LOCUS       sequence-254730-        7601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A nuclear plasmid encoding an error-prone mutant TP-DNAP1 (I777K, 
            L900S) for OrthoRep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7601)
  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 7601)
  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-Ec611
FEATURES             Location/Qualifiers
     source          1..7601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      3726..3891
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3781..3805)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     rep_origin      complement(3934..4522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4014..4033)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4736..4755)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4747..5562
                     /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             4747..5562
                     /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(4820..4839)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    5600..6103
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6144..6162
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6200..6222)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6322..6341
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6534..6720
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             6721..7383
                     /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"
ORIGIN
        1 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat cagggagctc gtgttgttat
       61 ccgatacaac cggatatttt tcttttaatg agtctaaacc gtgatagctt caggttaata
      121 caatcaaaaa aagctcaaat attcttttaa tgccgcgttc acagattcca attgaataca
      181 actaggtagt tcattatatg aagcctttgc tactattttt cactatagtc tgccttcacc
      241 ttaatgcaga catccacata ttttaatcac tttaaaataa aaaggaagat atattagaag
      301 ctatgatcca atctgtaagc cagattaaaa ttcacgaact cttctttcat ttgaattgaa
      361 tgctttgagt tggggtagat tatcgcaaat tactcatcac atttattgac tacgaacttg
      421 ctgatgtcct ttttttattt atatttttct tcagtgaagc gatttttttt tacacagacc
      481 aagacggaaa aaagtagcta aggaagaaaa caaaatcatg aaaaaaatgt gaagtgatca
      541 tgcacatcgc atcaacttaa acattggctt agagatatat agagttagag tttacggcaa
      601 cctttaagca ccaatacctt ttggcatagt ctaaagacct ggttcttaat tttaaacaaa
      661 tttaactaaa gatttcccta tcaaagaagt aacgagttga cagattttct caaaataaat
      721 cgatactgca tttctaggca tatccagcga gatctatgga ctacaaagac aaagcattaa
      781 acgacttgag aaacgtatac gcagacttcg actccttgcc attggacttc agacaaatct
      841 taattaagga tagagctaca ttgttgcaaa aggaagacgt tgaaaagaaa attttggaaa
      901 gacaagaaga tgccaaaaag tacgctgaat acttgaagca atcagaaatc ccagaaagaa
      961 tctccttacc taacatcaag agacataagg gtgtatcaat ctcctttgaa gaaacatctg
     1021 aagatatggt cttggaacca agacctttta tcttcgacgg tttgaacatc agatgtttca
     1081 gaagagaaac catattcagt ttgaaaaata agattttgaa catggttaag gaatcttcat
     1141 cctttaaaaa cgtttcaaga caaagtgtat cttttatgta cttcaaaatt tttaataagg
     1201 gtaaagttat tgcaagtaca aagtctgtta acatctatga agataagatc gacgaaagat
     1261 tggaagattt gtgtaacaac ttcgatgacg tattgaaaaa gattatagac gtcacctatg
     1321 gttacgaatc cttattcgtt agtgaaactt actcttacgt aatattctat gctaaatcaa
     1381 tctactttcc acaacctaga tgcgttaaca actggggtaa caacatccca aacatcttga
     1441 cattcgattc ctttaagctt ttcaccgcaa acaaaaacaa cgttagttgt atcaagcaat
     1501 gttcaagatt tttgtggcaa aaagatttca acactttaga agaaatgata gaatacaaga
     1561 acggtaatat ttgtatcgtc acaccacaat tgcatatcaa cgatgttaga gacatcaagt
