LOCUS       sequence-494842-        7769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of OsTIR1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7769)
  AUTHORS   Hubbe P, Sharma C, Pajonk O, Peters N, Guschtschin-Schmidt N, 
            Friemel N, Schuck S
  TITLE     An optimised system for rapid auxin-inducible protein degradation in
            budding yeast
  JOURNAL   bioRxiv 2025.09.24.678194
REFERENCE   2  (bases 1 to 7769)
  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"; journalName: "bioRxiv 
            2025.09.24.678194"
COMMENT     Sequence Label: pRS406-Padh-OsTIR1(F74G)
FEATURES             Location/Qualifiers
     source          1..7769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2241..2261
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2243..2261
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3030..3734
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             3760..5487
                     /codon_start=1
                     /product="rice transport inhibitor response 1 protein, 
                     mutated for use in the AID2 system of inducible degradation
                     (Yesbolatova et al., 2020)"
                     /label=OsTIR1(F74G)
                     /note="F-box protein that mediates auxin-dependent 
                     interaction of a ubiquitin ligase with an auxin-inducible 
                     degron (Tan et al., 2007)"
                     /translation="MTYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIERLSRRGVFV
                     GNCYAVRAGRVAARFPNVRALTVKGKPHGADFNLVPPDWGGYAGPWIEAAARGCHGLEE
                     LRMKRMVVSDESLELLARSFPRFRALVLISCEGFSTDGLAAVASHCKLLRELDLQENEV
                     EDRGPRWLSCFPDSCTSLVSLNFACIKGEVNAGSLERLVSRSPNLRSLRLNRSVSVDTL
                     AKILLRTPNLEDLGTGNLTDDFQTESYFKLTSALEKCKMLRSLSGFWDASPVCLSFIYP
                     LCAQLTGLNLSYAPTLDASDLTKMISRCVKLQRLWVLDCISDKGLQVVASSCKDLQELR
                     VFPSDFYVAGYSAVTEEGLVAVSLGCPKLNSLLYFCHQMTNAALVTVAKNCPNFTRFRL
                     CILEPGKPDVVTSQPLDEGFGAIVRECKGLQRLSISGLLTDKVFMYIGKYAKQLEMLSI
                     AFAGDSDKGMMHVMNGCKNLRKLEIRDSPFGDAALLGNFARYETMRSLWMSSCNVTLKG
                     CQVLASKMPMLNVEVINERDGSNEMEENHGDLPKVEKLYVYRTTAGARDDAPNFVKIL"
     primer_bind     complement(5501..5517)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5502..5518)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      5520..5767
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5536..5554)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5785..5804)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5786..5804)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5814..5831)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5814..5830)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5823..5845)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5971..6426
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6058..6077)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6268..6289
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6557..7360)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(7361..7576)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7600..7619)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7719..7741
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctcg
     2281 ccgggatcga agaaatgatg gtaaatgaaa taggaaatca aggagcatga aggcaaaaga
     2341 caaatataag ggtcgaacga aaaataaagt gaaaagtgtt gatatgatgt atttggcttt
     2401 gcggcgccga aaaaacgagt ttacgcaatt gcacaatcat gctgactctg tggcggaccc
     2461 gcgctcttgc cggcccggcg ataacgctgg gcgtgaggct gtgcccggcg gagttttttg
     2521 cgcctgcatt ttccaaggtt taccctgcgc taaggggcga gattggagaa gcaataagaa
     2581 tgccggttgg ggttgcgatg atgacgacca cgacaactgg tgtcattatt taagttgccg
     2641 aaagaacctg agtgcatttg caacatgagt atactagaag aatgagccaa gacttgcgag
     2701 acgcgagttt gccggtggtg cgaacaatag agcgaccatg accttgaagg tgagacgcgc
     2761 ataaccgcta gagtactttg aagaggaaac agcaataggg ttgctaccag tataaataga
     2821 caggtacata caacactgga aatggttgtc tgtttgagta cgctttcaat tcatttgggt
     2881 gtgcacttta ttatgttaca atatggaagg gaactttaca cttctcctat gcacatatat
     2941 taattaaagt ccaatgctag tagagaaggg gggtaacacc cctccgcgct cttttccgat
     3001 ttttttctaa accgtggaat atttcggata tccttttgtt gtttccgggt gtacaatatg
     3061 gacttcctct tttctggcaa ccaaacccat acatcgggat tcctataata ccttcgttgg
     3121 tctccctaac atgtaggtgg cggaggggag atatacaata gaacagatac cagacaagac
     3181 ataatgggct aaacaagact acaccaatta cactgcctca ttgatggtgg tacataacga
     3241 actaatactg tagccctaga cttgatagcc atcatcatat cgaagtttca ctaccctttt
     3301 tccatttgcc atctattgaa gtaataatag gcgcatgcaa cttcttttct ttttttttct
     3361 tttctctctc ccccgttgtt gtctcaccat atccgcaatg acaaaaaaat gatggaagac
     3421 actaaaggaa aaaattaacg acaaagacag caccaacaga tgtcgttgtt ccagagctga
     3481 tgaggggtat ctcgaagcac acgaaacttt ttccttcctt cattcacgca cactactctc
     3541 taatgagcaa cggtatacgg ccttccttcc agttacttga atttgaaata aaaaaaagtt
     3601 tgctgtcttg ctatcaagta taaatagacc tgcaattatt aatcttttgt ttcctcgtca
