LOCUS Exported 5816 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Yeast integrative vector for expression of His-tagged POL30 (PCNA) mutant K164R; Leu2 marker. ACCESSION . VERSION . KEYWORDS YIp128-P30-His-pol30(K164R) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5816) AUTHORS Davies AA, Ulrich HD TITLE Detection of PCNA modifications in Saccharomyces cerevisiae. JOURNAL Methods Mol Biol. 2012;920:543-67. doi: 10.1007/978-1-61779-998-3_36. PUBMED 22941627 REFERENCE 2 (bases 1 to 5816) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5816 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..17 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 576..593 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" terminator 1401..1588 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind complement(1456..1480) /label=pHybLex-R /note="ADH terminator, reverse primer" primer_bind complement(1612..1628) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1612..1628) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1625..1647) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 1636..1652 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(1660..1690) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1705..1726 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(1843..1860) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2014..2602) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2094..2113) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2773..3633) /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 3396..3415 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3634..3738) /gene="bla" /label=AmpR promoter primer_bind 3806..3824 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3826..4231 /gene="S. cerevisiae LEU2" /label=LEU2 promoter CDS 4232..5326 /codon_start=1 /gene="S. cerevisiae LEU2" /product="3-isopropylmalate dehydrogenase, required for leucine biosynthesis" /label=LEU2 /note="yeast auxotrophic marker" /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG DLGGSNSTTEVGDAVAEEVKKILA" primer_bind complement(5467..5489) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 5589..5608 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5802..8 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 5816..17 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" ORIGIN 1 gtaaaacgac ggccagtgaa ttcgccctta ttccttaact gctgaagaaa agcaacccta 61 tgaatctaag gctcaagcag acaagaagag atacgaatct gaaaaggaat tgtacaatgc 121 tacacgtgct tgatctaaca actcaatgtt ctctacctcc catacaacgt acttttgaaa 181 cttcagaaag aagttgagta ggttgcatta tgtgttatta ttattactat tatcattatt 241 ttacttatat atccttcttt tcttttatgt attctcttcc tttttcgcac aacttatgct 301 gattcttcaa tatctaatta tttagcattt ttcttctcca tccgacgcac caaaacgcga 361 aacgcgtaac tttttttttg gatttcaact gatagttttc gtactttgct tcctctggta 421 cataaaatta tatataagaa acacttttgc tttagccttc ctttctttcc acttgcacct 481 ttcactttcg ccgtcctttt tcactcacag caacaagcag caagcactaa gtacgcagtc 541 aaaagagaga aaaaggatcc aatatgagag gatctcacca tcaccatcac catggcccgg 601 gaatgttaga agcaaaattt gaagaagcat cccttttcaa gagaataatt gatggtttca 661 aagattgtgt ccagttggtc aatttccaat gtaaagaaga tggtatcatt gcacaagctg 721 tcgatgactc aagagttcta ttggtctcct tggaaatagg tgtcgaagcc ttccaagaat 781 atagatgtga ccatcctgtt acgttaggta tggatctaac ctcactaagt aaaatcctac 841 gttgtggtaa caacaccgat acattaacac taattgctga caacacaccg gattccatca 901 tcttattatt tgaggatacc aagaaagacc gtatagccga atactctctg aaattgatgg 961 atatcgatgc tgatttctta aagattgaag aattacagta cgactccacc ctgtcattgc 1021 catcttccga attctctaaa attgttcgtg acttgtccca attgagtgat tctattaata 1081 tcatgataac cagagaaaca ataaagtttg tagctgacgg tgatatcgga tcaggttcag 1141 tcataataaa accattcgtg gatatggaac atcctgaaac aagcatcaaa cttgaaatgg 1201 atcaacctgt cgacttgacg ttcggagcta aatatttatt ggacatcatt aagggctcct 1261 ccctttctga tagagttggt atcaggctct ccagcgaagc tcctgcttta ttccaatttg 1321 atttgaagag tgggttccta cagtttttct tggctcctaa atttaatgac gaagaataac 1381 tgcagccaag ctaattccgg gcgaatttct tatgatttat gatttttatt attaaataag 1441 ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat 1501 tcttattctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta tagcatgagg 1561 tcgctcttat tgaccacacc tctaccggca tgcaagcttg gcgtaatcat ggtcatagct 1621 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 1681 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 