LOCUS       sequence-430391-        7085 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TIR1-9Myc. TIR1 mediates auxin-induced ubiquitination of 
            auxin-inducible degron (AID)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7085)
  AUTHORS   He CW, Cui XF, Ma SJ, Xu Q, Ran YP, Chen WZ, Mu JX, Li H, Zhu J, 
            Gong Q, Xie Z
  TITLE     Membrane recruitment of Atg8 by Hfl1 facilitates turnover of 
            vacuolar membrane proteins in yeast cells approaching stationary 
            phase.
  JOURNAL   BMC Biol. 2021 Jun 4;19(1):117. doi: 10.1186/s12915-021-01048-7.
  PUBMED    34088313
REFERENCE   2  (bases 1 to 7085)
  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: "BMC Biol.";
            date: "2021-06-4"; volume: "19(1):117. doi"; pages: " 
            10.1186/s12915-021-01048-7"
COMMENT     Sequence Label: PADH1-TIR1-9myc-URA3
FEATURES             Location/Qualifiers
     source          1..7085
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     255..274
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     508..525
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    642..663
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(678..683,684..701,702..708)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    716..732
                     /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     721..743
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     740..756
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     740..756
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        777..795
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1141..1542
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             1561..3285
                     /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             3313..3342
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3349..3378
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3385..3414
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3433..3462
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3469..3498
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3505..3534
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3638..3657)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3639..3657)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3664..3681)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3664..3680)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3673..3695)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3821..4276
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3908..3927)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4118..4139
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4407..5210)
                     /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(5211..5426)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5450..5469)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5569..5591
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5629..5647)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5715..5819
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5820..5888,5889..6680)
                     /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(6038..6057)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6851..7085,1..354)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
       61 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
      121 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
      181 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
      241 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
      301 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
      361 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
      421 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
      481 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
      541 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
      601 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
      661 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
      721 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcgaaatt
      781 aaccctcact aaagggaaca aaagctggta ccgggccccc cctcgacggt atcgataagc
      841 ttgatatcga attcgggtgt acaatatgga cttcctcttt tctggcaacc aaacccatac
      901 atcgggattc ctataatacc ttcgttggtc tccctaacat gtaggtggcg gaggggagat
      961 atacaataga acagatacca gacaagacat aatgggctaa acaagactac accaattaca
     1021 ctgcctcatt gatggtggta cataacgaac taatactgta gccctagact tgatagccat
     1081 catcatatcg aagtttcact accctttttc catttgccat ctattgaagt aataataggc
     1141 gcatgcaact tcttttcttt ttttttcttt tctctctccc ccgttgttgt ctcaccatat
     1201 ccgcaatgac aaaaaaatga tggaagacac taaaggaaaa aattaacgac aaagacagca
     1261 ccaacagatg tcgttgttcc agagctgatg aggggtatct cgaagcacac gaaacttttt
     1321 ccttccttca ttcacgcaca ctactctcta atgagcaacg gtatacggcc ttccttccag
     1381 ttacttgaat ttgaaataaa aaaaagtttg ctgtcttgct atcaagtata aatagacctg
     1441 caattattaa tcttttgttt cctcgtcatt gttctcgttc cctttcttcc ttgtttcttt
     1501 ttctgcacaa tatttcaagc tataccaagc atacaatcaa ctatctcata tacaactaga
     1561 atgacgtact tcccggagga ggtggtggag cacatcttca gcttcctgcc ggcgcagcgc
     1621 gaccgcaaca cggtctcgct cgtctgcaag gtgtggtacg agatcgagag gctgagccgc
     1681 cgcggcgtct tcgtgggcaa ctgctacgcc gtgcgcgccg gccgcgtcgc cgcgcggttc
     1741 cccaacgtgc gggcgctcac ggtgaagggg aagccccact tcgccgactt caacctcgtg
     1801 ccccccgact ggggcggcta cgcggggccg tggatcgagg cggccgcgag gggatgccac
     1861 ggcctggagg agctcaggat gaagcggatg gtggtgtccg acgagagcct cgagctgctg
     1921 gctcgctcgt tcccgcggtt cagggctctt gttcttatca gctgcgaggg gttcagcact
     1981 gacgggctag ccgccgtcgc gagccattgc aagcttctga gggagttgga tttgcaggaa