     3661 ttgttctcgt tccctttctt ccttgtttct ttttctgcac aatatttcaa gctataccaa
     3721 gcatacaatc aactccaagc tggccgctct agaactagta tgacttattt tcctgaagaa
     3781 gttgtagaac acatattttc ctttttacct gctcaaagag atagaaacac cgtctccttg
     3841 gtctgtaagg tttggtacga aatcgaaaga ttgtctagaa gaggtgtttt tgttggtaat
     3901 tgttacgcag ttagagctgg tagagttgct gcaagattcc caaacgttag agcattgact
     3961 gttaaaggta aacctcatgg tgctgatttc aatttggttc cacctgattg gggtggttat
     4021 gctggtccat ggattgaagc tgcagctaga ggttgtcacg gtttggaaga attgagaatg
     4081 aagagaatgg ttgtttctga tgaatcattg gaattgttag caagatcttt tcctagattc
     4141 agagctttgg ttttgatctc atgtgaaggt ttttccaccg atggtttagc agctgttgct
     4201 tcacattgta agttgttgag agaattggat ttgcaagaaa acgaagttga agatagaggt
     4261 ccaagatggt tatcttgttt tcctgattcc tgtacaagtt tggtttcttt gaacttcgca
     4321 tgtatcaagg gtgaagttaa cgctggtagt ttggaaagat tagtttctag atcaccaaat
     4381 ttgagatctt tgagattgaa cagatccgtt agtgttgata ccttggcaaa gatcttgttg
     4441 agaactccta atttggaaga tttgggtact ggtaacttga cagatgattt ccaaacagaa
     4501 tcatacttca agttgacctc cgctttggaa aagtgtaaga tgttgagatc tttgtcaggt
     4561 ttttgggatg caagtccagt ttgtttgtct tttatatatc ctttgtgtgc tcaattgact
     4621 ggtttgaatt tgtcttacgc accaactttg gatgctagtg atttgacaaa gatgatctct
     4681 agatgtgtta agttgcaaag attgtgggtt ttggattgta tctcagataa aggtttgcaa
     4741 gttgttgctt cttcttgtaa ggatttgcaa gaattgagag tttttccaag tgatttctat
     4801 gttgcaggtt actctgctgt tacagaagaa ggtttagttg cagtttcatt gggttgtcct
     4861 aagttgaact ccttgttgta cttctgtcat caaatgacca acgcagcttt ggttactgtt
     4921 gctaaaaatt gtccaaactt cactagattc agattgtgta tcttggaacc aggtaaacct
     4981 gatgttgtta catcacaacc tttagatgaa ggttttggtg caatcgttag agagtgtaag
     5041 ggtttgcaaa gattgtccat cagtggtttg ttgacagata aggtttttat gtacatcggt
     5101 aaatacgcta agcaattgga aatgttgtca atcgcattcg ctggtgactc cgataagggt
     5161 atgatgcatg ttatgaacgg ttgtaagaac ttaagaaagt tggaaatcag agattctcca
     5221 tttggtgacg cagctttgtt gggtaatttc gcaagatatg aaacaatgag atctttgtgg
     5281 atgtccagtt gtaacgttac cttgaaaggt tgtcaagttt tagcttcaaa gatgccaatg
     5341 ttgaacgttg aagttattaa cgaaagagat ggttccaatg aaatggaaga aaaccatggt
     5401 gacttgccta aagttgaaaa gttatacgtc tacagaacaa ccgcaggtgc cagagatgat
     5461 gctcctaact tcgtcaagat tttatgagcg gcaagcttat cgataccgtc gacctcgagt
     5521 catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga
     5581 aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt
     5641 agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta
     5701 cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt
     5761 aatttgcggc cggtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     5821 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     5881 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     5941 caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg
     6001 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     6061 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     6121 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     6181 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     6241 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     6301 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     6361 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     6421 caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatagg
     6481 gtaataactg atataattaa attgaagctc taatttgtga gtttagtata catgcattta
     6541 cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct tcccagcctg
     6601 cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat agtcctcttc
     6661 caacaataat aatgtcagat cctgtagaga ccacatcatc cacggttcta tactgttgac
     6721 ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt cataatcaac caatcgtaac
     6781 cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa tctttgtcgc
     6841 tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag cccttgcatg
     6901 acaattctgc taacatcaaa aggcctctag gttcctttgt tacttcttct gccgcctgct
     6961 tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg tctgcccatt
     7021 ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca atgtcagcaa
     7081 attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt agcggcttaa
     7141 ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt aaacaaattt
     7201 tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca tccaatgaag
     7261 cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca acaggactag
     7321 gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt cgtttcctgc
     7381 aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt tcttcaacac
     7441 tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct tccttctgtt
     7501 cggagattac cgaatcaaaa aaatttcaag gaaaccgaaa tcaaaaaaaa gaataaaaaa
     7561 aaaatgatga attgaaaagg tggtatggtg cactctcagt acaatctgct ctgatgccgc
     7621 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     7681 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     7741 gttttcaccg tcatcaccga aacgcgcga
//