     1621 cattcaacga tatcagattg tactcagaat cccctattaa aacattttcc gttattgata
     1681 acactatcac atatttgttt tacttcaagg aacatttggg tgttattttt aacatcacta
     1741 agtcaagaca tgatagaaga gtaacaaaat tttcaccatt gtccaagttc agtgacgtca
     1801 aaaatatcac tgtttgtttc gatatcgaaa gttacttcga cccagaaaag gaatctaacc
     1861 aagttaacat ccctttcatt tgttgcgctt caatcatcta taataaggtt attggtaaca
     1921 tcgtcgattt tgaaggtaga gactgtgtcg cacaaatgat cgaatacgtt gtagatattt
     1981 gcggtgaatt gaacataagt tctgttgaat tgatcgccca taacggtggt ggttatgatt
     2041 tccactacat cttgtcatcc atgtataatc cagctgcaat taaaaacata ttgatcagaa
     2101 acaactcttt tatttctttt aatttcgcac atgatggtgt caagttctct gttaaggaca
     2161 gttactcatt tttgttgtgt agtttggcca acgcttctaa agcattttta aacgaagaaa
     2221 cttttaagaa aactgatttc cctcatcacg acttgaaaac tgccgatgac ttatataagg
     2281 tctacaagga atggagttct gttaacacag aaattaacca tgtcgttgaa aaggaaaagt
     2341 tgttgatcac ctctgaacac atcgttaact tcaccaagaa cgataagagt aagactttga
     2401 tcgaatggtc taaggattac tgtagaaacg acgtattggt cttgtctaag gtttggttgg
     2461 agtttaaaaa cgctgtagaa gatattttta actgcgaatt ggtagaccaa accatgactt
     2521 tggctggttt gtcatacaag ttgttccaag caaacatgcc attcgatgtc gaattgagac
     2581 atcctaataa ggaagactac ttcaacatga gagaagcatt gattggtggt agatgtatct
     2641 ctgttaacgg tatctacaag gatgttttgt gcttagacgt aaaatcatta tatccagctt
     2701 ctatggcttt ctatgatcaa ccttacggtt cttttaagag agtttcatcc agaccaaaag
     2761 atgaattggg tatctattac gttagagtaa ccccaaacag aaacaacaag tcaaactttt
     2821 tccctatcag atcccataat aagatcactt acaacaactt cgaagaatca acttatattg
     2881 cttggtacac aaacgttgat atcgacattg gtttgtcaga aggtcacaac atcgaataca
     2941 tccctttcga ttcctatggt aacattggtt actcatggtc caaaaagggt aaaatttttg
     3001 aaaagtacat caaggatgtt ttgtacaaat tgaagataaa atatgaaaag caaaataaca
     3061 aggtaaaaag aaatgtcatc aaaaagatca tgaactcatt gtggggtaaa ttcgcccaaa
     3121 agtgggttaa cttcgaatac ttcatcaagt cagaagatga cattgatttc gaatccgaag
     3181 aagcatataa aatttgggat acagacttca tgttgatcaa aaagattaag gaatctacct
     3241 acagttctaa gccaatacaa aacggtgttt ttactttaag ttgggccaga tatcacatga
     3301 agtctatttg ggatgcaggt gccaaagaag gtgctgaatg tatctattca gatacagact
     3361 ccatattcgt tcataaggaa cacttcaaaa agaatgcaaa gtttatgttg aacggtttga
     3421 aagtaccaat catcggttct gaagtcggtc aatctgaatt agaatgtgaa tttgataaat
     3481 tgttgtgcgc cggtaaaaag caatacatgg gtttctacac ttacttccaa gatggtaaac
     3541 cttgcatcaa ggaaaagaaa agattcaaag gtataccaag taactacatc atacctgaat
     3601 tgtatgctca cttgttatct ggtgcagata aggaagccaa aattcaattt ttaaaattca
     3661 gaagagaatg gggttccgtc aagggttaca tagaaaataa gactgtcaaa gccacctaac
     3721 tcgaggcgaa tttcttatga tttatgattt ttattattaa ataagttata aaaaaaataa
     3781 gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta ttcttgagta
     3841 actctttcct gtaggtcagg ttgctttctc aggtatagca tgaggtcgct cactagtcca
     3901 tggacgcgtg gtaccggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3961 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4021 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4081 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4141 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4201 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4261 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4321 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4381 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4441 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4501 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4561 gtggaacgaa aactcacgtt aagggatttt ggtcatgact gatccttcaa ctcagcaaaa
     4621 gttcgattta ttcaacaaag ccacgttgtg tctcaaaatc tctgatgtta cattgcacaa
     4681 gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     4741 ggtgttatga gccatattca acgggaaacg tcttgctcca ggccgcgatt aaattccaac
     4801 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     4861 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     4921 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     4981 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     5041 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     5101 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     5161 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     5221 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     5281 tggaaagaaa tgcataagct tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     5341 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     5401 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     5461 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     5521 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg
     5581 ttgtaacacg gtccttttca tcacgtgcta taaaaataat tataatttaa attttttaat
     5641 ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg
     5701 ttttccgaag atgtaaaaga ctctaggggg atcgccaaca aatactacct tttatcttgc
     5761 tcttcctgct ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta gaagaccaca
     5821 cacgaaaatc ctgtgatttt acattttact tatcgttaat cgaatgtata tctatttaat
     5881 ctgcttttct tgtctaataa atatatatgt aaagtacgct ttttgttgaa attttttaaa
     5941 cctttgttta tttttttttc ttcattccgt aactcttcta ccttctttat ttactttcta
     6001 aaatccaaat acaaaacata aaaataaata aacacagagt aaattcccaa attattccat
     6061 cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg atccgtctaa gaaaccatta
     6121 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     6181 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     6241 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagtgggt gttggcgggt
     6301 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatacca
     6361 ccttttcaat tcatcatttt ttttttattc ttttttttga ttttaattcc gttttaagag
     6421 cttggtgagc gctaggagtc actgccaggt atcgtttgaa cacggcatta gtcagggaag
     6481 tcataacaca gtcctttccc gcaattttct ttttctatta ctcttggcct cctctagtac
     6541 actctatatt tttttatgcc tcggtaatga ttttcatttt ttttttccac ctagcggatg
     6601 actctttttt tttcttagcg attggcatta tcacataatg aattatacat tatataaagt
     6661 aatgtgattt cttcgaagaa tatactaaaa aatgagcagg caagataaac gaaggcaaag
     6721 atgacagagc agaaagccct agtaaagcgt attacaaatg aaaccaagat tcagattgcg
     6781 atctctttaa agggtggtcc cctagcgata gagcactcga tcttcccaga aaaagaggca
     6841 gaagcagtag cagaacaggc cacacaatcg caagtgatta acgtccacac aggtataggg
     6901 tttctggacc atatgataca tgctctggcc aagcattccg gctggtcgct aatcgttgag
     6961 tgcattggtg acttacacat agacgaccat cacaccactg aagactgcgg gattgctctc
     7021 ggtcaagctt ttaaagaggc cctaggggcc gtgcgtggag taaaaaggtt tggatcagga
     7081 tttgcgcctt tggatgaggc actttccaga gcggtggttg atctttcgaa caggccgtac
     7141 gcagttgtcg aacttggttt gcaaagggag aaagtaggtg atctctcttg cgagatgatc
     7201 ccgcattttc ttgaaagctt tgcagaggct agcagaatta ccctccacgt tgattgtctg
     7261 cgaggcaaga atgatcatca ccgtagtgag agtgcgttca aggctcttgc ggttgccata
     7321 agagaagcca cctcgcccaa tggtaccaac gatgttccct ccaccaaagg tgttcttatg
     7381 tagtgacacc gattatttaa agctgctgca tacgatatat atacatgtgt atatatgtat
     7441 acctatgaat gtcagtaagt atgtatacga acagtatgat actgaagatg acaaggtaat
     7501 gcatcattct atacgtgtca ttctgaacga ggcgcgcttt ccttttttct ttttgctttt
     7561 tctttttttt tctcttgaac tcgacggatc atatatgcgg t
//
