LOCUS       sequence-192402-        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"
     rep_origin      12..600
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     501..520
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     754..771
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    786..926
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(852..871)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1012..1034
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1028..1219)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1362..1379)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1532..1618
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1710..1737
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4708..5499)
                     /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(5500..5571)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 caaataggcc gttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
       61 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccacctct ttttccgaag
      121 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
      181 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
      241 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
      301 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
      361 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
      421 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
      481 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
      541 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
      601 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
      661 ttctttcatg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      721 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      781 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg
      841 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
      901 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
      961 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     1021 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     1081 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     1141 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     1201 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     1261 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacaagagg
     1321 acatccggtg gctgttttgg cggatgagag aagattttca gcctgataca gattaaatca
     1381 gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca
     1441 cctgacccca tgccgaactc agaagtgaaa ctccgtagcg ccgatggtag tgtggggtct
     1501 ccccatgcga gagtagggaa ctgccagtca tcaaataaaa cgaaaggctc agtcgaaaga
     1561 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
     1621 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc
     1681 gccataaact gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc
     1741 gtttctacaa actcgctgag gattctagag cacagcttga tgaaattcgc cacttgttag
     1801 tgtaattcgc taactcatcc tggcatgttg ctgttgattc ttcaatcaga tctttataaa
     1861 tcaaaaagat aaaaaattgg cacgcaaatt gtattaacag ttcagcagga caatcctgaa
     1921 cgcagaaatc aagaggacaa cattaagaaa agtgaaatga atagttaagg aggaaaaatg
     1981 aacacgatta acatcgctaa gaacgacttc taggacatcg aactggctgc tatcccgttc
     2041 aacactctgg ctgaccatta cggtgagcgt ttagctcgcg aacagttggc ccttgagcat
     2101 gagtcttacg agatgggtga agcacgcttc cgcaagatgt ttgagcgtca acttaaagct
     2161 ggtgaggttg cggataacgc tgccgccaag cctctcatca ctaccctact ccctaagatg
     2221 attgcacgca tcaacgactg gtttgaggaa gtgaaagcta agcgcggcaa gcgcccgaca
     2281 gccttccagt tcctgcaaga aatcaagccg gaagccgtag cgtacatcac cattaagacc
     2341 actctggctt gcctaaccag tgctgacaat acaaccgttc aggctgtagc aagcgcaatc
     2401 ggtcgggcca ttgaggacga ggctcgcttc ggtcgtatcc gtgaccttga agctaagcac
     2461 ttcaagaaaa acgttgagga acaactcaac aagcgcgtag ggcacgtcta caagaaagca
     2521 tttatgcaag ttgtcgaggc tgacatgctc tctaagggtc tactcggtgg cgaggcgtgg
