LOCUS       sequence-254740-        7602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A nuclear plasmid encoding an error-prone mutant TP-DNAP1 (L477V, 
            L640Y, I777K, W814N) for OrthoRep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7602)
  AUTHORS   Ravikumar A, Arzumanyan GA, Obadi MKA, Javanpour AA, Liu CC
  TITLE     Scalable, Continuous Evolution of Genes at Mutation Rates above 
            Genomic Error Thresholds.
  JOURNAL   Cell. 2018 Dec 13;175(7):1946-1957.e13. doi: 
            10.1016/j.cell.2018.10.021. Epub 2018 Nov 8.
  PUBMED    30415839
REFERENCE   2  (bases 1 to 7602)
  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"; doi: 
            "10.1016/j.cell.2018.10.021"; journalName: "Cell"; date: 
            "2018-12-13- 13"; volume: "175"; issue: "7"; pages: "1946-1957.e13"
COMMENT     Sequence Label: pAR-Ec633
FEATURES             Location/Qualifiers
     source          1..7602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    271..774
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     815..833
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(871..893)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     993..1012
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1205..1391
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             1392..2054
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     terminator      5999..6164
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(6054..6078)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     rep_origin      complement(6207..6795)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6287..6306)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(7009..7028)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             join(7020..7602,1..233)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             join(7020..7602,1..233)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(7093..7112)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
       61 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
      121 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
      181 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat
      241 tggttaattg gttgtaacac ggtccttttc atcacgtgct ataaaaataa ttataattta
      301 aattttttaa tataaatata taaattaaaa atagaaagta aaaaaagaaa ttaaagaaaa
      361 aatagttttt gttttccgaa gatgtaaaag actctagggg gatcgccaac aaatactacc
      421 ttttatcttg ctcttcctgc tctcaggtat taatgccgaa ttgtttcatc ttgtctgtgt
      481 agaagaccac acacgaaaat cctgtgattt tacattttac ttatcgttaa tcgaatgtat
      541 atctatttaa tctgcttttc ttgtctaata aatatatatg taaagtacgc tttttgttga
      601 aattttttaa acctttgttt attttttttt cttcattccg taactcttct accttcttta
      661 tttactttct aaaatccaaa tacaaaacat aaaaataaat aaacacagag taaattccca
      721 aattattcca tcattaaaag atacgaggcg cgtgtaagtt acaggcaagc gatccgtcta
      781 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
      841 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
      901 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagtggg
      961 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     1021 gcaccatacc accttttcaa ttcatcattt tttttttatt cttttttttg attttaattc
     1081 cgttttaaga gcttggtgag cgctaggagt cactgccagg tatcgtttga acacggcatt
     1141 agtcagggaa gtcataacac agtcctttcc cgcaattttc tttttctatt actcttggcc
     1201 tcctctagta cactctatat ttttttatgc ctcggtaatg attttcattt tttttttcca
     1261 cctagcggat gactcttttt ttttcttagc gattggcatt atcacataat gaattataca
     1321 ttatataaag taatgtgatt tcttcgaaga atatactaaa aaatgagcag gcaagataaa
     1381 cgaaggcaaa gatgacagag cagaaagccc tagtaaagcg tattacaaat gaaaccaaga
     1441 ttcagattgc gatctcttta aagggtggtc ccctagcgat agagcactcg atcttcccag
