LOCUS       sequence-192369-        8301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Accessory plasmid for PACE of PylRS variants; negative selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8301)
  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 8301)
  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: pDB007ns2a
FEATURES             Location/Qualifiers
     source          1..8301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             257..1531
                     /codon_start=1
                     /gene="III"
                     /product="pIII"
                     /label=M13 gene III
                     /note="minor coat protein"
                     /translation="MKKLLFAIPLVVPFYSHSAETVESCLAKPHTENSFTNVWKDDKTL
                     DRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGG
                     TKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQ
                     GALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQ
                     GQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKM
                     ANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGS
                     NSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVF
                     AFLLYVATFMYVFSTFANILRNKES"
     primer_bind     complement(2214..2231)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2384..2470
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2562..2589
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    2649..2667
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        2668..2722
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    2673..2691
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    2703..2721
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             complement(join(5508..6299,6300..6368))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6131..6150
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6369..6473)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6541..6559
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(6580..7530)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      7578..7800
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttgc tgaggagact tagggaccct
       61 gatgaaattc gccacttgtt agtgtaattc gctaactcat cctggcatgt tgctgttgat
      121 tcttcaatca gatctttata aatcaaaaag ataaaaaatt ggcacgcaaa ttgtattaac
      181 agttcagcag gacaatcctg aacgcagaaa tcaagaggac aacattacct gcaggtgcag
      241 taaggaggaa aaaaaaatga aaaaattatt attcgcaatt cctttagttg ttcctttcta
      301 ttctcactcc gctgaaactg ttgaaagttg tttagcaaaa ccccatacag aaaattcatt
      361 tactaacgtc tggaaagacg acaaaacttt agatcgttac gctaactatg agggctgtct
      421 gtggaatgct acaggcgttg tagtttgtac tggtgacgaa actcagtgtt acggtacatg
      481 ggttcctatt gggcttgcta tccctgaaaa tgagggtggt ggctctgagg gtggcggttc
      541 tgagggtggc ggttctgagg gtggcggtac taaacctcct gagtacggtg atacacctat
      601 tccgggctat acttatatca accctctcga cggcacttat ccgcctggta ctgagcaaaa
      661 ccccgctaat cctaatcctt ctcttgagga gtctcagcct cttaatactt tcatgtttca
      721 gaataatagg ttccgaaata ggcagggggc attaactgtt tatacgggca ctgttactca
      781 aggcactgac cccgttaaaa cttattacca gtacactcct gtatcatcaa aagccatgta
      841 tgacgcttac tggaacggta aattcagaga ctgcgctttc cattctggct ttaatgagga
      901 tccattcgtt tgtgaatatc aaggccaatc gtctgacctg cctcaacctc ctgtcaatgc
      961 tggcggcggc tctggtggtg gttctggtgg cggctctgag ggtggtggct ctgagggtgg
     1021 cggttctgag ggtggcggct ctgagggagg cggttccggt ggtggctctg gttccggtga
     1081 ttttgattat gaaaagatgg caaacgctaa taagggggct atgaccgaaa atgccgatga
     1141 aaacgcgcta cagtctgacg ctaaaggcaa acttgattct gtcgctactg attacggtgc
     1201 tgctatcgat ggtttcattg gtgacgtttc cggccttgct aatggtaatg gtgctactgg
     1261 tgattttgct ggctctaatt cccaaatggc tcaagtcggt gacggtgata attcaccttt
     1321 aatgaataat ttccgtcaat atttaccttc cctccctcaa tcggttgaat gtcgcccttt
     1381 tgtctttggc gctggtaaac cttacgagtt cagtatcgac tgcgataaga tcaacctgtt
     1441 ccgcggtgtc tttgcgtttc ttttatatgt tgccaccttt atgtatgtat tttctacgtt
     1501 tgctaacata ctgcgtaata aggagtctta atcttgagta acggagccaa tgtacgcaaa
     1561 aaaccccgct tcagcggggt tttttcgcca aaaaaaaccc cgccctgtca ggggcggggt
     1621 ttttttttag ggccctactg actgtccgtg tgcttctcaa atgcctgagg ccagtttgct
     1681 caggctctcc ccgtggaggt aataattgac gatatgatca gtgcacggct aactaagcgg
     1741 cctgctgact ttctcgccga tcaaaaggca ttttgctatt aagggattga cgagggcgta
     1801 tctgcgcagt aagatgcgcc ccgcattccg gcggtagttc agcagggcag aacggcggac
     1861 tctaaatccg catggcaggg gttcaaatcc cctccgccgg accaaattcg aaaagcctgc
     1921 tcaacgagca ggcttttttg catgctcgag cagctcaggg tcgaatttgc tttcgaattt
     1981 ctgccattca tccgcttatt atcacttatt caggcgtagc aaccaggcgt ttaagggcac
     2041 caataactgc cttaaaaaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat
     2101 tcattaagca ttctgccgac gttcatcgga tcccgggccc gtcgactgca gaggcctgca
     2161 tgcaagcttg gcgtaatcag tggctgtttt ggcggatgag agaagatttt cagcctgata
     2221 cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc
     2281 gcggtggtcc cacctgaccc catgccgaac tcagaagtga aactccgtag cgccgatggt
     2341 agtgtggggt ctccccatgc gagagtaggg aactgccagt catcaaataa aacgaaaggc
     2401 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     2461 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
     2521 ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga
     2581 tggccttttt gcgtttctac aaactcgctg aggattctag agcacagctc catcgaatgg
     2641 ccagatgatc cctatcagtg atagagattg acactctatc attgatagag ttattttacc
     2701 actccctatc agtgatagag aaaagtgaaa tgaatagtta aggaggaaaa atgaacacga
     2761 ttaacatcgc taagaacgac ttctaggaca tcgaactggc tgctatcccg ttcaacactc
     2821 tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag catgagtctt
     2881 acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa gctggtgagg
     2941 ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag atgattgcac
     3001 gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg acagccttcc
     3061 agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag accactctgg
     3121 cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca atcggtcggg
     3181 ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag cacttcaaga
