LOCUS       sequence-494844-        8743 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 8743)
  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 8743)
  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: pNH604-Padh-OsTIR1(F74G)
FEATURES             Location/Qualifiers
     source          1..8743
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2162..2866
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             2892..4619
                     /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(4633..4649)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4634..4650)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(4668..4686)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      4698..4887
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5291..5310)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5292..5310)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5317..5334)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5317..5333)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5326..5348)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5474..5929
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5561..5580)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5771..5792
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6284..6303)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6403..6425
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6463..6481)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6566..6670
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6671..6739,6740..7531)
                     /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(6889..6908)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7702..8290
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8191..8210
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8444..8461
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8578..8599
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8614..8619,8620..8637,8638..8644)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8652..8668
                     /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     8657..8679
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8676..8692
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8676..8692
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        8713..8731
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ggccggccat ttaaatgttt aaacggccac gctggccata tttctaccaa tctctcaaca
       61 ctgagtaatg gtagttataa gaaagagacc gagttaggga cagttagagg cggtggagat
      121 attccttatg gcatgtctgg cgatgataaa acttttcaaa cggcagcccc gatctaaaag
      181 agctgacagg gaaatggtca gaaaaagaaa cgtgcacccg cccgtctgga cgcgccgctc
      241 acccgcacgg cagagaccaa tcagtaaaaa tcaacggtta acgacattac tatatatata
      301 atataggaag catttaatag aacagcatcg taatatatgt gtactttgca gttatgacgc
      361 cagatggcag tagtggaaga tattctttat tgaaaaatag cttgtcacct tacgtacaat
      421 cttgatccgg agcccgtcgc tttgagagaa atcagaagat tccaaaagtc tactgaactt
      481 ttgatcagaa agttgccttt ccaaagacta gtcagagaaa tcgcccaaga tttcaagacc
      541 gatctaagat tccaatcctc tgccatcggt gctctacagg aatccgttga agcttacttg
      601 gtctctttgt tcgaagacac caacttggcc gctatccacg ctaagcgtgt taccatccaa
      661 aagaaggata tcaagttggc tagaagattg agaggtgaaa gatcctaagc acacagggat
      721 tgttgtacaa ttacttttta tgattttaga tacacacaaa tcttttctac gattaccttt
      781 catttatttt cgtttcatgt tttattaatc attctttttg tatatttcac atcgggtggg
      841 gtttttatat taaattttaa taacataatc ttttaaagtt tctactttct atcctgtata
      901 ttatttccat atctatatct atattttcaa gcttgttcac atccttatcg gaattgttcc
      961 agcgtttttg tacagaaaat gatgttatgg taacgagcta tatcagacct actagtctta
     1021 tagagctgta cagagtcttg gaaacataac caacacacca acaagatgtc atttgattcg
     1081 ttactcgaca agaatgataa gctggtaaaa gtttgcggga ttcaaaccgt cgaggctgcc
     1141 gaaactgcgc ttcaagcagg cgctgatttg atagggatca tatgcgtccc caacaggaag
     1201 cggactatcg agagcgctgt ggctcgtgaa atatccaaat tgattcacaa atcaggtact
     1261 acaaagctgg tgggggtgtt caggaatcaa tctgttgagg acgtacatcg gctttctgag
     1321 gaatatgacc ttgacataat ccaattacat ggtgatgaat catggccaga gtactataac
     1381 gtcattaaga aaccaataat caaaagagtc atattcccta gagatgtcga tgttgtaaca
