LOCUS       sequence-192376-        8337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Accessory plasmid for PACE of MjTyrRS variants; negative selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8337)
  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 8337)
  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: pDB036a
FEATURES             Location/Qualifiers
     source          1..8337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             262..1536
                     /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(2219..2236)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2389..2475
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2567..2594
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(join(5549..6340,6341..6409))
                     /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     6172..6191
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6410..6514)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6582..6600
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(6621..7571)
                     /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      7619..7841
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttgctgagg agacttaggg
       61 accctgatga aattcgccac ttgttagtgt aattcgctaa ctcatcctgg catgttgctg
      121 ttgattcttc aatcagatct ttataaatca aaaagataaa aaattggcac gcaaattgta
      181 ttaacagttc agcaggacaa tcctgaacgc agaaatcaag aggacaacat tacctgcagg
      241 tgcagtaagg aggaaaaaaa aatgaaaaaa ttattattcg caattccttt agttgttcct
      301 ttctattctc actccgctga aactgttgaa agttgtttag caaaacccca tacagaaaat
      361 tcatttacta acgtctggaa agacgacaaa actttagatc gttacgctaa ctatgagggc
      421 tgtctgtgga atgctacagg cgttgtagtt tgtactggtg acgaaactca gtgttacggt
      481 acatgggttc ctattgggct tgctatccct gaaaatgagg gtggtggctc tgagggtggc
      541 ggttctgagg gtggcggttc tgagggtggc ggtactaaac ctcctgagta cggtgataca
      601 cctattccgg gctatactta tatcaaccct ctcgacggca cttatccgcc tggtactgag
      661 caaaaccccg ctaatcctaa tccttctctt gaggagtctc agcctcttaa tactttcatg
      721 tttcagaata ataggttccg aaataggcag ggggcattaa ctgtttatac gggcactgtt
      781 actcaaggca ctgaccccgt taaaacttat taccagtaca ctcctgtatc atcaaaagcc
      841 atgtatgacg cttactggaa cggtaaattc agagactgcg ctttccattc tggctttaat
      901 gaggatccat tcgtttgtga atatcaaggc caatcgtctg acctgcctca acctcctgtc
      961 aatgctggcg gcggctctgg tggtggttct ggtggcggct ctgagggtgg tggctctgag
     1021 ggtggcggtt ctgagggtgg cggctctgag ggaggcggtt ccggtggtgg ctctggttcc
     1081 ggtgattttg attatgaaaa gatggcaaac gctaataagg gggctatgac cgaaaatgcc
     1141 gatgaaaacg cgctacagtc tgacgctaaa ggcaaacttg attctgtcgc tactgattac
     1201 ggtgctgcta tcgatggttt cattggtgac gtttccggcc ttgctaatgg taatggtgct
     1261 actggtgatt ttgctggctc taattcccaa atggctcaag tcggtgacgg tgataattca
     1321 cctttaatga ataatttccg tcaatattta ccttccctcc ctcaatcggt tgaatgtcgc
     1381 ccttttgtct ttggcgctgg taaaccttac gagttcagta tcgactgcga taagatcaac
     1441 ctgttccgcg gtgtctttgc gtttctttta tatgttgcca cctttatgta tgtattttct
     1501 acgtttgcta acatactgcg taataaggag tcttaatctt gagtaacgga gccaatgtac
     1561 gcaaaaaacc ccgcttcagc ggggtttttt cgccaaaaaa aaccccgccc tgtcaggggc
     1621 ggggtttttt tttagggccc tactgactgt ccgtgtgctt ctcaaatgcc tgaggccagt
     1681 ttgctcaggc tctccccgtg gaggtaataa ttgacgatat gatcagtgca cggctaacta
     1741 agcggcctgc tgactttctc gccgatcaaa aggcattttg ctattaaggg attgacgagg
     1801 gcgtatctgc gcagtaagat gcgccccgca ttccggcggt agttcagcag ggcagaacgg
     1861 cggactctaa atccgcatgg caggggttca aatcccctcc gccggaccaa attcgaaaag
     1921 cctgctcaac gagcaggctt ttttgcatgc tcgagcagct cagggtcgaa tttgctttcg
     1981 aatttctgcc attcatccgc ttattatcac ttattcaggc gtagcaacca ggcgtttaag
     2041 ggcaccaata actgccttaa aaaaattacg ccccgccctg ccactcatcg cagtactgtt