     3241 aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa gcatttatgc
     3301 aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg tggtcttagt
     3361 ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc attgagtcaa
     3421 ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac tctgagacta
     3481 tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg ctggctggca
     3541 tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc attactggtg
     3601 gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac agtaagaaag
     3661 cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt aacattgcgc
     3721 aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta atcaccaagt
     3781 ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc ccgatgaaac
     3841 cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct gccgctgctg
     3901 tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc atgcttgagc
     3961 aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg gactggcgcg
     4021 gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc aaaggactgc
     4081 ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg aaaatccacg
     4141 gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag ttcattgagg
     4201 aaaaccacga gaacatcatg gcttgcgcta agtctccact ggagaacact tggtgggctg
     4261 agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg gtacagcacc
     4321 acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc tctggcatcc
     4381 agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac ttgcttccta
     4441 gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag attctacaag
     4501 cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag aacactggtg
     4561 aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg ctggcttacg
     4621 gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggcttacggg tccaaagagt
     4681 tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat tccggcaagg
     4741 gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg atttgggaat
     4801 ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag tctgctgcta
     4861 agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc aagcgttgcg
     4921 ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag aagcctattc
     4981 agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc attaacacca
     5041 acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct aactttgtac
     5101 acagccaaga cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag aagtacggaa
     5161 tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac gctgcgaacc
     5221 tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat gtactggctg
     5281 atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa atgccagcac
     5341 ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc gcgttcgcgt
     5401 aacttaatta acggcactgc gaaaaaaccc cgccgaagcg gggttttttg cgaaatataa
     5461 tgaccctctt gataacccaa gagggcattt tttaatgccc atggcgttta ccaatgctta
     5521 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5581 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5641 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5701 agggccgagc gcagaagcgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5761 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5821 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5881 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5941 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6001 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6061 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccagcg
     6121 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6181 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6241 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6301 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6361 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6421 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6481 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     6541 aataggcgta tcacgaggcc cttaggccaa ataggccgtt cagatccttc cgtatttagc
     6601 cagtatgttc tctagtgtgg ttcgttgttt ttgcgtgagc catgagaacg aaccattgag
     6661 atcatgctta ctttgcatgt cactcaaaaa ttttgcctca aaactggtga gctgaatttt
     6721 tgcagttaaa gcatcgtgta gtgtttttct tagtccgtta cgtaggtagg aatctgatgt
     6781 aatggttgtt ggtattttgt caccattcat ttttatctgg ttgttctcaa gttcggttac
     6841 gagatccatt tgtctatcta gttcaacttg gaaaatcaac gtatcagtcg ggcggcctcg
     6901 cttatcaacc accaatttca tattgctgta agtgtttaaa tctttactta ttggtttcaa
     6961 aacccattgg ttaagccttt taaactcatg gtagttattt tcaagcatta acatgaactt
     7021 aaattcatca aggctaatct ctatatttgc cttgtgagtt ttcttttgtg ttagttcttt
     7081 taataaccac tcataaatcc tcatagagta tttgttttca aaagacttaa catgttccag
     7141 attatatttt atgaattttt ttaactggaa aagataaggc aatatctctt cactaaaaac
     7201 taattctaat ttttcgcttg agaacttggc atagtttgtc cactggaaaa tctcaaagcc
     7261 tttaaccaaa ggattcctga tttccacagt tctcgtcatc agctctctgg ttgctttagc
     7321 taatacacca taagcatttt ccctactgat gttcatcatc tgagcgtatt ggttataagt
     7381 gaacgatacc gtccgttctt tccttgtagg gttttcaatc gtggggttga gtagtgccac
     7441 acagcataaa attagcttgg tttcatgctc cgttaagtca tagcgactaa tcgctagttc
     7501 atttgctttg aaaacaacta attcagacat acatctcaat tggtctaggt gattttaatc
     7561 actataccaa ttgagatggg ctagtcaatg ataattacta gtccttttcc tttgagttgt
     7621 gggtatctgt aaattctgct agacctttgc tggaaaactt gtaaattctg ctagaccctc
     7681 tgtaaattcc gctagacctt tgtgtgtttt ttttgtttat attcaagtgg ttataattta
     7741 tagaataaag aaagaataaa aaaagataaa aagaatagat cccagccctg tgtataactc
     7801 actactttag tcagttccgc agtattacaa aaggatgtcg caaacgctgt ttgctcctct
     7861 acaaaacaga ccttaaaacc ctaaaggctt aagtagcacc ctcgcaagct cgggcaaatc
     7921 gctgaatatt ccttttgtct ccgaccatca ggcacctgag tcgctgtctt tttcgtgaca
     7981 ttcagttcgc tgcgctcacg gctctggcag tgaatggggg taaatggcac tacaggcgcc
     8041 ttttatggat tcatgcaagg aaactaccca taatacaaga aaagcccgtc acgggcttct
     8101 cagggcgttt tatggcgggt ctgctatgtg gtgctatctg actttttgct gttcagcagt
     8161 tcctgccctc tgattttcca gtctgaccac ttcggattat cccgtgacag gtcattcaga
     8221 ctggctaatg cacccagtaa ggcagcggta tcatcaacag gcttacccgt cttactgtca
     8281 agaggacatc cggtcaaata a
//
