LOCUS       sequence-299845-        7590 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 7590)
  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 7590)
  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..7590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1230..2954
                     /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             2955..3008
                     /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(3435..3454)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     misc_feature    join(3491..4065,4066..4068,4069..4077)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3658..3675)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3885..3904
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4120..4323
                     /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(4324..4326,4327..4329,4330..5043)
                     /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(4324..4326,4327..4329,4330..5040)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4369..4390)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4630..4649)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4977..4998
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5058..5179
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5095..5114)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5149..5168
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5333..5921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5413..5432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6148..6939,6940..7008))
                     /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    7113..7161
                     /note="synthetic polyadenylation signal"
     misc_feature    7175..7266
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     7215..7234
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        7286..7496
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        7308..7504
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7355..7490
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgaa ttgcggaagg ccgtcaaggc
     1201 cacgtgtctt gtccagaggc gctgccacca tgacatactt tcctgaagag gtcgtcgaac
     1261 acatttttag cttcctgcct gcacagagag atagaaacac agtgagcctg gtctgcaaag
     1321 tgtggtacga gatcgaacgc ctgagccgga gaggagtgtt cgtcggcaac tgctatgctg
     1381 tgagagcagg cagggtcgcc gctaggtttc caaatgtgcg cgcactgacc gtcaagggga
     1441 aaccccactt cgccgacttt aacctggtgc cccctgattg gggaggatac gccggccctt
     1501 ggatcgaggc agccgctcgc ggctgtcatg gactggagga actgcgcatg aagcgaatgg
     1561 tggtctctga cgaaagtctg gagctgctgg ctcggagctt ccctaggttt cgcgcactgg
     1621 tgctgatttc ttgcgaaggc ttcagcaccg atggactggc agccgtggcc tcccactgta
     1681 agctgctgcg ggagctggac ctccaggaga atgaagtgga ggatagaggc cccagatggc
     1741 tgtcttgctt cccagactca tgtaccagcc tggtgtccct gaactttgcc tgcatcaaag
     1801 gcgaagtgaa tgctgggtcc ctggagcggc tggtctcaag aagccccaac ctgaggtctc
     1861 tgcggctgaa ccggagcgtg agcgtggaca ctctggctaa gattctgctg agaaccccta
     1921 acctggagga tctgggaacc ggcaatctga cagacgattt ccagacagaa tcctacttta
     1981 aactgacttc tgccctggag aagtgtaaaa tgctgaggag tctgtcagga ttctgggatg
     2041 cttcacccgt gtgcctgagc tttatctacc ctctgtgtgc acagctgaca ggcctgaacc
     2101 tgagctatgc accaaccctg gacgccagtg atctgacaaa gatgatctca cgctgcgtga
     2161 aactccagcg actgtgggtg ctggactgta tttccgataa ggggctccag gtggtcgcca
     2221 gctcctgcaa ggacctccag gagctgagag tgttcccatc tgatttttac gtggccggat
     2281 atagtgctgt cactgaggaa ggcctggtgg cagtctcact gggatgccca aagctgaaca
     2341 gcctgctgta tttctgtcat cagatgacta atgctgcact ggtgaccgtc gccaagaact
     2401 gccctaattt cacccgattt cggctgtgta ttctggaacc aggcaaaccc gacgtggtca
     2461 catcccagcc actggatgaa gggtttggag ctatcgtgag agagtgcaag ggactccaga
     2521 ggctgagcat ttccggcctg ctgacagaca aagtgttcat gtacatcggc aagtatgcta
     2581 agcagctgga gatgctgagc attgcatttg ccggagactc cgataagggc atgatgcacg
     2641 tgatgaacgg gtgtaagaat ctgcgaaaac tggaaatccg ggacagccct ttcggggatg
     2701 ccgctctgct gggaaacttt gccagatacg agacaatgag gagcctgtgg atgtctagtt
     2761 gcaatgtgac tctgaagggc tgtcaggtcc tggctagtaa aatgcctatg ctgaacgtgg
     2821 aagtcattaa tgagcgggac gggtctaacg aaatggagga aaatcatggc gacctgccaa
     2881 aggtggagaa actgtatgtg tatcggacca ccgcaggggc aagagatgat gctcccaact
     2941 ttgtgaagat tctggagggc cgcggcagcc tgctgacctg cggcgacgtg gaggagaacc
     3001 ccggccccat gaaaacattt aacatttctc aacaagatct agaattagta gaagtagcga
     3061 cagagaagat tacaatgctt tatgaggata ataaacatca tgtgggagcg gcaattcgta