     2101 gtaattcatt aagcattctg ccgacgttca tcggatcccg ggcccgtcga ctgcagaggc
     2161 ctgcatgcaa gcttggcgta atcagtggct gttttggcgg atgagagaag attttcagcc
     2221 tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca
     2281 gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaactc cgtagcgccg
     2341 atggtagtgt ggggtctccc catgcgagag tagggaactg ccagtcatca aataaaacga
     2401 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     2461 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
     2521 tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg
     2581 acggatggcc tttttgcgtt tctacaaact cgctgaggat tctagagcac agctaacacc
     2641 acgtcgtccc tatctgctgc cctaggtcta tgagtggttg ctggataact ttacgggcat
     2701 gcataaggct cgtataatat attcagggag accacaacgg tttccctcta caaataattt
     2761 tgtttaactt ttactagagt aaggaggaaa aatgaacacg attaacatcg ctaagaacga
     2821 cttctaggac atcgaactgg ctgctatccc gttcaacact ctggctgacc attacggtga
     2881 gcgtttagct cgcgaacagt tggcccttga gcatgagtct tacgagatgg gtgaagcacg
     2941 cttccgcaag atgtttgagc gtcaacttaa agctggtgag gttgcggata acgctgccgc
     3001 caagcctctc atcactaccc tactccctaa gatgattgca cgcatcaacg actggtttga
     3061 ggaagtgaaa gctaagcgcg gcaagcgccc gacagccttc cagttcctgc aagaaatcaa
     3121 gccggaagcc gtagcgtaca tcaccattaa gaccactctg gcttgcctaa ccagtgctga
     3181 caatacaacc gttcaggctg tagcaagcgc aatcggtcgg gccattgagg acgaggctcg
     3241 cttcggtcgt atccgtgacc ttgaagctaa gcacttcaag aaaaacgttg aggaacaact
     3301 caacaagcgc gtagggcacg tctacaagaa agcatttatg caagttgtcg aggctgacat
     3361 gctctctaag ggtctactcg gtggcgaggc gtggtcttag tggcataagg aagactctat
     3421 tcatgtagga gtacgctgca tcgagatgct cattgagtca accggaatgg ttagcttaca
     3481 ccgccaaaat gctggcgtag taggtcaaga ctctgagact atcgaactcg cacctgaata
     3541 cgctgaggct atcgcaaccc gtgcaggtgc gctggctggc atctctccga tgttccaacc
     3601 ttgcgtagtt cctcctaagc cgtggactgg cattactggt ggtggctatt gggctaacgg
     3661 tcgtcgtcct ctggcgctgg tgcgtactca cagtaagaaa gcactgatgc gctacgaaga
     3721 cgtttacatg cctgaggtgt acaaagcgat taacattgcg caaaacaccg catggaaaat
     3781 caacaagaaa gtcctagcgg tcgccaacgt aatcaccaag tggaagcatt gtccggtcga
     3841 ggacatccct gcgattgagc gtgaagaact cccgatgaaa ccggaagaca tcgacatgaa
     3901 tcctgaggct ctcaccgcgt ggaaacgtgc tgccgctgct gtgtaccgca aggacaaggc
     3961 tcgcaagtct cgccgtatca gccttgagtt catgcttgag caagccaata agtttgctaa
     4021 ccataaggcc atctggttcc cttacaacat ggactggcgc ggtcgtgttt acgctgtgtc
     4081 aatgttcaac ccgcaaggta acgatatgac caaaggactg cttacgctgg cgaaaggtaa
     4141 accaatcggt aaggaaggtt actactggct gaaaatccac ggtgcaaact gtgcgggtgt
     4201 cgataaggtt ccgttccctg agcgcatcaa gttcattgag gaaaaccacg agaacatcat
     4261 ggcttgcgct aagtctccac tggagaacac ttggtgggct gagcaagatt ctccgttctg
     4321 cttccttgcg ttctgctttg agtacgctgg ggtacagcac cacggcctga gctataactg
     4381 ctcccttccg ctggcgtttg acgggtcttg ctctggcatc cagcacttct ccgcgatgct
     4441 ccgagatgag gtaggtggtc gcgcggttaa cttgcttcct agtgaaaccg ttcaggacat
     4501 ctacgggatt gttgctaaga aagtcaacga gattctacaa gcagacgcaa tcaatgggac
     4561 cgataacgaa gtagttaccg tgaccgatga gaacactggt gaaatctctg agaaagtcaa
     4621 gctgggcact aaggcactgg ctggtcaatg gctggcttac ggtgttactc gcagtgtgac
     4681 taagcgttca gtcatgacgc tggcttacgg gtccaaagag ttcggcttcc gtcaacaagt
     4741 gctggaagat accattcagc cagctattga ttccggcaag ggtctgatgt tcactcagcc
     4801 gaatcaggct gctggataca tggctaagct gatttgggaa tctgtgagcg tgacggtggt
     4861 agctgcggtt gaagcaatga actggcttaa gtctgctgct aagctgctgg ctgctgaggt
     4921 caaagataag aagactggag agattcttcg caagcgttgc gctgtgcatt gggtaactcc
     4981 tgatggtttc cctgtgtggc aggaatacaa gaagcctatt cagacgcgct tgaacctgat
     5041 gttcctcggt cagttccgct tacagcctac cattaacacc aacaaagata gcgagattga
     5101 tgcacacaaa caggagtctg gtatcgctcc taactttgta cacagccaag acggtagcca
