LOCUS       sequence-182102-        8302 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 8302)
  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 8302)
  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..8302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             279..1553
                     /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(2237..2254)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2407..2493
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2585..2612
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    2672..2690
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        2691..2745
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    2696..2714
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    2726..2744
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             complement(join(5531..6322,6323..6391))
                     /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     6154..6173
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6392..6496)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6564..6582
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(6603..7553)
                     /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      7601..7823
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 aagaggacat ccggtcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
       61 gctgaggaga cttagggacc ctgatgaaat tcgccacttg ttagtgtaat tcgctaactc
      121 atcctggcat gttgctgttg attcttcaat cagatcttta taaatcaaaa agataaaaaa
      181 ttggcacgca aattgtatta acagttcagc aggacaatcc tgaacgcaga aatcaagagg
      241 acaacattac ctgcaggtgc agtaaggagg aaaaaaaaat gaaaaaatta ttattcgcaa
      301 ttcctttagt tgttcctttc tattctcact ccgctgaaac tgttgaaagt tgtttagcaa
      361 aaccccatac agaaaattca tttactaacg tctggaaaga cgacaaaact ttagatcgtt
      421 acgctaacta tgagggctgt ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg
      481 aaactcagtg ttacggtaca tgggttccta ttgggcttgc tatccctgaa aatgagggtg
      541 gtggctctga gggtggcggt tctgagggtg gcggttctga gggtggcggt actaaacctc
      601 ctgagtacgg tgatacacct attccgggct atacttatat caaccctctc gacggcactt
      661 atccgcctgg tactgagcaa aaccccgcta atcctaatcc ttctcttgag gagtctcagc
      721 ctcttaatac tttcatgttt cagaataata ggttccgaaa taggcagggg gcattaactg
      781 tttatacggg cactgttact caaggcactg accccgttaa aacttattac cagtacactc
      841 ctgtatcatc aaaagccatg tatgacgctt actggaacgg taaattcaga gactgcgctt
      901 tccattctgg ctttaatgag gatccattcg tttgtgaata tcaaggccaa tcgtctgacc
      961 tgcctcaacc tcctgtcaat gctggcggcg gctctggtgg tggttctggt ggcggctctg
     1021 agggtggtgg ctctgagggt ggcggttctg agggtggcgg ctctgaggga ggcggttccg
     1081 gtggtggctc tggttccggt gattttgatt atgaaaagat ggcaaacgct aataaggggg
     1141 ctatgaccga aaatgccgat gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt
     1201 ctgtcgctac tgattacggt gctgctatcg atggtttcat tggtgacgtt tccggccttg
     1261 ctaatggtaa tggtgctact ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg
     1321 gtgacggtga taattcacct ttaatgaata atttccgtca atatttacct tccctccctc
     1381 aatcggttga atgtcgccct tttgtctttg gcgctggtaa accttacgag ttcagtatcg
     1441 actgcgataa gatcaacctg ttccgcggtg tctttgcgtt tcttttatat gttgccacct
     1501 ttatgtatgt attttctacg tttgctaaca tactgcgtaa taaggagtct taatcttgag
     1561 taacggagcc aatgtacgca aaaaaccccg cttcagcggg gttttttcgc caaaaaaaac
     1621 cccgccctgt caggggcggg gttttttttt tagggcccta ctgactgtcc gtgtgcttct
     1681 caaatgcctg aggccagttt gctcaggctc tccccgtgga ggtaataatt gacgatatga
     1741 tcagtgcacg gctaactaag cggcctgctg actttctcgc cgatcaaaag gcattttgct
     1801 attaagggat tgacgagggc gtatctgcgc agtaagatgc gccccgcatt ccggcggtag
     1861 ttcagcaggg cagaacggcg gactctaaat ccgcatggca ggggttcaaa tcccctccgc
     1921 cggaccaaat tcgaaaagcc tgctcaacga gcaggctttt ttgcatgctc gagcagctca