     2041 aatgaagtgg aggatcgagg gcctaggtgg ctttcctgct tccctgattc ctgcacatca
     2101 cttgtctcat tgaattttgc ctgcatcaaa ggggaggtta atgctggttc actggagaga
     2161 cttgttagca ggtccccaaa cctgcggagt ttgaggctga atcgatctgt atcggtagat
     2221 acacttgcaa agatactact gcgtacccct aacttggagg atttggggac agggaatttg
     2281 acagatgact tccaaactga gtcctacttt aagcttacca gtgctctgga gaaatgcaag
     2341 atgttgagga gtttgtctgg attctgggat gcttctcctg tttgcctgtc atttatctac
     2401 cccctgtgtg ctcaactgac aggattgaac ttgagctatg cacccacact tgatgcttct
     2461 gaccttacaa aaatgattag ccgctgtgtg aagctccaac gcctttgggt actggattgt
     2521 atctcggaca aaggcttgca agtggtggcc tccagttgca aagacttgca agaactcagg
     2581 gtatttccat cagatttcta cgtagctggt tattctgcag tgacagagga gggacttgtt
     2641 gcagtatcct tgggctgtcc aaaactgaac tcactactgt acttctgtca ccaaatgact
     2701 aatgctgcac tagttactgt cgccaagaac tgtccaaatt tcacacgatt cagactttgt
     2761 attcttgagc cagggaagcc tgatgttgtg acaagccaac cattagatga aggctttgga
     2821 gctattgttc gtgagtgcaa gggattacaa cgtttgtcaa tatctggtct tctcacagac
     2881 aaagttttca tgtatattgg gaaatatgca aaacaacttg agatgctttc tatagcattt
     2941 gctggtgaca gtgataaggg tatgatgcat gttatgaatg gatgcaagaa tttaaggaaa
     3001 ctggagataa gagatagccc gtttggtgat gctgcactct tggggaattt tgctaggtac
     3061 gagacaatgc gatccctttg gatgtcatct tgcaatgtca cgttaaaggg gtgccaagtc
     3121 cttgcgtcaa agatgccgat gctcaatgtt gaggtcataa atgagcggga tggtagcaat
     3181 gaaatggagg aaaaccatgg agatctgcct aaagtggaga aattatatgt gtaccgcaca
     3241 actgctgggg cgagggatga tgcaccaaat tttgttaaaa tcctagtcga ctccggttct
     3301 gctgctagtg gtgaacaaaa gttgatttct gaagaagatt tgaacggtga acaaaagcta
     3361 atctccgagg aagacttgaa cggtgaacaa aaattaatct cagaagaaga cttgaacgga
     3421 tcctctagag gtgaacaaaa gttgatttct gaagaagatt tgaacggtga acaaaagcta
     3481 atctccgagg aagacttgaa cggtgaacaa aaattaatct cagaagaaga cttgaacgga
     3541 tccactagct aaaaagtgga acgatcattc aagagatccc cgggttaatt aaggcgcgcc
     3601 agatctgcgg ccgccaccgc ggtggagctc caattcgccc tatagtgagt cgtattacaa
     3661 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     3721 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     3781 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca
     3841 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3901 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3961 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4021 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4081 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4141 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     4201 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4261 ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4321 accgcatagg gtaataactg atataattaa attgaagctc taatttgtga gtttagtata
     4381 catgcattta cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct
     4441 tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat
     4501 agtcctcttc caacaataat aatgtcagat cctgtagaga ccacatcatc cacggttcta
     4561 tactgttgac ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt cataatcaac
     4621 caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa
     4681 tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag
     4741 cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt tacttcttct
     4801 gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg
     4861 tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca
     4921 atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt
     4981 agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt
     5041 aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca
     5101 tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca
     5161 acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt
     5221 cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt
     5281 tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct
     5341 tccttctgtt cggagattac cgaatcaaaa aaatttcaag gaaaccgaaa tcaaaaaaaa
     5401 gaataaaaaa aaaatgatga attgaaaagg tggtatggtg cactctcagt acaatctgct
     5461 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     5521 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5581 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     5641 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     5701 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5761 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5821 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5881 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5941 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     6001 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     6061 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     6121 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     6181 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     6241 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6301 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6361 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6421 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6481 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6541 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6601 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6661 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6721 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6781 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6841 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6901 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6961 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     7021 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     7081 cagtg
//
