LOCUS Exported 8902 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses OsTIR1 via the ADH1 promoter in yeast. The cassette is integrated in the HO locus. Assembly and characterization of the auxin-inducible degradation.. ACCESSION . VERSION . KEYWORDS pOsTIR1w/oGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8902) AUTHORS Papagiannakis A, Niebel B, Wit EC, Heinemann M TITLE Autonomous Metabolic Oscillations Robustly Gate the Early and Late Cell Cycle. JOURNAL Mol Cell. 2017 Jan 19;65(2):285-295. doi: 10.1016/j.molcel.2016.11.018. Epub 2016 Dec 15. PUBMED 27989441 REFERENCE 2 (bases 1 to 8902) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8902 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..397 /gene="S. cerevisiae ADH1" /label=ADH1 promoter /note="promoter for alcohol dehydrogenase 1" CDS 416..2140 /codon_start=1 /product="rice transport inhibitor response 1 protein" /label=OsTIR1 /note="F-box protein that mediates auxin-dependent interaction of a ubiquitin ligase with an auxin-inducible degron (Tan et al., 2007)" /translation="MTYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIERLSRRGVFV GNCYAVRAGRVAARFPNVRALTVKGKPHFADFNLVPPDWGGYAGPWIEAAARGCHGLEE LRMKRMVVSDESLELLARSFPRFRALVLISCEGFSTDGLAAVASHCKLLRELDLQENEV EDRGPRWLSCFPDSCTSLVSLNFACIKGEVNAGSLERLVSRSPNLRSLRLNRSVSVDTL AKILLRTPNLEDLGTGNLTDDFQTESYFKLTSALEKCKMLRSLSGFWDASPVCLSFIYP LCAQLTGLNLSYAPTLDASDLTKMISRCVKLQRLWVLDCISDKGLQVVASSCKDLQELR VFPSDFYVAGYSAVTEEGLVAVSLGCPKLNSLLYFCHQMTNAALVTVAKNCPNFTRFRL CILEPGKPDVVTSQPLDEGFGAIVRECKGLQRLSISGLLTDKVFMYIGKYAKQLEMLSI AFAGDSDKGMMHVMNGCKNLRKLEIRDSPFGDAALLGNFARYETMRSLWMSSCNVTLKG CQVLASKMPMLNVEVINERDGSNEMEENHGDLPKVEKLYVYRTTAGARDDAPNFVKIL" CDS 2168..2197 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2204..2233 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 2240..2269 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" terminator 2419..2584 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind complement(2474..2498) /label=pHybLex-R /note="ADH terminator, reverse primer" gene 2632..3988 /label=kanMX /note="yeast selectable marker conferring kanamycin resistance (Wach et al., 1994)" promoter 2632..2975 /label=TEF promoter /note="Ashbya gossypii TEF promoter" CDS 2976..3785 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin" /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(3043..3062) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" terminator 3791..3988 /label=TEF terminator /note="Ashbya gossypii TEF terminator" primer_bind complement(4596..4615) /label=T7 /note="T7 promoter, forward primer" promoter complement(4597..4615) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4622..4639) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4622..4638) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4631..4653) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(4938..4957) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5070..5091 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5245..5264) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5364..5386 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5424..5442) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5510..5614 /gene="bla" /label=AmpR promoter CDS 5615..6475 /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 complement(5833..5852) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6646..7234 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7135..7154 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7388..7405 /label=L4440 /note="L4440 vector, forward primer" protein_bind 7522..7543 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." protein_bind 7598..7614 /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)." primer_bind 7603..7625 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7622..7638 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7622..7638 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 caacttcttt tctttttttt tcttttctct ctcccccgtt gttgtctcac catatccgca 61 atgacaaaaa aatgatggaa gacactaaag gaaaaaatta acgacaaaga cagcaccaac 121 agatgtcgtt gttccagagc tgatgagggg tatctcgaag cacacgaaac tttttccttc 181 cttcattcac gcacactact ctctaatgag caacggtata cggccttcct tccagttact 241 tgaatttgaa ataaaaaaaa gtttgctgtc ttgctatcaa gtataaatag acctgcaatt 301 attaatcttt tgtttcctcg tcattgttct cgttcccttt cttccttgtt tctttttctg 361 cacaatattt caagctatac caagcataca atcaactatc tcatatacaa ctagaatgac 421 gtacttcccg gaggaggtgg tggagcacat cttcagcttc ctgccggcgc agcgcgaccg 481 caacacggtc tcgctcgtct gcaaggtgtg gtacgagatc gagaggctga gccgccgcgg 541 cgtcttcgtg ggcaactgct acgccgtgcg