LOCUS       sequence-296037-        6880 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GAL-OsTIR1(F74G).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6880)
  AUTHORS   Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano 
            R, Nakaoka H, Fukui K, Gamo K, Tominari Y, Takeuchi H, Saga Y, 
            Hayashi KI, Kanemaki MT
  TITLE     The auxin-inducible degron 2 technology provides sharp degradation 
            control in yeast, mammalian cells, and mice.
  JOURNAL   Nat Commun. 2020 Nov 11;11(1):5701. doi: 10.1038/s41467-020-19532-z.
  PUBMED    33177522
REFERENCE   2  (bases 1 to 6880)
  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: "Nat 
            Commun."; date: "2020-11-11"; volume: "11(1):5701. doi"; pages: " 
            10.1038/s41467-020-19532-z"
COMMENT     Sequence Label: pMK419 (GAL-OsTIR1(F74G))
FEATURES             Location/Qualifiers
     source          1..6880
                     /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"
     promoter        2243..2261
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2289..2305
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2290..2306
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2472..3124
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    2676..2793
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3088..3111
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             3140..4867
                     /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(4899..4918)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4900..4918)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4925..4942)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4925..4941)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4934..4956)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5082..5537
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5169..5188)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5379..5400
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5668..6471)
                     /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(6472..6687)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6711..6730)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6830..6852
                     /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 cgccaagctc gaaattaacc ctcactaaag ggaacaaaag ctggtaccgg
     2281 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcgaca ggttatcagc
     2341 aacaacacag tcatatccat tctcaattag ctctaccaca gtgtgtgaac caatgtatcc
     2401 agcaccacct gtaaccaaaa caattttaga agtactttca ctttgtaact gagctgtcat
     2461 ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata
     2521 atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg
     2581 tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag
     2641 taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggtg
     2701 acagccctcc gaaggaagac tctcctccgt gcgtcctcgt cttcaccggt cgcgttcctg
     2761 aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct
     2821 tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaatgaac
     2881 gaatcaaatt aacaaccata ggatgataat gcgattagtt ttttagcctt atttctgggg
     2941 taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat gcaaaaactg
     3001 cataaccact ttaactaata ctttcaacat tttcggtttg tattacttct tattcaaatg
     3061 taataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa
     3121 aaaccccgga tccactagta tgacttattt tcctgaagaa gttgtagaac acatattttc
     3181 ctttttacct gctcaaagag atagaaacac cgtctccttg gtctgtaagg tttggtacga
     3241 aatcgaaaga ttgtctagaa gaggtgtttt tgttggtaat tgttacgcag ttagagctgg
     3301 tagagttgct gcaagattcc caaacgttag agcattgact gttaaaggta aacctcatgg
     3361 tgctgatttc aatttggttc cacctgattg gggtggttat gctggtccat ggattgaagc
     3421 tgcagctaga ggttgtcacg gtttggaaga attgagaatg aagagaatgg ttgtttctga
     3481 tgaatcattg gaattgttag caagatcttt tcctagattc agagctttgg ttttgatctc
     3541 atgtgaaggt ttttccaccg atggtttagc agctgttgct tcacattgta agttgttgag
