LOCUS       sequence-495248-        8248 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 8248)
  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 8248)
  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: pRS303N-Padh-OsTIR1(F74G)
FEATURES             Location/Qualifiers
     source          1..8248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..47)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     147..166
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        313..500
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     terminator      complement(856..1043)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     964..988
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(1051..1623)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(1624..1967)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        2046..2750
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             2802..4529
                     /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"
     terminator      4536..4783
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4552..4570)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(5308..5763)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5445..5466)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5657..5676
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(5740..6027)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSSKLTLTKGNKSWYRAPPSNSPYSESYYNSLAVVLQRRD
                     WENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTRPVAAH"
     primer_bind     5889..5911
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5903..5920
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5904..5920
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5927..5946
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5927..5945
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5984..6002)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6023..6039)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6023..6039)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6036..6058)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6047..6063
                     /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(join(6071..6077,6078..6095,6096..6101))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6116..6137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6254..6271)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6425..7013)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6505..6524)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7184..7975,7976..8044))
                     /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     7807..7826
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8045..8149)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8217..8235
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
       61 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
      121 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
      181 taaattcccg ttttaagagc ttggtgagcg ctaggagtca ctgccaggta tcgtttgaac
      241 acggcattag tcagggaagt cataacacag tcctttcccg caattttctt tttctattac
      301 tcttggcctc ctctagtaca ctctatattt ttttatgcct cggtaatgat tttcattttt
      361 ttttttccac ctagcggatg actctttttt tttcttagcg attggcatta tcacataatg
      421 aattatacat tatataaagt aatgtgattt cttcgaagaa tatactaaaa aatgagcagg
      481 caagataaac gaaggcaaag atgacagagc agaaagccct agtaaagcgt attacaaatg
      541 aaaccaagat tcagattgcg atctctttaa agggtggtcc cctagcgata gagcactcga
      601 tcttcccaga aaaagaggca gaagcagtag cagaacaggc cacacaatcg caagtgatta
      661 acgtccacac aggtataggg tttctggacc atatgataca tgctctggat catcgatgaa
      721 ttcgagctcg attacaacag gtgttgtcct ctgaggacat aaaatacaca ccgagattca
      781 tcaactcatt gctggagtta gcatatctac aattgggtga aatggggagc gatttgcagg
      841 catttgctcg gcatgccggt agaggtgtgg tcaataagag cgacctcatg ctatacctga
      901 gaaagcaacc tgacctacag gaaagagtta ctcaagaata agaattttcg ttttaaaacc
      961 taagagtcac tttaaaattt gtatacactt atttttttta taacttattt aataataaaa
     1021 atcataaatc ataagaaatt cgcctcgaga ttaggggcag ggcatgctca tgtagagcgc
     1081 ctgctcgccg tccgaggcgg tgccgtcgta cagggcggtg tccaggccgc agagggtgaa
     1141 ccccatccgc cggtacgcgt ggatcgccgg tgcgttgacg ttggtgacct ccagccagag
     1201 gtgcccggcg ccccgctcgc gggcgaactc cgtcgcgagc cccatcaacg cgcgcccgac
     1261 cccgtgcccc cggtgctccg gggcgacctc gatgtcctcg acggtcagcc ggcggttcca
     1321 gccggagtac gagacgacca cgaagcccgc caggtcgccg tcgtccccgt acgcgacgaa
     1381 cgtccgggag tccgggtcgc cgtcctcccc gtcgtccgat tcgtcgtccg attcgtcgtc
     1441 ggggaacacc ttggtcaggg gcgggtccac cggcacctcc cgcagggtga agccgtcccc
     1501 ggtggcggtg acgcggaaga cggtgtcggt ggtgaaggac ccatccagtg cctcgatggc
     1561 ctcggcgtcc cccgggacac tggtgcggta ccggtaagcc gtgtcgtcaa gagtggtacc
     1621 catggttgtt tatgttcgga tgtgatgtga gaactgtatc ctagcaagat tttaaaagga
     1681 agtatatgaa agaagaacct cagtggcaaa tcctaacctt ttatatttct ctacaggggc
     1741 gcggcgtggg gacaattcaa cgcgtctgtg aggggagcgt ttccctgctc gcaggtctgc
     1801 agcgaggagc cgtaattttt gcttcgcgcc gtgcggccat caaaatgtat ggatgcaaat
     1861 gattatacat ggggatgtat gggctaaatg tacgggcgac agtcacatca tgcccctgag
     1921 ctgcgcacgt caagactgtc aaggagggta ttctgggcct ccatgtcgct ggccgggtga
     1981 cccggcgggg acgaggcaag ctaaacagat ctggcgcgcc ttaattaacc cggggatccg
     2041 agctcatcct tttgttgttt ccgggtgtac aatatggact tcctcttttc tggcaaccaa
     2101 acccatacat cgggattcct ataatacctt cgttggtctc cctaacatgt aggtggcgga
     2161 ggggagatat acaatagaac agataccaga caagacataa tgggctaaac aagactacac
     2221 caattacact gcctcattga tggtggtaca taacgaacta atactgtagc cctagacttg
     2281 atagccacca tcatatcgaa gtttcactac cctttttcca tttgccatct attgaagtaa
     2341 taataggcgc atgcaacttc ttttcttttt ttttcttttc tctctccccc gttgttgtct
     2401 caccatatcc gcaatgacaa aaaaatgatg gaagacacta aaggaaaaaa ttaacgacaa
     2461 agacagcacc aacagatgtc gttgttccag agctgatgag gggtatctcg aagcacacga
     2521 aactttttcc ttccttcatt cacgcacact actctctaat gagcaacggt atacggcctt
     2581 ccttccagtt acttgaattt gaaataaaaa aaagtttgct gtcttgctat caagtataaa
     2641 tagacctgca attattaatc ttttgtttcc tcgtcattgt tctcgttccc tttcttcctt
     2701 gtttcttttt ctgcacaata tttcaagcta taccaagcat acaatcaact ccaagctggc
