LOCUS       sequence-250432-        7596 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 7596)
  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 7596)
  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..7596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      3683..3848
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3738..3762)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     rep_origin      complement(3891..4479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4693..4712)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4704..5519
                     /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             4704..5519
                     /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(4777..4796)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    5557..6060
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6102..6120
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6158..6180)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6280..6299
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6484..6671
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             6672..7334
                     /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 gagctcgtgt tgttatccga tacaaccgga tatttttctt ttaatgagtc taaaccgtga
       61 tagcttcagg ttaatacaat caaaaaaagc tcaaatattc ttttaatgcc gcgttcacag
      121 attccaattg aatacaacta ggtagttcat tatatgaagc ctttgctact atttttcact
      181 atagtctgcc ttcaccttaa tgcagacatc cacatatttt aatcacttta aaataaaaag
      241 gaagatatat tagaagctat gatccaatct gtaagccaga ttaaaattca cgaactcttc
      301 tttcatttga attgaatgct ttgagttggg gtagattatc gcaaattact catcacattt
      361 attgactacg aacttgctga tgtccttttt ttatttatat ttttcttcag tgaagcgatt
      421 ttttttttac acagaccaag acggaaaaaa gtagctaagg aagaaaacaa aatcatgaaa
      481 aaaatgtgaa gtgatcatgc acatcgcatc aacttaaaca ttggcttaga gatatataga
      541 gttagagttt acggcaacct ttaagcacca ataccttttg gcatagtcta aagacctggt
      601 tcttaatttt aaacaaattt aactaaagat ttccctatca aagaagtaac gagttgacag
      661 attttctcaa aataaatcga tactgcattt ctaggcatat ccagcgagat ctatggacta
      721 caaagacaaa gcattaaacg acttgagaaa cgtatacgca gacttcgact ccttgccatt
      781 ggacttcaga caaatcttaa ttaaggatag agctacattg ttgcaaaagg aagacgttga
      841 aaagaaaatt ttggaaagac aagaagatgc caaaaagtac gctgaatact tgaagcaatc
      901 agaaatccca gaaagaatct ccttacctaa catcaagaga cataagggtg tatcaatctc
      961 ctttgaagaa acatctgaag atatggtctt ggaaccaaga ccttttatct tcgacggttt
     1021 gaacatcaga tgtttcagaa gagaaaccat attcagtttg aaaaataaga ttttgaacat
     1081 ggttaaggaa tcttcatcct ttaaaaacgt ttcaagacaa agtgtatctt ttatgtactt
     1141 caaaattttt aataagggta aagttattgc aagtacaaag tctgttaaca tctatgaaga
     1201 taagatcgac gaaagattgg aagatttgtg taacaacttc gatgacgtat tgaaaaagat
     1261 tatagacgtc acctatggtt acgaatcctt attcgttagt gaaacttact cttacgtaat
     1321 attctatgct aaatcaatct actttccaca acctagatgc gttaacaact ggggtaacaa
     1381 catcccaaac atcttgacat tcgattcctt taagcttttc accgcaaaca aaaacaacgt
     1441 tagttgtatc aagcaatgtt caagattttt gtggcaaaaa gatttcaaca ctttagaaga
     1501 aatgatagaa tacaagaacg gtaatatttg tatcgtcaca ccacaattgc atatcaacga
     1561 tgttagagac atcaagtcat tcaacgatat cagattgtac tcagaatccc ctattaaaac
     1621 attttccgtt attgataaca ctatcacata tttgttttac ttcaaggaac atttgggtgt
     1681 tatttttaac atcactaagt caagacatga tagaagagta acaaaatttt caccattgtc
     1741 caagttcagt gacgtcaaaa atatcactgt ttgtttcgat atcgaaagtt acttcgaccc