cgccggccgc gtcgccgcgc ggttccccaa 601 cgtgcgggcg ctcacggtga aggggaagcc ccacttcgcc gacttcaacc tcgtgccccc 661 cgactggggc ggctacgcgg ggccgtggat cgaggcggcc gcgaggggat gccacggcct 721 ggaggagctc aggatgaagc ggatggtggt gtccgacgag agcctcgagc tgctggctcg 781 ctcgttcccg cggttcaggg ctcttgttct tatcagctgc gaggggttca gcactgacgg 841 gctagccgcc gtcgcgagcc attgcaagct tctgagggag ttggatttgc aggaaaatga 901 agtggaggat cgagggccta ggtggctttc ctgcttccct gattcctgca catcacttgt 961 ctcattgaat tttgcctgca tcaaagggga ggttaatgct ggttcactgg agagacttgt 1021 tagcaggtcc ccaaacctgc ggagtttgag gctgaatcga tctgtatcgg tagatacact 1081 tgcaaagata ctactgcgta cccctaactt ggaggatttg gggacaggga atttgacaga 1141 tgacttccaa actgagtcct actttaagct taccagtgct ctggagaaat gcaagatgtt 1201 gaggagtttg tctggattct gggatgcttc tcctgtttgc ctgtcattta tctaccccct 1261 gtgtgctcaa ctgacaggat tgaacttgag ctatgcaccc acacttgatg cttctgacct 1321 tacaaaaatg attagccgct gtgtgaagct ccaacgcctt tgggtactgg attgtatctc 1381 ggacaaaggc ttgcaagtgg tggcctccag ttgcaaagac ttgcaagaac tcagggtatt 1441 tccatcagat ttctacgtag ctggttattc tgcagtgaca gaggagggac ttgttgcagt 1501 atccttgggc tgtccaaaac tgaactcact actgtacttc tgtcaccaaa tgactaatgc 1561 tgcactagtt actgtcgcca agaactgtcc aaatttcaca cgattcagac tttgtattct 1621 tgagccaggg aagcctgatg ttgtgacaag ccaaccatta gatgaaggct ttggagctat 1681 tgttcgtgag tgcaagggat tacaacgttt gtcaatatct ggtcttctca cagacaaagt 1741 tttcatgtat attgggaaat atgcaaaaca acttgagatg ctttctatag catttgctgg 1801 tgacagtgat aagggtatga tgcatgttat gaatggatgc aagaatttaa ggaaactgga 1861 gataagagat agcccgtttg gtgatgctgc actcttgggg aattttgcta ggtacgagac 1921 aatgcgatcc ctttggatgt catcttgcaa tgtcacgtta aaggggtgcc aagtccttgc 1981 gtcaaagatg ccgatgctca atgttgaggt cataaatgag cgggatggta gcaatgaaat 2041 ggaggaaaac catggagatc tgcctaaagt ggagaaatta tatgtgtacc gcacaactgc 2101 tggggcgagg gatgatgcac caaattttgt taaaatccta gtcgactccg gttctgctgc 2161 tagtggtgaa caaaagttga tttctgaaga agatttgaac ggtgaacaaa agctaatctc 2221 cgaggaagac ttgaacggtg aacaaaaatt aatctcagaa gaagacttga acggatccac 2281 tagctaaaac aattcttcgc cagaggtttg gtcaagtctc caatcaaggt tgtcggcttg 2341 tctaccttgc cagaaattta cgaaaagatg gaaaagggtc aaatcgttgg tagatacgtt 2401 gttgacactt ctaaataagc gaatttctta tgatttatga tttttattat taaataagtt 2461 ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa acgaaaattc 2521 ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata gcatgaggtc 2581 gctcgtagat ctgtttagct tgcctcgtcc ccgccgggtc acccggccag cgacatggag 2641 gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt 2701 cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat acattttgat 2761 ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc agacctgcga gcagggaaac 2821 gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa 2881 ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga 2941 tacagttctc acatcacatc cgaacataaa caaccatggg taaggaaaag actcacgttt 3001 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg 3061 ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag 3121 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca 3181 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc 3241 ctgatgatgc atggttactc accactgcga tccccggcaa aacagcattc caggtattag 3301 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt 3361 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc 3421 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta 3481 atggctggcc tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg 3541 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat 3601 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca 3661 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat 3721 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt 3781 tctaatcagt actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt 3841 tttttatatt gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg 3901 cctcgacatc atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa 3961 tcgtatgtga atgctggtcg ctatactgct gtcgattcga tactaacgcc gccatccagt 4021 gtcgaaaacg agctcgaatt aattcctggg ggaacaactt cacagaatgt tttgtcatat 4081 tgtcgaagtg gtcacaaaac aagagaagtt ccgccaatta taaaaaggga acccgtatat 4141 ttcagcttca cggatgattt ccagggtgag agtactgtat atgggcttac gatagaaggc 4201 cataaaaatt tcttgcttgg caacaaaata gaagtgaaat catgtcgagg ctgctgtgtg 4261 ggagaacagc ataaaatatc acaaaaaaag aatctaaaac actgtgttgc ttgtcccaga 4321 aagggaatca agtattttta taaagattgg agtggtaaaa atcgagtatg tgctagatgc 4381 tatggaagat acaaattcag cggtcatcac tgtataaatt gcaagtatgt accagaagca 4441 cgtgaagtga aaaaggcaaa agacaaaggc gaaaaattgg gcattacgcc cgaaggtttg 4501 ccagttaaag gaccagagtg tataaaatgt ggcggaatct tacagtggcc tatgcggccg 4561 