     1501 aaaaagaggc agaagcagta gcagaacagg ccacacaatc gcaagtgatt aacgtccaca
     1561 caggtatagg gtttctggac catatgatac atgctctggc caagcattcc ggctggtcgc
     1621 taatcgttga gtgcattggt gacttacaca tagacgacca tcacaccact gaagactgcg
     1681 ggattgctct cggtcaagct tttaaagagg ccctaggggc cgtgcgtgga gtaaaaaggt
     1741 ttggatcagg atttgcgcct ttggatgagg cactttccag agcggtggtt gatctttcga
     1801 acaggccgta cgcagttgtc gaacttggtt tgcaaaggga gaaagtaggt gatctctctt
     1861 gcgagatgat cccgcatttt cttgaaagct ttgcagaggc tagcagaatt accctccacg
     1921 ttgattgtct gcgaggcaag aatgatcatc accgtagtga gagtgcgttc aaggctcttg
     1981 cggttgccat aagagaagcc acctcgccca atggtaccaa cgatgttccc tccaccaaag
     2041 gtgttcttat gtagtgacac cgattattta aagctgctgc atacgatata tatacatgtg
     2101 tatatatgta tacctatgaa tgtcagtaag tatgtatacg aacagtatga tactgaagat
     2161 gacaaggtaa tgcatcattc tatacgtgtc attctgaacg aggcgcgctt tccttttttc
     2221 tttttgcttt ttcttttttt ttctcttgaa ctcgacggat catatatgcg gtgtgaaata
     2281 ccgcacagat gcgtaaggag aaaataccgc atcagggagc tcgtgttgtt atccgataca
     2341 accggatatt tttcttttaa tgagtctaaa ccgtgatagc ttcaggttaa tacaatcaaa
     2401 aaaagctcaa atattctttt aatgccgcgt tcacagattc caattgaata caactaggta
     2461 gttcattata tgaagccttt gctactattt ttcactatag tctgccttca ccttaatgca
     2521 gacatccaca tattttaatc actttaaaat aaaaaggaag atatattaga agctatgatc
     2581 caatctgtaa gccagattaa aattcacgaa ctcttctttc atttgaattg aatgctttga
     2641 gttggggtag attatcgcaa attactcatc acatttattg actacgaact tgctgatgtc
     2701 ctttttttat ttatattttt cttcagtgaa gcgatttttt ttttacacag accaagacgg
     2761 aaaaaagtag ctaaggaaga aaacaaaatc atgaaaaaaa tgtgaagtga tcatgcacat
     2821 cgcatcaact taaacattgg cttagagata tatagagtta gagtttacgg caacctttaa
     2881 gcaccaatac cttttggcat agtctaaaga cctggttctt aattttaaac aaatttaact
     2941 aaagatttcc ctatcaaaga agtaacgagt tgacagattt tctcaaaata aatcgatact
     3001 gcatttctag gcatatccag cgagatctat ggactacaaa gacaaagcat taaacgactt
     3061 gagaaacgta tacgcagact tcgactcctt gccattggac ttcagacaaa tcttaattaa
     3121 ggatagagct acattgttgc aaaaggaaga cgttgaaaag aaaattttgg aaagacaaga
     3181 agatgccaaa aagtacgctg aatacttgaa gcaatcagaa atcccagaaa gaatctcctt
     3241 acctaacatc aagagacata agggtgtatc aatctccttt gaagaaacat ctgaagatat
     3301 ggtcttggaa ccaagacctt ttatcttcga cggtttgaac atcagatgtt tcagaagaga
     3361 aaccatattc agtttgaaaa ataagatttt gaacatggtt aaggaatctt catcctttaa
     3421 aaacgtttca agacaaagtg tatcttttat gtacttcaaa atttttaata agggtaaagt
     3481 tattgcaagt acaaagtctg ttaacatcta tgaagataag atcgacgaaa gattggaaga
     3541 tttgtgtaac aacttcgatg acgtattgaa aaagattata gacgtcacct atggttacga
     3601 atccttattc gttagtgaaa cttactctta cgtaatattc tatgctaaat caatctactt
     3661 tccacaacct agatgcgtta acaactgggg taacaacatc ccaaacatct tgacattcga
     3721 ttcctttaag cttttcaccg caaacaaaaa caacgttagt tgtatcaagc aatgttcaag
     3781 atttttgtgg caaaaagatt tcaacacttt agaagaaatg atagaataca agaacggtaa
     3841 tatttgtatc gtcacaccac aattgcatat caacgatgtt agagacatca agtcattcaa
     3901 cgatatcaga ttgtactcag aatcccctat taaaacattt tccgttattg ataacactat
     3961 cacatatttg ttttacttca aggaacattt gggtgttatt tttaacatca ctaagtcaag
     4021 acatgataga agagtaacaa aattttcacc attgtccaag ttcagtgacg tcaaaaatat
     4081 cactgtttgt ttcgatatcg aaagttactt cgacccagaa aaggaatcta accaagttaa
     4141 catccctttc atttgttgcg cttcaatcat ctataataag gttattggta acatcgtcga
     4201 ttttgaaggt agagactgtg tcgcacaaat gatcgaatac gttgtagata tttgcggtga
     4261 attgaacata agttctgttg aattgatcgc ccataacggt ggtggttatg atttccacta
     4321 catcttgtca tccatgtata atccagctgc aattaaaaac atattgatca gaaacaactc
     4381 ttttatttct tttaatttcg cacatgatgg tgtcaagttc tctgttaagg acagttactc