     1801 agaaaaggaa tctaaccaag ttaacatccc tttcatttgt tgcgcttcaa tcatctataa
     1861 taaggttatt ggtaacatcg tcgattttga aggtagagac tgtgtcgcac aaatgatcga
     1921 atacgttgta gatatttgcg gtgaattgaa cataagttct gttgaattga tcgcccataa
     1981 cggtggtggt tatgatttcc actacatctt gtcatccatg tataatccag ctgcaattaa
     2041 aaacatattg atcagaaaca actcttttat ttcttttaat ttcgcacatg atggtgtcaa
     2101 gttctctgtt aaggacagtt actcattttt gttgtgtagt ttggccaacg cttctaaagc
     2161 atttttaaac gaagaaactt ttaagaaaac tgatttccct catcacgact tgaaaactgc
     2221 cgatgactta tataaggtct acaaggaatg gagttctgtt aacacagaaa ttaaccatgt
     2281 cgttgaaaag gaaaagttgt tgatcacctc tgaacacatc gttaacttca ccaagaacga
     2341 taagagtaag actttgatcg aatggtctaa ggattactgt agaaacgacg tattggtctt
     2401 gtctaaggtt tggttggagt ttaaaaacgc tgtagaagat atttttaact gcgaattggt
     2461 agaccaaacc atgactttgg ctggtttgtc atacaagttg ttccaagcaa acatgccatt
     2521 cgatgtcgaa ttgagacatc ctaataagga agactacttc aacatgagag aagcattgat
     2581 tggtggtaga tgtatctctg ttaacggtat ctacaaggat gttttgtgct tagacgtaaa
     2641 atcattatat ccagcttcta tggctttcta tgatcaacct tacggttctt ttaagagagt
     2701 ttcatccaga ccaaaagatg aattgggtat ctattacgtt agagtaaccc caaacagaaa
     2761 caacaagtca aactttttcc ctatcagatc ccataataag atcacttaca acaacttcga
     2821 agaatcaact tatattgctt ggtacacaaa cgttgatatc gacattggtt tgtcagaagg
     2881 tcacaacatc gaatacatcc ctttcgattc ctatggtaac attggttact catggtccaa
     2941 aaagggtaaa atttttgaaa agtacatcaa ggatgttttg tacaaattga agataaaata
     3001 tgaaaagcaa aataacaagg taaaaagaaa tgtcatcaaa aagatcatga actcattgtg
     3061 gggtaaattc gcccaaaagt gggttaactt cgaatacttc atcaagtcag aagatgacat
     3121 tgatttcgaa tccgaagaag catataaaat ttgggataca gacttcatgt tgatcaaaaa
     3181 gattaaggaa tctacctaca gttctaagcc aatacaaaac ggtgttttta ctttaagttg
     3241 ggccagatat cacatgaagt ctatttggga tgcaggtgcc aaagaaggtg ctgaatgtat
     3301 ctattcagat acagactcca tattcgttca taaggaacac ttcaaaaaga atgcaaagtt
     3361 tatgttgaac ggtttgaaag taccaatcat cggttctgaa gtcggtcaat ctgaattaga
     3421 atgtgaattt gataaattgt tgtgcgccgg taaaaagcaa tacatgggtt tctacactta
     3481 cttccaagat ggtaaacctt gcatcaagga aaagaaaaga ttcaaaggta taccaagtaa
     3541 ctacatcata cctgaattgt atgctcactt gttatctggt gcagataagg aagccaaaat
     3601 tcaattttta aaattcagaa gagaatgggg ttccgtcaag ggttacatag aaaataagac
     3661 tgtcaaagcc acctaactcg aggcgaattt cttatgattt atgattttta ttattaaata
     3721 agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa
     3781 attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg tatagcatga
     3841 ggtcgctcac tagtccatgg acgcgtggta ccggccgcgt tgctggcgtt tttccatagg
     3901 ctcggccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3961 acaggactat aaagatacca ggcgttcccc cctggaagct ccctcgtgcg ctctcctgtt
     4021 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4081 tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4141 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4201 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4261 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4321 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4381 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4441 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4501 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgactgat
     4561 ccttcaactc agcaaaagtt cgatttattc aacaaagcca cgttgtgtct caaaatctct
     4621 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