     1441 caagtgtgtc aaagaaaacc cttggtatgt ctaccattgt tcgactccga ggcaggcggt
     1501 acaggtgaaa agctggactg gtccagtatt tcctcctggg caagcgaaca gaataacgta
     1561 ttttatatac tagcgggtgg actaactgca gaaaacgtca tggaggcggt ggggttgcct
     1621 ggtgtaattg gtgttgacgt ctcaggtggt gtagagactg acggcgtcaa agacaatgac
     1681 aagataatta aatatgtaca aaatgcaaag aaacaatgaa ttacgaagag aagtctcaat
     1741 gaggttttac aaacaatcct ataatacata tatgccactt agatatactt tgaatactga
     1801 atatactgac tcattatatt ctttaattgt agtttctcaa aaatattcat acaggttagc
     1861 tatgaattca tgctataatt tccattttgt tcatgtaaga tacacatagg gataaaatgt
     1921 gataactaat cagcggtacc gggcccttat attgaatttt caaaaattct tacttttttt
     1981 tggatggacg caaagaagtt taataatcat attacatggc attaccacca tatacatatc
     2041 catatctaat cttacttata tgttgtggaa atgtaaagag ccccattatc ttagcctaaa
     2101 aaaaccttct ctttggaact ttcagtaata cgcttaactg ctcattgcta tattgaagta
     2161 catccttttg ttgtttccgg gtgtacaata tggacttcct cttttctggc aaccaaaccc
     2221 atacatcggg attcctataa taccttcgtt ggtctcccta acatgtaggt ggcggagggg
     2281 agatatacaa tagaacagat accagacaag acataatggg ctaaacaaga ctacaccaat
     2341 tacactgcct cattgatggt ggtacataac gaactaatac tgtagcccta gacttgatag
     2401 ccatcatcat atcgaagttt cactaccctt tttccatttg ccatctattg aagtaataat
     2461 aggcgcatgc aacttctttt cttttttttt cttttctctc tcccccgttg ttgtctcacc
     2521 atatccgcaa tgacaaaaaa atgatggaag acactaaagg aaaaaattaa cgacaaagac
     2581 agcaccaaca gatgtcgttg ttccagagct gatgaggggt atctcgaagc acacgaaact
     2641 ttttccttcc ttcattcacg cacactactc tctaatgagc aacggtatac ggccttcctt
     2701 ccagttactt gaatttgaaa taaaaaaaag tttgctgtct tgctatcaag tataaataga
     2761 cctgcaatta ttaatctttt gtttcctcgt cattgttctc gttccctttc ttccttgttt
     2821 ctttttctgc acaatatttc aagctatacc aagcatacaa tcaactccaa gctggccgct
     2881 ctagaactag tatgacttat tttcctgaag aagttgtaga acacatattt tcctttttac
     2941 ctgctcaaag agatagaaac accgtctcct tggtctgtaa ggtttggtac gaaatcgaaa
     3001 gattgtctag aagaggtgtt tttgttggta attgttacgc agttagagct ggtagagttg
     3061 ctgcaagatt cccaaacgtt agagcattga ctgttaaagg taaacctcat ggtgctgatt
     3121 tcaatttggt tccacctgat tggggtggtt atgctggtcc atggattgaa gctgcagcta
     3181 gaggttgtca cggtttggaa gaattgagaa tgaagagaat ggttgtttct gatgaatcat
     3241 tggaattgtt agcaagatct tttcctagat tcagagcttt ggttttgatc tcatgtgaag
     3301 gtttttccac cgatggttta gcagctgttg cttcacattg taagttgttg agagaattgg
     3361 atttgcaaga aaacgaagtt gaagatagag gtccaagatg gttatcttgt tttcctgatt
     3421 cctgtacaag tttggtttct ttgaacttcg catgtatcaa gggtgaagtt aacgctggta
     3481 gtttggaaag attagtttct agatcaccaa atttgagatc tttgagattg aacagatccg
     3541 ttagtgttga taccttggca aagatcttgt tgagaactcc taatttggaa gatttgggta
     3601 ctggtaactt gacagatgat ttccaaacag aatcatactt caagttgacc tccgctttgg
     3661 aaaagtgtaa gatgttgaga tctttgtcag gtttttggga tgcaagtcca gtttgtttgt
     3721 cttttatata tcctttgtgt gctcaattga ctggtttgaa tttgtcttac gcaccaactt
     3781 tggatgctag tgatttgaca aagatgatct ctagatgtgt taagttgcaa agattgtggg
     3841 ttttggattg tatctcagat aaaggtttgc aagttgttgc ttcttcttgt aaggatttgc
     3901 aagaattgag agtttttcca agtgatttct atgttgcagg ttactctgct gttacagaag
     3961 aaggtttagt tgcagtttca ttgggttgtc ctaagttgaa ctccttgttg tacttctgtc
     4021 atcaaatgac caacgcagct ttggttactg ttgctaaaaa ttgtccaaac ttcactagat
     4081 tcagattgtg tatcttggaa ccaggtaaac ctgatgttgt tacatcacaa cctttagatg
     4141 aaggttttgg tgcaatcgtt agagagtgta agggtttgca aagattgtcc atcagtggtt
     4201 tgttgacaga taaggttttt atgtacatcg gtaaatacgc taagcaattg gaaatgttgt
     4261 caatcgcatt cgctggtgac tccgataagg gtatgatgca tgttatgaac ggttgtaaga
     4321 acttaagaaa gttggaaatc agagattctc catttggtga cgcagctttg ttgggtaatt
     4381 tcgcaagata tgaaacaatg agatctttgt ggatgtccag ttgtaacgtt accttgaaag
     4441 gttgtcaagt tttagcttca aagatgccaa tgttgaacgt tgaagttatt aacgaaagag
     4501 atggttccaa tgaaatggaa gaaaaccatg gtgacttgcc taaagttgaa aagttatacg
     4561 tctacagaac aaccgcaggt gccagagatg atgctcctaa cttcgtcaag attttatgag
     4621 cggcaagctt atcgataccg tcgacctcga gtcatgtaat tagttatgtc acgcttacat
     4681 tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc
     4741 taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa
     4801 atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg
     4861 cttgagaagg ttttgggacg ctcgaaggct ttatttgctg gttccgatgc tgacttactg
     4921 ggtattatat gtgtgcccaa tagaaagaga acaattgacc cggttattgc aaggaaaatt
     4981 tcaagtcttg taaaagcata taaaaatagt tcaggcactc cgaaatactt ggttggcgtg
     5041 tttcgtaatc aacctaagga ggatgttttg gctctggtca atgattacgg cattgatatc
     5101 gtccaactgc atggagatga gtcgtggcga gaataccaag agttcctcgg tttgccagtt
     5161 attaaaagac tcgtatttcc aaaagactgc aacatactac tcagtgcagc ttcacagaaa
     5221 cctcattcgt ttattccctt gtttgattca gaaggccggc catttaaatg tttaaacggc
     5281 cacgctggcc ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
     5341 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     5401 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     5461 gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     5521 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     5581 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5641 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5701 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5761 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5821 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5881 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt
     5941 attttctcct tacgcatctg tgcggtattt cacaccgcat agggtaataa ctgatataat
     6001 taaattgaag ctctaatttg tgagtttagt attaggatct ctacccttgg cgaaaagtcc
     6061 tctgccaaca atgatgatat ctgatccacc acttacaact tcgtcgacgg ttctgtactg
     6121 ctgacccaat atgcgtatat ataccaatct aagtctgtgc tccttccttc gttcttcctt
     6181 ctgttcggag attaccgaat caaaaaaatt tcaaggaaac cgaaatcaaa aaaagaataa
     6241 aaaaaaaatg atgaattgaa aaggtggtat ggtgcactct cagtacaatc tgctctgatg
     6301 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     6361 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     6421 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     6481 ttttataggt taatgtcatg ataataatgg tttcttagac gtcggcgcgc cgaggacgtc
     6541 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     6601 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     6661 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     6721 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     6781 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     6841 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     6901 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     6961 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     7021 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     7081 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     7141 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     7201 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     7261 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     7321 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     7381 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     7441 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     7501 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     7561 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     7621 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     7681 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     7741 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     7801 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7861 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7921 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7981 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     8041 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     8101 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     8161 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     8221 cgggtttcgc cacctccgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     8281 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     8341 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     8401 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     8461 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     8521 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     8581 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     8641 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     8701 cgccaagctc ggaattaacc ctcactaaag ggaacaaaag ctg
//
