agttcctcct aagccgtgga ctggcattac tggtggtggc
     2821 tattgggcta acggtcgtcg tcctctggcg ctggtgcgta ctcacagtaa gaaagcactg
     2881 atgcgctacg aagacgttta catgcctgag gtgtacaaag cgattaacat tgcgcaaaac
     2941 accgcatgga aaatcaacaa gaaagtccta gcggtcgcca acgtaatcac caagtggaag
     3001 cattgtccgg tcgaggacat ccctgcgatt gagcgtgaag aactcccgat gaaaccggaa
     3061 gacatcgaca tgaatcctga ggctctcacc gcgtggaaac gtgctgccgc tgctgtgtac
     3121 cgcaaggaca aggctcgcaa gtctcgccgt atcagccttg agttcatgct tgagcaagcc
     3181 aataagtttg ctaaccataa ggccatctgg ttcccttaca acatggactg gcgcggtcgt
     3241 gtttacgctg tgtcaatgtt caacccgcaa ggtaacgata tgaccaaagg actgcttacg
     3301 ctggcgaaag gtaaaccaat cggtaaggaa ggttactact ggctgaaaat ccacggtgca
     3361 aactgtgcgg gtgtcgataa ggttccgttc cctgagcgca tcaagttcat tgaggaaaac
     3421 cacgagaaca tcatggcttg cgctaagtct ccactggaga acacttggtg ggctgagcaa
     3481 gattctccgt tctgcttcct tgcgttctgc tttgagtacg ctggggtaca gcaccacggc
     3541 ctgagctata actgctccct tccgctggcg tttgacgggt cttgctctgg catccagcac
     3601 ttctccgcga tgctccgaga tgaggtaggt ggtcgcgcgg ttaacttgct tcctagtgaa
     3661 accgttcagg acatctacgg gattgttgct aagaaagtca acgagattct acaagcagac
     3721 gcaatcaatg ggaccgataa cgaagtagtt accgtgaccg atgagaacac tggtgaaatc
     3781 tctgagaaag tcaagctggg cactaaggca ctggctggtc aatggctggc ttacggtgtt
     3841 actcgcagtg tgactaagcg ttcagtcatg acgctggctt acgggtccaa agagttcggc
     3901 ttccgtcaac aagtgctgga agataccatt cagccagcta ttgattccgg caagggtctg
     3961 atgttcactc agccgaatca ggctgctgga tacatggcta agctgatttg ggaatctgtg
     4021 agcgtgacgg tggtagctgc ggttgaagca atgaactggc ttaagtctgc tgctaagctg
     4081 ctggctgctg aggtcaaaga taagaagact ggagagattc ttcgcaagcg ttgcgctgtg
     4141 cattgggtaa ctcctgatgg tttccctgtg tggcaggaat acaagaagcc tattcagacg
     4201 cgcttgaacc tgatgttcct cggtcagttc cgcttacagc ctaccattaa caccaacaaa
     4261 gatagcgaga ttgatgcaca caaacaggag tctggtatcg ctcctaactt tgtacacagc
     4321 caagacggta gccaccttcg taagactgta gtgtgggcac acgagaagta cggaatcgaa
     4381 tcttttgcac tgattcacga ctccttcggt accattccgg ctgacgctgc gaacctgttc
     4441 aaagcagtgc gcgaaactat ggttgacaca tatgagtctt gtgatgtact ggctgatttc
     4501 tacgaccagt tcgctgacca gttgcacgag tctcaattgg acaaaatgcc agcacttccg
     4561 gctaaaggta acttgaacct ccgtgacatc ttagagtcgg acttcgcgtt cgcgtaactt
     4621 aattaacggc actcctcagc aaatataatg accctcttga taacccaaga gggcattttt
     4681 taatgcccat ggcgtttatt tgccgactac cttggtgatc tcgcctttca cgtagtggac
     4741 aaattcttcc aactgatctg cgcgcgaggc caagcgatct tcttcttgtc caagataagc
     4801 ctgtctagct tcaagtatga cgggctgata ctgggccggc aggcgctcca ttgcccagtc
     4861 ggcagcgaca tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa
     4921 cgtaagcact acatttcgct catcgccagc ccagtcgggc ggcgagttcc atagcgttaa
     4981 ggtttcattt agcgcctcaa atagatcctg ttcaggaacc ggatcaaaga gttcctccgc
     5041 cgctggacct accaaggcaa cgctatgttc tcttgctttt gtcagcaaga tagccagatc
     5101 aatgtcgatc gtggctggct cgaagatacc tgcaagaatg tcattgcgct gccattctcc
     5161 aaattgcagt tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt gcacaacaat
     5221 ggtgacttct acagcgcgga gaatctcgct ctctccaggg gaagccgaag tttccaaaag
     5281 gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt acggtcaccg taaccagcaa
     5341 atcaatatca ctgtgtggct tcaggccgcc atccactgcg gagccgtaca aatgtacggc
     5401 cagcaacgtc ggttcgagat ggcgctcgat gacgccaact acctctgata gttgagtcga
     5461 tacttcggcg atcaccgctt ccctcatact cttccttttt caatattatt gaagcattta
     5521 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa atattaattg
     5581 cgttgcgctc actgcccgct ttccaggcca aataggccgt
//