     4681 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctccaggc
     4741 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     4801 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt
     4861 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     4921 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     4981 atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat
     5041 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     5101 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     5161 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     5221 ggcctgttga acaagtctgg aaagaaatgc ataagctttt gccattctca ccggattcag
     5281 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     5341 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     5401 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     5461 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     5521 cagaattggt taattggttg taacacggtc cttttcatca cgtgctataa aaataattat
     5581 aatttaaatt ttttaatata aatatataaa ttaaaaatag aaagtaaaaa aagaaattaa
     5641 agaaaaaata gtttttgttt tccgaagatg taaaagactc tagggggatc gccaacaaat
     5701 actacctttt atcttgctct tcctgctctc aggtattaat gccgaattgt ttcatcttgt
     5761 ctgtgtagaa gaccacacac gaaaatcctg tgattttaca ttttacttat cgttaatcga
     5821 atgtatatct atttaatctg cttttcttgt ctaataaata tatatgtaaa gtacgctttt
     5881 tgttgaaatt ttttaaacct ttgtttattt ttttttcttc attccgtaac tcttctacct
     5941 tctttattta ctttctaaaa tccaaataca aaacataaaa ataaataaac acagagtaaa
     6001 ttcccaaatt attccatcat taaaagatac gaggcgcgtg taagttacag gcaagcgatc
     6061 cgtcctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     6121 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     6181 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     6241 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     6301 tgagagtgca ccacgctttt caattcaatt catcattttt tttttattct tttttttgat
     6361 tttttaagag cttggtgagc gctaggagtc actgccaggt atcgtttgaa cacggcatta
     6421 gtcagggaag tcataacaca gtcctttccc gcaattttct ttttctatta ctcttggcct
     6481 cctctagtac actctatatt tttttatgcc tcggtaatga ttttcatttt tttttttcca
     6541 cctagcggat gactcttttt ttttcttagc gattggcatt atcacataat gaattataca
     6601 ttatataaag taatgtgatt tcttcgaaga atatactaaa aaatgagcag gcaagataaa
     6661 cgaaggcaaa gatgacagag cagaaagccc tagtaaagcg tattacaaat gaaaccaaga
     6721 ttcagattgc gatctcttta aagggtggtc ccctagcgat agagcactcg atcttcccag
     6781 aaaaagaggc agaagcagta gcagaacagg ccacacaatc gcaagtgatt aacgtccaca
     6841 caggtatagg gtttctggac catatgatac atgctctggc caagcattcc ggctggtcgc
     6901 taatcgttga gtgcattggt gacttacaca tagacgacca tcacaccact gaagactgcg
     6961 ggattgctct cggtcaagct tttaaagagg ccctaggggc cgtgcgtgga gtaaaaaggt
     7021 ttggatcagg atttgcgcct ttggatgagg cactttccag agcggtggtt gatctttcga
     7081 acaggccgta cgcagttgtc gaacttggtt tgcaaaggga gaaagtaggt gatctctctt
     7141 gcgagatgat cccgcatttt cttgaaagct ttgcagaggc tagcagaatt accctccacg
     7201 ttgattgtct gcgaggcaag aatgatcatc accgtagtga gagtgcgttc aaggctcttg
     7261 cggttgccat aagagaagcc acctcgccca atggtaccaa cgatgttccc tccaccaaag
     7321 gtgttcttat gtagtgacac cgattattta aagctgctgc atacgatata tatacatgtg
     7381 tatatatgta tacctatgaa tgtcagtaag tatgtatacg aacagtatga tactgaagat
     7441 gacaaggtaa tgcatcattc tatacgtgtc attctgaacg aggcgcgctt tccttttttc
     7501 tttttgcttt ttcttttttt ttctcttgaa ctcgagaaaa aaaatatgcg gtgtgaaata
     7561 ccgcacagat gcgtaaggag aaaataccgc atcagg
//