     3601 agaattggat ttgcaagaaa acgaagttga agatagaggt ccaagatggt tatcttgttt
     3661 tcctgattcc tgtacaagtt tggtttcttt gaacttcgca tgtatcaagg gtgaagttaa
     3721 cgctggtagt ttggaaagat tagtttctag atcaccaaat ttgagatctt tgagattgaa
     3781 cagatccgtt agtgttgata ccttggcaaa gatcttgttg agaactccta atttggaaga
     3841 tttgggtact ggtaacttga cagatgattt ccaaacagaa tcatacttca agttgacctc
     3901 cgctttggaa aagtgtaaga tgttgagatc tttgtcaggt ttttgggatg caagtccagt
     3961 ttgtttgtct tttatatatc ctttgtgtgc tcaattgact ggtttgaatt tgtcttacgc
     4021 accaactttg gatgctagtg atttgacaaa gatgatctct agatgtgtta agttgcaaag
     4081 attgtgggtt ttggattgta tctcagataa aggtttgcaa gttgttgctt cttcttgtaa
     4141 ggatttgcaa gaattgagag tttttccaag tgatttctat gttgcaggtt actctgctgt
     4201 tacagaagaa ggtttagttg cagtttcatt gggttgtcct aagttgaact ccttgttgta
     4261 cttctgtcat caaatgacca acgcagcttt ggttactgtt gctaaaaatt gtccaaactt
     4321 cactagattc agattgtgta tcttggaacc aggtaaacct gatgttgtta catcacaacc
     4381 tttagatgaa ggttttggtg caatcgttag agagtgtaag ggtttgcaaa gattgtccat
     4441 cagtggtttg ttgacagata aggtttttat gtacatcggt aaatacgcta agcaattgga
     4501 aatgttgtca atcgcattcg ctggtgactc cgataagggt atgatgcatg ttatgaacgg
     4561 ttgtaagaac ttaagaaagt tggaaatcag agattctcca tttggtgacg cagctttgtt
     4621 gggtaatttc gcaagatatg aaacaatgag atctttgtgg atgtccagtt gtaacgttac
     4681 cttgaaaggt tgtcaagttt tagcttcaaa gatgccaatg ttgaacgttg aagttattaa
     4741 cgaaagagat ggttccaatg aaatggaaga aaaccatggt gacttgccta aagttgaaaa
     4801 gttatacgtc tacagaacaa ccgcaggtgc cagagatgat gctcctaact tcgtcaagat
     4861 tttatgagcg gccgccaccg cggtggagct ccaattcgcc ctatagtgag tcgtattaca
     4921 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     4981 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     5041 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gacgcgccct gtagcggcgc
     5101 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     5161 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5221 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5281 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5341 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5401 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     5461 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5521 attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     5581 caccgcatag ggtaataact gatataatta aattgaagct ctaatttgtg agtttagtat
     5641 acatgcattt acttataata cagtttttta gttttgctgg ccgcatcttc tcaaatatgc
     5701 ttcccagcct gcttttctgt aacgttcacc ctctacctta gcatcccttc cctttgcaaa
     5761 tagtcctctt ccaacaataa taatgtcaga tcctgtagag accacatcat ccacggttct
     5821 atactgttga cccaatgcgt ctcccttgtc atctaaaccc acaccgggtg tcataatcaa
     5881 ccaatcgtaa ccttcatctc ttccacccat gtctctttga gcaataaagc cgataacaaa
     5941 atctttgtcg ctcttcgcaa tgtcaacagt acccttagta tattctccag tagataggga
     6001 gcccttgcat gacaattctg ctaacatcaa aaggcctcta ggttcctttg ttacttcttc
     6061 tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc acaccgtgtg cattcgtaat
     6121 gtctgcccat tctgctattc tgtatacacc cgcagagtac tgcaatttga ctgtattacc
     6181 aatgtcagca aattttctgt cttcgaagag taaaaaattg tacttggcgg ataatgcctt
     6241 tagcggctta actgtgccct ccatggaaaa atcagtcaag atatccacat gtgtttttag
     6301 taaacaaatt ttgggaccta atgcttcaac taactccagt aattccttgg tggtacgaac
     6361 atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg atattaaata gcttggcagc
     6421 aacaggacta ggatgagtag cagcacgttc cttatatgta gctttcgaca tgatttatct
     6481 tcgtttcctg caggtttttg ttctgtgcag ttgggttaag aatactgggc aatttcatgt
     6541 ttcttcaaca ctacatatgc gtatatatac caatctaagt ctgtgctcct tccttcgttc
     6601 ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa ggaaaccgaa atcaaaaaaa
     6661 agaataaaaa aaaaatgatg aattgaaaag gtggtatggt gcactctcag tacaatctgc
     6721 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     6781 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     6841 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
//