     1981 gggtcgaatt tgctttcgaa tttctgccat tcatccgctt attatcactt attcaggcgt
     2041 agcaaccagg cgtttaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc
     2101 actcatcgca gtactgttgt aattcattaa gcattctgcc gacgttcatc ggatcccggg
     2161 cccgtcgact gcagaggcct gcatgcaagc ttggcgtaat cagtggctgt tttggcggat
     2221 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
     2281 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     2341 tgaaactccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     2401 agtcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     2461 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     2521 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
     2581 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactcg ctgaggattc
     2641 tagagcacag ctccatcgaa tggccagatg atccctatca gtgatagaga ttgacactct
     2701 atcattgata gagttatttt accactccct atcagtgata gagaaaagtg aaatgaatag
     2761 ttaaggagga aaaatgaaca cgattaacat cgctaagaac gacttctagg acatcgaact
     2821 ggctgctatc ccgttcaaca ctctggctga ccattacggt gagcgtttag ctcgcgaaca
     2881 gttggccctt gagcatgagt cttacgagat gggtgaagca cgcttccgca agatgtttga
     2941 gcgtcaactt aaagctggtg aggttgcgga taacgctgcc gccaagcctc tcatcactac
     3001 cctactccct aagatgattg cacgcatcaa cgactggttt gaggaagtga aagctaagcg
     3061 cggcaagcgc ccgacagcct tccagttcct gcaagaaatc aagccggaag ccgtagcgta
     3121 catcaccatt aagaccactc tggcttgcct aaccagtgct gacaatacaa ccgttcaggc
     3181 tgtagcaagc gcaatcggtc gggccattga ggacgaggct cgcttcggtc gtatccgtga
     3241 ccttgaagct aagcacttca agaaaaacgt tgaggaacaa ctcaacaagc gcgtagggca
     3301 cgtctacaag aaagcattta tgcaagttgt cgaggctgac atgctctcta agggtctact
     3361 cggtggcgag gcgtggtctt agtggcataa ggaagactct attcatgtag gagtacgctg
     3421 catcgagatg ctcattgagt caaccggaat ggttagctta caccgccaaa atgctggcgt
     3481 agtaggtcaa gactctgaga ctatcgaact cgcacctgaa tacgctgagg ctatcgcaac
     3541 ccgtgcaggt gcgctggctg gcatctctcc gatgttccaa ccttgcgtag ttcctcctaa
     3601 gccgtggact ggcattactg gtggtggcta ttgggctaac ggtcgtcgtc ctctggcgct
     3661 ggtgcgtact cacagtaaga aagcactgat gcgctacgaa gacgtttaca tgcctgaggt
     3721 gtacaaagcg attaacattg cgcaaaacac cgcatggaaa atcaacaaga aagtcctagc
     3781 ggtcgccaac gtaatcacca agtggaagca ttgtccggtc gaggacatcc ctgcgattga
     3841 gcgtgaagaa ctcccgatga aaccggaaga catcgacatg aatcctgagg ctctcaccgc
     3901 gtggaaacgt gctgccgctg ctgtgtaccg caaggacaag gctcgcaagt ctcgccgtat
     3961 cagccttgag ttcatgcttg agcaagccaa taagtttgct aaccataagg ccatctggtt
     4021 cccttacaac atggactggc gcggtcgtgt ttacgctgtg tcaatgttca acccgcaagg
     4081 taacgatatg accaaaggac tgcttacgct ggcgaaaggt aaaccaatcg gtaaggaagg
     4141 ttactactgg ctgaaaatcc acggtgcaaa ctgtgcgggt gtcgataagg ttccgttccc
     4201 tgagcgcatc aagttcattg aggaaaacca cgagaacatc atggcttgcg ctaagtctcc
     4261 actggagaac acttggtggg ctgagcaaga ttctccgttc tgcttccttg cgttctgctt
     4321 tgagtacgct ggggtacagc accacggcct gagctataac tgctcccttc cgctggcgtt
     4381 tgacgggtct tgctctggca tccagcactt ctccgcgatg ctccgagatg aggtaggtgg
     4441 tcgcgcggtt aacttgcttc ctagtgaaac cgttcaggac atctacggga ttgttgctaa
     4501 gaaagtcaac gagattctac aagcagacgc aatcaatggg accgataacg aagtagttac
     4561 cgtgaccgat gagaacactg gtgaaatctc tgagaaagtc aagctgggca ctaaggcact
     4621 ggctggtcaa tggctggctt acggtgttac tcgcagtgtg actaagcgtt cagtcatgac
     4681 gctggcttac gggtccaaag agttcggctt ccgtcaacaa gtgctggaag ataccattca
     4741 gccagctatt gattccggca agggtctgat gttcactcag ccgaatcagg ctgctggata
     4801 catggctaag ctgatttggg aatctgtgag cgtgacggtg gtagctgcgg ttgaagcaat
     4861 gaactggctt aagtctgctg ctaagctgct ggctgctgag gtcaaagata agaagactgg
     4921 agagattctt cgcaagcgtt gcgctgtgca ttgggtaact cctgatggtt tccctgtgtg
     4981 gcaggaatac aagaagccta ttcagacgcg cttgaacctg atgttcctcg gtcagttccg
