LOCUS       sequence-272988-        7364 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of OsTIR1 and mAID-GFP 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 7364)
  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 7364)
  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-CGFP-mAID
FEATURES             Location/Qualifiers
     source          1..7364
                     /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             join(3118..3120,3121..3123,3124..3837)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3118..3120,3121..3123,3124..3834)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3163..3184)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3424..3443)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3771..3792
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3852..3973
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3889..3908)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3943..3962
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4127..4715)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4207..4226)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4942..5733,5734..5802))
                     /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    5907..5955
                     /note="synthetic polyadenylation signal"
     misc_feature    5969..6060
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6009..6028
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        6155..7337
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6386..7328
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7285..7305
                     /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 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     3181 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     3241 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     3301 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     3361 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     3421 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     3481 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     3541 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     3601 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     3661 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     3721 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     3781 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtgacaa
     3841 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3901 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3961 tcaatgtatc ttagcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4021 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4081 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4141 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4201 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4261 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4321 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4381 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4441 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4501 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4561 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4621 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4681 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4741 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4801 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4861 tatatgagta aacttggtct gacagcccct gcgcaacgtg gcaggaagcg cgcgctggca
     4921 aatgctaaac cactgcagtg gttaccagtg cttgatcagt gaggcaccga tctcagcgat
     4981 ctgcctattt cgttcgtcca tagtggcctg actccccgtc gtgtagatca ctacgattcg
     5041 tgagggctta ccatcaggcc ccagcgcagc aatgatgccg cgagagccgc gttcaccggc
     5101 ccccgatttg tcagcaatga accagccagc agggagggcc gagcgaagaa gtggtcctgc
     5161 tactttgtcc gcctccatcc agtctatgag ctgctgtcgt gatgctagag taagaagttc
     5221 gccagtgagt agtttccgaa gagttgtggc cattgctact ggcatcgtgg tatcacgctc
     5281 gtcgttcggt atggcttcgt tcaactctgg ttcccagcgg tcaagccggg tcacatgatc
     5341 acccatatta tgaagaaatg cagtcagctc cttagggcct ccgatcgttg tcagaagtaa
     5401 gttggccgcg gtgttgtcgc tcatggtaat ggcagcacta cacaattctc ttaccgtcat
     5461 gccatccgta agatgctttt ccgtgaccgg cgagtactca accaagtcgt tttgtgagta
     5521 gtgtatacgg cgaccaagct gctcttgccc ggcgtctata cgggacaaca ccgcgccaca
     5581 tagcagtact ttgaaagtgc tcatcatcgg gaatcgttct tcggggcgga aagactcaag
     5641 gatcttgccg ctattgagat ccagttcgat atagcccact cttgcaccca gttgatcttc
     5701 agcatctttt actttcacca gcgtttcggg gtgtgcaaaa acaggcaagc aaaatgccgc
     5761 aaagaaggga atgagtgcga cacgaaaatg ttggatgctc atactcgtcc tttttcaata
     5821 ttattgaagc atttatcagg gttactagta cgtctctcaa ggataagtaa gtaatattaa
     5881 ggtacgggag gtattggaca ggccgcaata aaatatcttt attttcatta catctgtgtg
     5941 ttggtttttt gtgtgaatcg atagtactaa catacgctct ccatcaaaac aaaacgaaac
     6001 aaaacaaact agcaaaatag gctgtcccca gtgcaagtgc aggtgccaga acatttctct
     6061 ggcctaacta agctttgcaa agatggataa agttttaaac agagaggaat ctttgcagct
     6121 aatggacctt ctaggtcttg aaaggagtgc gtgaggctcc ggtgcccgtc agtgggcaga
     6181 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc
     6241 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     6301 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     6361 caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct
     6421 ctttacgggt tatggccctt gcgtgccttg aattacttcc acgcccctgg ctgcagtacg
     6481 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
     6541 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
     6601 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
     6661 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     6721 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     6781 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     6841 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
     6901 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     6961 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     7021 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     7081 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     7141 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     7201 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     7261 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     7321 catttcaggt gtcgtgaatt gcggaaggcc gtcaaggcca cgtg
//