     2761 cgctctagaa gaacttagtt tcgacggatt ctagaactag tatgacttat tttcctgaag
     2821 aagttgtaga acacatattt tcctttttac ctgctcaaag agatagaaac accgtctcct
     2881 tggtctgtaa ggtttggtac gaaatcgaaa gattgtctag aagaggtgtt tttgttggta
     2941 attgttacgc agttagagct ggtagagttg ctgcaagatt cccaaacgtt agagcattga
     3001 ctgttaaagg taaacctcat ggtgctgatt tcaatttggt tccacctgat tggggtggtt
     3061 atgctggtcc atggattgaa gctgcagcta gaggttgtca cggtttggaa gaattgagaa
     3121 tgaagagaat ggttgtttct gatgaatcat tggaattgtt agcaagatct tttcctagat
     3181 tcagagcttt ggttttgatc tcatgtgaag gtttttccac cgatggttta gcagctgttg
     3241 cttcacattg taagttgttg agagaattgg atttgcaaga aaacgaagtt gaagatagag
     3301 gtccaagatg gttatcttgt tttcctgatt cctgtacaag tttggtttct ttgaacttcg
     3361 catgtatcaa gggtgaagtt aacgctggta gtttggaaag attagtttct agatcaccaa
     3421 atttgagatc tttgagattg aacagatccg ttagtgttga taccttggca aagatcttgt
     3481 tgagaactcc taatttggaa gatttgggta ctggtaactt gacagatgat ttccaaacag
     3541 aatcatactt caagttgacc tccgctttgg aaaagtgtaa gatgttgaga tctttgtcag
     3601 gtttttggga tgcaagtcca gtttgtttgt cttttatata tcctttgtgt gctcaattga
     3661 ctggtttgaa tttgtcttac gcaccaactt tggatgctag tgatttgaca aagatgatct
     3721 ctagatgtgt taagttgcaa agattgtggg ttttggattg tatctcagat aaaggtttgc
     3781 aagttgttgc ttcttcttgt aaggatttgc aagaattgag agtttttcca agtgatttct
     3841 atgttgcagg ttactctgct gttacagaag aaggtttagt tgcagtttca ttgggttgtc
     3901 ctaagttgaa ctccttgttg tacttctgtc atcaaatgac caacgcagct ttggttactg
     3961 ttgctaaaaa ttgtccaaac ttcactagat tcagattgtg tatcttggaa ccaggtaaac
     4021 ctgatgttgt tacatcacaa cctttagatg aaggttttgg tgcaatcgtt agagagtgta
     4081 agggtttgca aagattgtcc atcagtggtt tgttgacaga taaggttttt atgtacatcg
     4141 gtaaatacgc taagcaattg gaaatgttgt caatcgcatt cgctggtgac tccgataagg
     4201 gtatgatgca tgttatgaac ggttgtaaga acttaagaaa gttggaaatc agagattctc
     4261 catttggtga cgcagctttg ttgggtaatt tcgcaagata tgaaacaatg agatctttgt
     4321 ggatgtccag ttgtaacgtt accttgaaag gttgtcaagt tttagcttca aagatgccaa
     4381 tgttgaacgt tgaagttatt aacgaaagag atggttccaa tgaaatggaa gaaaaccatg
     4441 gtgacttgcc taaagttgaa aagttatacg tctacagaac aaccgcaggt gccagagatg
     4501 atgctcctaa cttcgtcaag attttatgac tcgagtcatg taattagtta tgtcacgctt
     4561 acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga
     4621 agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt
     4681 tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac
     4741 cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcggccggt cgacctgcag
     4801 cgtacgcagt tgtcgaactt ggtttgcaaa gggagaaagt aggagatctc tcttgcgaga
     4861 tgatcccgca ttttcttgaa agctttgcag aggctagcag aattaccctc cacgttgatt
     4921 gtctgcgagg caagaatgat catcaccgta gtgagagtgc gttcaaggct cttgcggttg
     4981 ccataagaga agccacctcg cccaatggta ccaacgatgt tccctccacc aaaggtgttc
     5041 ttatgtagtg acaccgatta tttaaagctg cagcatacga tatatataca tgtgtatata
     5101 tgtataccta tgaatgtcag taagtatgta tacgaacagt atgatactga agatgacaag
     5161 gtaatgcatc attctatacg tgtcattctg aacgaggcgc gctttccttt tttctttttg
     5221 ctttttcttt ttttttctct tgaactcgac ggatctatgc ggtgtgaaat accgcacaga
     5281 tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg
     5341 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     5401 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     5461 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     5521 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     5581 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     5641 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     5701 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     5761 cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     5821 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     5881 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact
     5941 atagggcgaa ttggaggggg gggcccggta ccagcttttg ttccctttag tgagggttaa
     6001 tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     6061 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     6121 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6181 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6241 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     6301 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     6361 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6421 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6481 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6541 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6601 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6661 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6721 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6781 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6841 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     6901 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6961 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     7021 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     7081 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     7141 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     7201 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7261 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     7321 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7381 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7441 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7501 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7561 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7621 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7681 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7741 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7801 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7861 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7921 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7981 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     8041 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     8101 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     8161 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     8221 ggcgtatcac gaggcccttt cgtctcgc
//