     5041 cttacagcct accattaaca ccaacaaaga tagcgagatt gatgcacaca aacaggagtc
     5101 tggtatcgct cctaactttg tacacagcca agacggtagc caccttcgta agactgtagt
     5161 gtgggcacac gagaagtacg gaatcgaatc ttttgcactg attcacgact ccttcggtac
     5221 cattccggct gacgctgcga acctgttcaa agcagtgcgc gaaactatgg ttgacacata
     5281 tgagtcttgt gatgtactgg ctgatttcta cgaccagttc gctgaccagt tgcacgagtc
     5341 tcaattggac aaaatgccag cacttccggc taaaggtaac ttgaacctcc gtgacatctt
     5401 agagtcggac ttcgcgttcg cgtaacttaa ttaacggcac tgcgaaaaaa ccccgccgaa
     5461 gcggggtttt ttgcgaaata taatgaccct cttgataacc caagagggca ttttttaatg
     5521 cccatggcgt ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5581 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5641 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5701 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5761 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5821 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5881 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5941 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6001 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6061 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6121 gaccgagttg ctcttgccca gcgtcaatac gggataatac cgcgccacat agcagaactt
     6181 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6241 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6301 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6361 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6421 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6481 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     6541 ttatcatgac attaacctat aaaaataggc gtatcacgag gcccttaggc caaataggcc
     6601 gttcagatcc ttccgtattt agccagtatg ttctctagtg tggttcgttg tttttgcgtg
     6661 agccatgaga acgaaccatt gagatcatgc ttactttgca tgtcactcaa aaattttgcc
     6721 tcaaaactgg tgagctgaat ttttgcagtt aaagcatcgt gtagtgtttt tcttagtccg
     6781 ttacgtaggt aggaatctga tgtaatggtt gttggtattt tgtcaccatt catttttatc
     6841 tggttgttct caagttcggt tacgagatcc atttgtctat ctagttcaac ttggaaaatc
     6901 aacgtatcag tcgggcggcc tcgcttatca accaccaatt tcatattgct gtaagtgttt
     6961 aaatctttac ttattggttt caaaacccat tggttaagcc ttttaaactc atggtagtta
     7021 ttttcaagca ttaacatgaa cttaaattca tcaaggctaa tctctatatt tgccttgtga
     7081 gttttctttt gtgttagttc ttttaataac cactcataaa tcctcataga gtatttgttt
     7141 tcaaaagact taacatgttc cagattatat tttatgaatt tttttaactg gaaaagataa
     7201 ggcaatatct cttcactaaa aactaattct aatttttcgc ttgagaactt ggcatagttt
     7261 gtccactgga aaatctcaaa gcctttaacc aaaggattcc tgatttccac agttctcgtc
     7321 atcagctctc tggttgcttt agctaataca ccataagcat tttccctact gatgttcatc
     7381 atctgagcgt attggttata agtgaacgat accgtccgtt ctttccttgt agggttttca
     7441 atcgtggggt tgagtagtgc cacacagcat aaaattagct tggtttcatg ctccgttaag
     7501 tcatagcgac taatcgctag ttcatttgct ttgaaaacaa ctaattcaga catacatctc
     7561 aattggtcta ggtaatttta atcactatac caattgagat gggctagtca atgataatta
     7621 ctagtccttt tcctttgagt tgtgggtatc tgtaaattct gctagacctt tgctggaaaa
     7681 cttgtaaatt ctgctagacc ctctgtaaat tccgctagac ctttgtgtgt tttttttgtt
     7741 tatattaaag tggttataat ttatagaata aagaaagaat aaaaaaagat aaaaagaata
     7801 gatcccagcc ctgtgtataa ctcactactt tagtcagttc cgcagtatta caaaaggatg
     7861 tcgcaaacgc tgtttgctcc tctacaaaac agaccttaaa accctaaagg cttaagtagc
     7921 accctcgcaa gctcgggcaa atcgctgaat attccttttg tctccaacca tcaggcacct
     7981 gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc acggctctgg cagtgaatgg
     8041 gggtaaatgg cactacaggc gccttttatg gattcatgca aggaaactac ccataataca
     8101 agaaaagccc gtcacgggct tctcagggcg ttttatggcg ggtctgctat gtggtgctat
     8161 ctgacttttt gctgttcagc agttcctgcc ctctgatttt ccagtctgac cacttcggat
     8221 tatcccgtga caggtcattc agactggcta atgcacccag taaggcagcg gtatcatcaa
     8281 caggcttacc cgtcttactg tc
//