     4441 atttttgttg tgtagtgttg ccaacgcttc taaagcattt ttaaacgaag aaacttttaa
     4501 gaaaactgat ttccctcatc acgacttgaa aactgccgat gacttatata aggtctacaa
     4561 ggaatggagt tctgttaaca cagaaattaa ccatgtcgtt gaaaaggaaa agttgttgat
     4621 cacctctgaa cacatcgtta acttcaccaa gaacgataag agtaagactt tgatcgaatg
     4681 gtctaaggat tactgtagaa acgacgtatt ggtcttgtct aaggtttggt tggagtttaa
     4741 aaacgctgta gaagatattt ttaactgcga attggtagac caaaccatga ctttggctgg
     4801 tttgtcatac aagttgttcc aagcaaacat gccattcgat gtcgaattga gacatcctaa
     4861 taaggaagac tacttcaaca tgagagaagc attgattggt ggtagatgta tctctgttaa
     4921 cggtatctac aaggatgttt tgtgctatga cgtaaaatca ttatatccag cttctatggc
     4981 tttctatgat caaccttacg gttcttttaa gagagtttca tccagaccaa aagatgaatt
     5041 gggtatctat tacgttagag taaccccaaa cagaaacaac aagtcaaact ttttccctat
     5101 cagatcccat aataagatca cttacaacaa cttcgaagaa tcaacttata ttgcttggta
     5161 cacaaacgtt gatatcgaca ttggtttgtc agaaggtcac aacatcgaat acatcccttt
     5221 cgattcctat ggtaacattg gttactcatg gtccaaaaag ggtaaaattt ttgaaaagta
     5281 catcaaggat gttttgtaca aattgaagat aaaatatgaa aagcaaaata acaaggtaaa
     5341 aagaaatgtc atcaaaaaga tcatgaactc attgtggggt aaattcgccc aaaagtgggt
     5401 taacttcgaa tacttcatca agtcagaaga tgacattgat ttcgaatccg aagaagcata
     5461 taaaattaac gatacagact tcatgttgat caaaaagatt aaggaatcta cctacagttc
     5521 taagccaata caaaacggtg tttttacttt aagttgggcc agatatcaca tgaagtctat
     5581 ttgggatgca ggtgccaaag aaggtgctga atgtatctat tcagatacag actccatatt
     5641 cgttcataag gaacacttca aaaagaatgc aaagtttatg ttgaacggtt tgaaagtacc
     5701 aatcatcggt tctgaagtcg gtcaattgga attagaatgt gaatttgata aattgttgtg
     5761 cgccggtaaa aagcaataca tgggtttcta cacttacttc caagatggta aaccttgcat
     5821 caaggaaaag aaaagattca aaggtatacc aagtaactac atcatacctg aattgtatgc
     5881 tcacttgtta tctggtgcag ataaggaagc caaaattcaa tttttaaaat tcagaagaga
     5941 atggggttcc gtcaagggtt acatagaaaa taagactgtc aaagccacct aactcgaggc
     6001 gaatttctta tgatttatga tttttattat taaataagtt ataaaaaaaa taagtgtata
     6061 caaattttaa agtgactctt aggttttaaa acgaaaattc ttattcttga gtaactcttt
     6121 cctgtaggtc aggttgcttt ctcaggtata gcatgaggtc gctcactagt ccatggacgc
     6181 gtggtaccgg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     6241 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6301 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6361 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6421 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6481 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6541 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6601 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6661 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6721 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6781 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6841 gaaaactcac gttaagggat tttggtcatg actgatcctt caactcagca aaagttcgat
     6901 ttattcaaca aagccacgtt gtgtctcaaa atctctgatg ttacattgca caagataaaa
     6961 atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta
     7021 tgagccatat tcaacgggaa acgtcttgct ccaggccgcg attaaattcc aacatggatg
     7081 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
     7141 atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
     7201 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc
     7261 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
     7321 tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg
     7381 ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt
     7441 ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg
     7501 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     7561 aaatgcataa gcttttgcca ttctcaccgg attcagtcgt ca
//
