LOCUS       sequence-272990-        7355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of OsTIR1 and mAID-mCherry protein in mammalian cells 
            under the control of EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7355)
  AUTHORS   Nishimura K, Yamada R, Hagihara S, Iwasaki R, Uchida N, Kamura T, 
            Takahashi K, Torii KU, Fukagawa T
  TITLE     A super-sensitive auxin-inducible degron system with an engineered 
            auxin-TIR1 pair.
  JOURNAL   Nucleic Acids Res. 2020 Oct 9;48(18):e108. doi: 10.1093/nar/gkaa748.
  PUBMED    32941625
REFERENCE   2  (bases 1 to 7355)
  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: "Nucleic 
            Acids Res."; date: "2020-10-9"; volume: "48(18):e108. doi"; pages: "
            10.1093/nar/gkaa748"
COMMENT     Sequence Label: pAID1.2-EF1a-CmCherry-mAID
FEATURES             Location/Qualifiers
     source          1..7355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             24..1748
                     /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             1749..1802
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(2229..2248)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     misc_feature    join(2285..2859,2860..2862,2863..2871)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2452..2469)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2679..2698
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2914..3117
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             3118..3828
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3263..3281)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3531..3550
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3843..3964
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3880..3899)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3934..3953
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4118..4706)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4198..4217)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4933..5724,5725..5793))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     polyA_signal    5898..5946
                     /note="synthetic polyadenylation signal"
     misc_feature    5960..6051
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6000..6019
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        6146..7328
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6377..7319
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7276..7296
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 tcttgtccag aggcgctgcc accatgacat actttcctga agaggtcgtc gaacacattt
       61 ttagcttcct gcctgcacag agagatagaa acacagtgag cctggtctgc aaagtgtggt
      121 acgagatcga acgcctgagc cggagaggag tgttcgtcgg caactgctat gctgtgagag
      181 caggcagggt cgccgctagg tttccaaatg tgcgcgcact gaccgtcaag gggaaacccc
      241 acttcgccga ctttaacctg gtgccccctg attggggagg atacgccggc ccttggatcg
      301 aggcagccgc tcgcggctgt catggactgg aggaactgcg catgaagcga atggtggtct
      361 ctgacgaaag tctggagctg ctggctcgga gcttccctag gtttcgcgca ctggtgctga
      421 tttcttgcga aggcttcagc accgatggac tggcagccgt ggcctcccac tgtaagctgc
      481 tgcgggagct ggacctccag gagaatgaag tggaggatag aggccccaga tggctgtctt
      541 gcttcccaga ctcatgtacc agcctggtgt ccctgaactt tgcctgcatc aaaggcgaag
      601 tgaatgctgg gtccctggag cggctggtct caagaagccc caacctgagg tctctgcggc
      661 tgaaccggag cgtgagcgtg gacactctgg ctaagattct gctgagaacc cctaacctgg
      721 aggatctggg aaccggcaat ctgacagacg atttccagac agaatcctac tttaaactga
      781 cttctgccct ggagaagtgt aaaatgctga ggagtctgtc aggattctgg gatgcttcac
      841 ccgtgtgcct gagctttatc taccctctgt gtgcacagct gacaggcctg aacctgagct
      901 atgcaccaac cctggacgcc agtgatctga caaagatgat ctcacgctgc gtgaaactcc
      961 agcgactgtg ggtgctggac tgtatttccg ataaggggct ccaggtggtc gccagctcct
     1021 gcaaggacct ccaggagctg agagtgttcc catctgattt ttacgtggcc ggatatagtg
     1081 ctgtcactga ggaaggcctg gtggcagtct cactgggatg cccaaagctg aacagcctgc
     1141 tgtatttctg tcatcagatg actaatgctg cactggtgac cgtcgccaag aactgcccta
     1201 atttcacccg atttcggctg tgtattctgg aaccaggcaa acccgacgtg gtcacatccc
     1261 agccactgga tgaagggttt ggagctatcg tgagagagtg caagggactc cagaggctga
     1321 gcatttccgg cctgctgaca gacaaagtgt tcatgtacat cggcaagtat gctaagcagc
     1381 tggagatgct gagcattgca tttgccggag actccgataa gggcatgatg cacgtgatga
     1441 acgggtgtaa gaatctgcga aaactggaaa tccgggacag ccctttcggg gatgccgctc
     1501 tgctgggaaa ctttgccaga tacgagacaa tgaggagcct gtggatgtct agttgcaatg
     1561 tgactctgaa gggctgtcag gtcctggcta gtaaaatgcc tatgctgaac gtggaagtca
     1621 ttaatgagcg ggacgggtct aacgaaatgg aggaaaatca tggcgacctg ccaaaggtgg
     1681 agaaactgta tgtgtatcgg accaccgcag gggcaagaga tgatgctccc aactttgtga
     1741 agattctgga gggccgcggc agcctgctga cctgcggcga cgtggaggag aaccccggcc
     1801 ccatgaaaac atttaacatt tctcaacaag atctagaatt agtagaagta gcgacagaga
     1861 agattacaat gctttatgag gataataaac atcatgtggg agcggcaatt cgtacgaaaa
     1921 caggagaaat catttcggca gtacatattg aagcgtatat aggacgagta actgtttgtg
     1981 cagaagccat tgcgattggt agtgcagttt cgaatggaca aaaggatttt gacacgattg
     2041 tagctgttag acacccttat tctgacgaag tagatagaag tattcgagtg gtaagtcctt
     2101 gtggtatgtg tagggagttg atttcagact atgcaccaga ttgttttgtg ttaatagaaa
     2161 tgaatggcaa gttagtcaaa actacgattg aagaactcat tccactcaaa tatacccgaa
     2221 attaaggatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgaggga
     2281 tccgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     2341 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     2401 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     2461 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     2521 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     2581 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     2641 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     2701 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     2761 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     2821 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac catgtgcgat
     2881 atcggagctg gtgcaggcgc tggagcgggt gccaaggaga agagtgcttg tcctaaagat
     2941 ccagccaaac ctccggccaa ggcacaagtt gtgggatggc caccggtgag atcataccgg
     3001 aagaacgtga tggtttcctg ccaaaaatca agcggtggcc cggaggcggc ggcgttcgtg
     3061 aaggtatcaa tggacggagc accgtacttg aggaaaatcg atttgaggat gtataaaatg
     3121 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     3181 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     3241 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     3301 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     3361 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     3421 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     3481 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     3541 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     3601 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     3661 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     3721 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     3781 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtgaca attgttgttg
     3841 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3901 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3961 cttagcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4021 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4081 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4141 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4201 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4261 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4321 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4381 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4441 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4501 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4561 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4621 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4681 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4741 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4801 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4861 aaacttggtc tgacagcccc tgcgcaacgt ggcaggaagc gcgcgctggc aaatgctaaa
     4921 ccactgcagt ggttaccagt gcttgatcag tgaggcaccg atctcagcga tctgcctatt
     4981 tcgttcgtcc atagtggcct gactccccgt cgtgtagatc actacgattc gtgagggctt
     5041 accatcaggc cccagcgcag caatgatgcc gcgagagccg cgttcaccgg cccccgattt
     5101 gtcagcaatg aaccagccag cagggagggc cgagcgaaga agtggtcctg ctactttgtc
     5161 cgcctccatc cagtctatga gctgctgtcg tgatgctaga gtaagaagtt cgccagtgag
     5221 tagtttccga agagttgtgg ccattgctac tggcatcgtg gtatcacgct cgtcgttcgg
     5281 tatggcttcg ttcaactctg gttcccagcg gtcaagccgg gtcacatgat cacccatatt
     5341 atgaagaaat gcagtcagct ccttagggcc tccgatcgtt gtcagaagta agttggccgc
     5401 ggtgttgtcg ctcatggtaa tggcagcact acacaattct cttaccgtca tgccatccgt
     5461 aagatgcttt tccgtgaccg gcgagtactc aaccaagtcg ttttgtgagt agtgtatacg
     5521 gcgaccaagc tgctcttgcc cggcgtctat acgggacaac accgcgccac atagcagtac
     5581 tttgaaagtg ctcatcatcg ggaatcgttc ttcggggcgg aaagactcaa ggatcttgcc
     5641 gctattgaga tccagttcga tatagcccac tcttgcaccc agttgatctt cagcatcttt
     5701 tactttcacc agcgtttcgg ggtgtgcaaa aacaggcaag caaaatgccg caaagaaggg
     5761 aatgagtgcg acacgaaaat gttggatgct catactcgtc ctttttcaat attattgaag
     5821 catttatcag ggttactagt acgtctctca aggataagta agtaatatta aggtacggga
     5881 ggtattggac aggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt
     5941 tgtgtgaatc gatagtacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac
     6001 tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc tggcctaact
     6061 aagctttgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct
     6121 tctaggtctt gaaaggagtg cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc
     6181 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     6241 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     6301 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     6361 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
     6421 ttatggccct tgcgtgcctt gaattacttc cacgcccctg gctgcagtac gtgattcttg
     6481 atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc
     6541 cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
     6601 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt
     6661 gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc
     6721 tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc
     6781 gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt
     6841 ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct
     6901 gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
     6961 gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt
     7021 cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg
     7081 agtaccgggc gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt
     7141 taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg
     7201 aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg
     7261 gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg
     7321 tgtcgtgaat tgcggaaggc cgtcaaggcc acgtg
//