     2581 tcttagtggc ataaggaaga ctctattcat gtaggagtac gctgcatcga gatgctcatt
     2641 gagtcaaccg gaatggttag cttacaccgc caaaatgctg gcgtagtagg tcaagactct
     2701 gagactatcg aactcgcacc tgaatacgct gaggctatcg caacccgtgc aggtgcgctg
     2761 gctggcatct ctccgatgtt ccaaccttgc gtagttcctc ctaagccgtg gactggcatt
     2821 actggtggtg gctattgggc taacggtcgt cgtcctctgg cgctggtgcg tactcacagt
     2881 aagaaagcac tgatgcgcta cgaagacgtt tacatgcctg aggtgtacaa agcgattaac
     2941 attgcgcaaa acaccgcatg gaaaatcaac aagaaagtcc tagcggtcgc caacgtaatc
     3001 accaagtgga agcattgtcc ggtcgaggac atccctgcga ttgagcgtga agaactcccg
     3061 atgaaaccgg aagacatcga catgaatcct gaggctctca ccgcgtggaa acgtgctgcc
     3121 gctgctgtgt accgcaagga caaggctcgc aagtctcgcc gtatcagcct tgagttcatg
     3181 cttgagcaag ccaataagtt tgctaaccat aaggccatct ggttccctta caacatggac
     3241 tggcgcggtc gtgtttacgc tgtgtcaatg ttcaacccgc aaggtaacga tatgaccaaa
     3301 ggactgctta cgctggcgaa aggtaaacca atcggtaagg aaggttacta ctggctgaaa
     3361 atccacggtg caaactgtgc gggtgtcgat aaggttccgt tccctgagcg catcaagttc
     3421 attgaggaaa accacgagaa catcatggct tgcgctaagt ctccactgga gaacacttgg
     3481 tgggctgagc aagattctcc gttctgcttc cttgcgttct gctttgagta cgctggggta
     3541 cagcaccacg gcctgagcta taactgctcc cttccgctgg cgtttgacgg gtcttgctct
     3601 ggcatccagc acttctccgc gatgctccga gatgaggtag gtggtcgcgc ggttaacttg
     3661 cttcctagtg aaaccgttca ggacatctac gggattgttg ctaagaaagt caacgagatt
     3721 ctacaagcag acgcaatcaa tgggaccgat aacgaagtag ttaccgtgac cgatgagaac
     3781 actggtgaaa tctctgagaa agtcaagctg ggcactaagg cactggctgg tcaatggctg
     3841 gcttacggtg ttactcgcag tgtgactaag cgttcagtca tgacgctggc ttacgggtcc
     3901 aaagagttcg gcttccgtca acaagtgctg gaagatacca ttcagccagc tattgattcc
     3961 ggcaagggtc tgatgttcac tcagccgaat caggctgctg gatacatggc taagctgatt
     4021 tgggaatctg tgagcgtgac ggtggtagct gcggttgaag caatgaactg gcttaagtct
     4081 gctgctaagc tgctggctgc tgaggtcaaa gataagaaga ctggagagat tcttcgcaag
     4141 cgttgcgctg tgcattgggt aactcctgat ggtttccctg tgtggcagga atacaagaag
     4201 cctattcaga cgcgcttgaa cctgatgttc ctcggtcagt tccgcttaca gcctaccatt
     4261 aacaccaaca aagatagcga gattgatgca cacaaacagg agtctggtat cgctcctaac
     4321 tttgtacaca gccaagacgg tagccacctt cgtaagactg tagtgtgggc acacgagaag
     4381 tacggaatcg aatcttttgc actgattcac gactccttcg gtaccattcc ggctgacgct
     4441 gcgaacctgt tcaaagcagt gcgcgaaact atggttgaca catatgagtc ttgtgatgta
     4501 ctggctgatt tctacgacca gttcgctgac cagttgcacg agtctcaatt ggacaaaatg
     4561 ccagcacttc cggctaaagg taacttgaac ctccgtgaca tcttagagtc ggacttcgcg
     4621 ttcgcgtaac ttaattaacg gcactcctca gcaaatataa tgaccctctt gataacccaa
     4681 gagggcattt tttaatgccc atggcgttta tttgccgact accttggtga tctcgccttt
     4741 cacgtagtgg acaaattctt ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg
     4801 tccaagataa gcctgtctag cttcaagtat gacgggctga tactgggccg gcaggcgctc
     4861 cattgcccag tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca
     4921 aatgcgggac aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt
     4981 ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa
     5041 gagttcctcc gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa
     5101 gatagccaga tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg
     5161 ctgccattct ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc
     5221 gtgcacaaca atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga
     5281 agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac
     5341 cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg ccatccactg cggagccgta
     5401 caaatgtacg gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga
     5461 tagttgagtc gatacttcgg cgatcaccgc ttccctcata ctcttccttt ttcaatatta
     5521 ttgaagcatt tatcagggtt attgtttcat gagcgtatac atatttgaat gtatttagaa
     5581 aaatattaat tgcgttgcgc tcactgcccg ctttccaggc
//