     5161 ccttcgtaag actgtagtgt gggcacacga gaagtacgga atcgaatctt ttgcactgat
     5221 tcacgactcc ttcggtacca ttccggctga cgctgcgaac ctgttcaaag cagtgcgcga
     5281 aactatggtt gacacatatg agtcttgtga tgtactggct gatttctacg accagttcgc
     5341 tgaccagttg cacgagtctc aattggacaa aatgccagca cttccggcta aaggtaactt
     5401 gaacctccgt gacatcttag agtcggactt cgcgttcgcg taacttaatt aacggcactg
     5461 cgaaaaaacc ccgccgaagc ggggtttttt gcgaaatata atgaccctct tgataaccca
     5521 agagggcatt ttttaatgcc catggcgttt accaatgctt aatcagtgag gcacctatct
     5581 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5641 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5701 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagcg
     5761 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5821 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5881 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5941 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6001 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6061 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6121 gagaatagtg tatgcggcga ccgagttgct cttgcccagc gtcaatacgg gataataccg
     6181 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6241 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6301 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6361 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6421 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6481 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6541 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     6601 ccttaggcca aataggccgt tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg
     6661 gttcgttgtt tttgcgtgag ccatgagaac gaaccattga gatcatgctt actttgcatg
     6721 tcactcaaaa attttgcctc aaaactggtg agctgaattt ttgcagttaa agcatcgtgt
     6781 agtgtttttc ttagtccgtt acgtaggtag gaatctgatg taatggttgt tggtattttg
     6841 tcaccattca tttttatctg gttgttctca agttcggtta cgagatccat ttgtctatct
     6901 agttcaactt ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac caccaatttc
     6961 atattgctgt aagtgtttaa atctttactt attggtttca aaacccattg gttaagcctt
     7021 ttaaactcat ggtagttatt ttcaagcatt aacatgaact taaattcatc aaggctaatc
     7081 tctatatttg ccttgtgagt tttcttttgt gttagttctt ttaataacca ctcataaatc
     7141 ctcatagagt atttgttttc aaaagactta acatgttcca gattatattt tatgaatttt
     7201 tttaactgga aaagataagg caatatctct tcactaaaaa ctaattctaa tttttcgctt
     7261 gagaacttgg catagtttgt ccactggaaa atctcaaagc ctttaaccaa aggattcctg
     7321 atttccacag ttctcgtcat cagctctctg gttgctttag ctaatacacc ataagcattt
     7381 tccctactga tgttcatcat ctgagcgtat tggttataag tgaacgatac cgtccgttct
     7441 ttccttgtag ggttttcaat cgtggggttg agtagtgcca cacagcataa aattagcttg
     7501 gtttcatgct ccgttaagtc atagcgacta atcgctagtt catttgcttt gaaaacaact
     7561 aattcagaca tacatctcaa ttggtctagg tgattttaat cactatacca attgagatgg
     7621 gctagtcaat gataattact agtccttttc ctttgagttg tgggtatctg taaattctgc
     7681 tagacctttg ctggaaaact tgtaaattct gctagaccct ctgtaaattc cgctagacct
     7741 ttgtgtgttt tttttgttta tattcaagtg gttataattt atagaataaa gaaagaataa
     7801 aaaaagataa aaagaataga tcccagccct gtgtataact cactacttta gtcagttccg
     7861 cagtattaca aaaggatgtc gcaaacgctg tttgctcctc tacaaaacag accttaaaac
     7921 cctaaaggct taagtagcac cctcgcaagc tcgggcaaat cgctgaatat tccttttgtc
     7981 tccgaccatc aggcacctga gtcgctgtct ttttcgtgac attcagttcg ctgcgctcac
     8041 ggctctggca gtgaatgggg gtaaatggca ctacaggcgc cttttatgga ttcatgcaag
     8101 gaaactaccc ataatacaag aaaagcccgt cacgggcttc tcagggcgtt ttatggcggg
     8161 tctgctatgt ggtgctatct gactttttgc tgttcagcag ttcctgccct ctgattttcc
     8221 agtctgacca cttcggatta tcccgtgaca ggtcattcag actggctaat gcacccagta
     8281 aggcagcggt atcatcaaca ggcttacccg tcttactgtc aagaggacat ccggtca
//