1741 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 1801 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 1861 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 1921 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 1981 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 2041 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 2101 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 2161 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 2221 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 2281 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 2341 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 2401 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 2461 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 2521 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 2581 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 2641 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 2701 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 2761 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 2821 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 2881 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 2941 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 3001 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 3061 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 3121 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 3181 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 3241 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 3301 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 3361 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 3421 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 3481 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 3541 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 3601 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 3661 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 3721 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat 3781 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccaactg tgggaatact 3841 caggtatcgt aagatgcaag agttcgaatc tcttagcaac cattattttt ttcctcaaca 3901 taacgagaac acacaggggc gctatcgcac agaatcaaat tcgatgactg gaaatttttt 3961 gttaatttca gaggtcgcct gacgcatata cctttttcaa ctgaaaaatt gggagaaaaa 4021 ggaaaggtga gagcgccgga accggctttt catatagaat agagaagcgt tcatgactaa 4081 atgcttgcat cacaatactt gaagttgaca atattattta aggacctatt gttttttcca 4141 ataggtggtt agcaatcgtc ttactttcta acttttctta ccttttacat ttcagcaata 4201 tatatatata tttcaaggat ataccattct aatgtctgcc cctaagaaga tcgtcgtttt 4261 gccaggtgac cacgttggtc aagaaatcac agccgaagcc attaaggttc ttaaagctat 4321 ttctgatgtt cgttccaatg tcaagttcga tttcgaaaat catttaattg gtggtgctgc 4381 tatcgatgct acaggtgttc cacttccaga tgaggcgctg gaagcctcca agaaggctga 4441 tgccgttttg ttaggtgctg tgggtggtcc taaatggggt acaggtagtg ttagacctga 4501 acaaggttta ctaaaaatcc gtaaagaact tcaattgtac gccaacttaa gaccatgtaa 4561 ctttgcatcc gactctcttt tagacttatc tccaatcaag ccacaatttg ctaaaggtac 4621 tgacttcgtt gttgtcagag aattagtggg aggtatttac tttggtaaga gaaaggaaga 4681 cgatggtgat ggtgtcgctt gggatagtga acaatacacc gttccagaag tgcaaagaat 4741 cacaagaatg gccgctttca tggccctaca acatgagcca ccattgccta tttggtcctt 4801 ggataaagct aatgttttgg cctcttcaag attatggaga aaaactgtgg aggaaaccat 4861 caagaacgaa tttcctacat tgaaggttca acatcaattg attgattctg ccgccatgat 4921 cctagttaag aacccaaccc acctaaatgg tattataatc accagcaaca tgtttggtga 4981 tatcatctcc gatgaagcct ccgttatccc aggttccttg ggtttgttgc catctgcgtc 5041 cttggcctct ttgccagaca agaacaccgc atttggtttg tacgaaccat gccacggttc 5101 tgctccagat ttgccaaaga ataaggtcaa ccctatcgcc actatcttgt ctgctgcaat 5161 gatgttgaaa ttgtcattga acttgcctga agaaggtaag gccattgaag atgcagttaa 5221 aaaggttttg gatgcaggta tcagaactgg tgatttaggt ggttccaaca gtaccaccga 5281 agtcggtgat gctgtcgccg aagaagttaa gaaaatcctt gcttaaaaag attctctttt 5341 tttatgatat ttgtacataa actttataaa tgaaattcat aatagaaacg acacgaaatt 5401 acaaaatgga atatgttcat agggtaggcc ctttcgtctc gcgcgtttcg gtgatgacgg 5461 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 5521 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct 5581 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc 5641 gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa 5701 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg 5761 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgtt //