LOCUS       sequence-265851-        3979 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Accessory plasmid for aaRS PACE; negative selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3979)
  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 3979)
  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: pDB016
FEATURES             Location/Qualifiers
     source          1..3979
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(841..860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1001..1023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1017..1208)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1826..1845
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1826..1844
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1948..1965
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3056..3847)
                     /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(3848..3919)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accacctctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
      841 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
      901 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
      961 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     1021 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     1081 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     1141 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     1201 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     1261 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacagaggac atccggtaag
     1321 gccaataggg gttcataagg tacggcccgt actgaacccg ggtaggctgc ttgagccagt
     1381 gagcgattgc tggcctagat gaatgactgt ccacgacaga acccggctta tcggtcagtt
     1441 tcacctgatt tacgtaaaaa cccgcttcgg cgggtttttg cttttggagg ggcagaaaga
     1501 tgaatgactg tccacgacgc tatacccaaa agaaagcggc ttatcggtca gtttcacctg
     1561 gtttacgtaa aaacccgctt cggcgggttt ttgcttttgg aggggcagaa agatgaatga
     1621 ctgtccacga cactataccc aaaagaaagc ggcttatcgg tcagtttcac ctgttttacg
     1681 taaaaacccg cttcggcggg tttttacttt tggaggggca gaaagatgaa tgactgtcca
     1741 cgacactata cccaaaagaa agcggcttat cggtcagttt tacctgatgt acgtaataaa
     1801 ccgttccggc gggtttggga caaattaata cgactcacta tagggtgaaa gacctgcagg
     1861 tgcagtaagg aggaaaaaaa aatgaaaaaa ttattattcg caattccttt agttgttcct
     1921 ttctattctc actccgctga aactgttcat caccatcacc atcacgctga aactgttgaa
     1981 agttgtttag caaaacccca tacagaaaat tcatttacta acgtctggaa agacgacaaa
     2041 actttagatc gttacgctaa ctatgagggc tgtctgtgga atgctacagg cgttgtagtt
     2101 tgtactggtg acgaaactca gtgttacggt acatgggttc ctattgggct tgctatccct
     2161 gaaaatgagg gtggtggctc tgagggtggc ggttctgagg gtggcggttc tgagggtggc
     2221 ggtactaaac ctcctgagta cggtgataca cctattccgg gctatactta tatcaaccct
     2281 ctcgacggca cttatccgcc tggtactgag caaaaccccg ctaatcctaa tccttctctt
     2341 gaggagtctc agcctcttaa tactttcatg tttcagaata ataggttccg aaataggcag
     2401 ggggcattaa ctgtttatac gggcactgtt actcaaggca ctgaccccgt taaaacttat
     2461 taccagtaca ctcctgtatc atcaaaagcc atgtatgacg cttactggaa cggtaaattc
     2521 agagactgcg ctttccattc tggctttaat gaggatccat tcgtttgtga atatcaaggc
     2581 caatcgtctg acctgcctca acctcctgtc aatgctggcg gcggctctgg tggtggttct
     2641 ggtggcggct ctgagggtgg tggctctgag ggtggcggtt ctgagggtgg cggctctgag
     2701 ggaggcggtt ccggtggtgg ctcttcccaa atggctcaag tcggtgacgg tgataattca
     2761 cctttaatga ataatttccg tcaatattta ccttccctcc ctcaatcggt tgaatgtcgc
     2821 ccttttgtct ttggcgctgg taaaccttac gagttcagta tcgactgcga taagatcaac
     2881 ctgttccgcg gtgtctttgc gtttctttta tatgttgcca cctttatgta tgtattttct
     2941 acgtttgcta acatactgcg taataaggag tcttaaactt aattaacggc actcctcagc
     3001 aaatataatg accctcttga taacccaaga gggcattttt taatgcccat ggcgtttatt
     3061 tgccgactac cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg
     3121 cgcgcgaggc caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga
     3181 cgggctgata ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg
     3241 cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct
     3301 catcgccagc ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa
     3361 atagatcctg ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa
     3421 cgctatgttc tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct
     3481 cgaagatacc tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag
     3541 ctggataacg ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga
     3601 gaatctcgct ctctccaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc
     3661 gcgttgtttc atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct
     3721 tcaggccgcc atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat
     3781 ggcgctcgat gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt
     3841 ccctcatact cttccttttt caatattatt gaagcattta tcagggttat tgtttcatga
     3901 gcgtatacat atttgaatgt atttagaaaa atattaattg cgttgcgctc actgcccgct
     3961 ttccaggcca aataggccg
//