ctctagctag aactagtgga tcgatcccca attcgcccta tagtgagtcg tattacaatt 4621 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 4681 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 4741 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc 4801 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg 4861 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 4921 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 4981 ctttccccgt caagctctaa atcggggtgg gccatcgccc tgatagacgg tttttcgccc 5041 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 5101 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg 5161 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt 5221 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc 5281 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 5341 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 5401 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 5461 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 5521 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 5581 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 5641 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 5701 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 5761 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 5821 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 5881 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 5941 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 6001 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 6061 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 6121 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 6181 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 6241 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 6301 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 6361 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 6421 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 6481 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 6541 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 6601 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 6661 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 6721 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 6781 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 6841 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 6901 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcac agcggtcggg 6961 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 7021 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 7081 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 7141 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 7201 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 7261 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 7321 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 7381 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 7441 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 7501 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 7561 acactttatg cttccgcggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 7621 acaggaaaca gctatgacca tgattacgcc actagtccga ggcctcgaga tccgatatcg 7681 ccgtggcggc cgccagctga agcttaatta tcctgggcac gagtgaaaca aagctaaaac 7741 ctttatttag catggccatt gaatgtaaca attatatata tcgcaagcac aaaaaatcaa 7801 ggagagagaa ctaccacttt gttcatgtgt acaatgttca ttatctccat aagcaaaaaa 7861 aaaaaaatag aaaacatatg ctataaggtt gatattctca cgagtaagcg gcacttgcta 7921 cttattgaca ttgcagattt ttggctacag aaatagtata ttagagatta taattgctaa 7981 tcaaatcaaa atataaaatt agtaaaccaa accatttata cccttcctta gtagttatgg 8041 attgtttttt aatgatattt ctgcaaacca aagaaagatt gttatccaga tagaatttag 8101 ttttgatatt catttttttg ttgaagattg aacgccatat ctgggcctca taattcaaaa 8161 gacggtgcca ttatcggtag cgtttcgcat tgtactggat ttcagaaatt tcacagttga 8221 tgaatcgaaa agaatggtct cattgcaaca cgtaaggtta agatgtccct ttttaccatt 8281 ataggcaata aatgaatcat aaaacgaccg tatactggtg aaatagtagg gagaacgagt 8341 acctgtagta aaaagtataa atcatagtta atcgggcaat gtccctcgat caaggagtat 8401 tgtgtcatgt tcgagacaaa cgccaacatt tttgtttctt ttggacaaat gttgtttgca 8461 tttatgatcc gttatatttt gatctaatgt agagttgcac gtagttctta ctggcaaaga 8521 aatcgatgca taccaaaaaa gaataaaggt gatatttgat ctttaccgtt tagttccaac 8581 gtaaaattgt gcctttggac ttaaaatggc ggggtgtaca atatggactt cctcttttct 8641 ggcaaccaaa cccatacatc gggattccta taataccttc gttggtctcc ctaacatgta 8701 ggtggcggag gggagatata caatagaaca gataccagac aagacataat gggctaaaca 8761 agactacacc aattacactg cctcattgat ggtggtacat aacgaactaa tactgtagcc 8821 ctagacttga tagccatcat catatcgaag tttcactacc ctttttccat ttgccatcta 8881 ttgaagtaat aataggcgca tg //