     3121 cgaaaacagg agaaatcatt tcggcagtac atattgaagc gtatatagga cgagtaactg
     3181 tttgtgcaga agccattgcg attggtagtg cagtttcgaa tggacaaaag gattttgaca
     3241 cgattgtagc tgttagacac ccttattctg acgaagtaga tagaagtatt cgagtggtaa
     3301 gtccttgtgg tatgtgtagg gagttgattt cagactatgc accagattgt tttgtgttaa
     3361 tagaaatgaa tggcaagtta gtcaaaacta cgattgaaga actcattcca ctcaaatata
     3421 cccgaaatta aggatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt
     3481 gagggatccg cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat
     3541 aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg
     3601 tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc
     3661 tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt
     3721 cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg
     3781 acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac
     3841 cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg
     3901 tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg
     3961 ggcctcggta cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc
     4021 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg
     4081 tgcgatatcg gagctggtgc aggcgctgga gcgggtgcca aggagaagag tgcttgtcct
     4141 aaagatccag ccaaacctcc ggccaaggca caagttgtgg gatggccacc ggtgagatca
     4201 taccggaaga acgtgatggt ttcctgccaa aaatcaagcg gtggcccgga ggcggcggcg
     4261 ttcgtgaagg tatcaatgga cggagcaccg tacttgagga aaatcgattt gaggatgtat
     4321 aaaatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     4381 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     4441 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     4501 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     4561 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     4621 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     4681 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     4741 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     4801 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     4861 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     4921 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     4981 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     5041 tgacaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5101 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5161 aactcatcaa tgtatcttag cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5221 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5281 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5341 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5401 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5461 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5521 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5581 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5641 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5701 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5761 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5821 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5881 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5941 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6001 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6061 aaagtatata tgagtaaact tggtctgaca gcccctgcgc aacgtggcag gaagcgcgcg
     6121 ctggcaaatg ctaaaccact gcagtggtta ccagtgcttg atcagtgagg caccgatctc
     6181 agcgatctgc ctatttcgtt cgtccatagt ggcctgactc cccgtcgtgt agatcactac
     6241 gattcgtgag ggcttaccat caggccccag cgcagcaatg atgccgcgag agccgcgttc
     6301 accggccccc gatttgtcag caatgaacca gccagcaggg agggccgagc gaagaagtgg
     6361 tcctgctact ttgtccgcct ccatccagtc tatgagctgc tgtcgtgatg ctagagtaag
     6421 aagttcgcca gtgagtagtt tccgaagagt tgtggccatt gctactggca tcgtggtatc
     6481 acgctcgtcg ttcggtatgg cttcgttcaa ctctggttcc cagcggtcaa gccgggtcac
     6541 atgatcaccc atattatgaa gaaatgcagt cagctcctta gggcctccga tcgttgtcag
     6601 aagtaagttg gccgcggtgt tgtcgctcat ggtaatggca gcactacaca attctcttac
     6661 cgtcatgcca tccgtaagat gcttttccgt gaccggcgag tactcaacca agtcgttttg
     6721 tgagtagtgt atacggcgac caagctgctc ttgcccggcg tctatacggg acaacaccgc
     6781 gccacatagc agtactttga aagtgctcat catcgggaat cgttcttcgg ggcggaaaga
     6841 ctcaaggatc ttgccgctat tgagatccag ttcgatatag cccactcttg cacccagttg
     6901 atcttcagca tcttttactt tcaccagcgt ttcggggtgt gcaaaaacag gcaagcaaaa
     6961 tgccgcaaag aagggaatga gtgcgacacg aaaatgttgg atgctcatac tcgtcctttt
     7021 tcaatattat tgaagcattt atcagggtta ctagtacgtc tctcaaggat aagtaagtaa
     7081 tattaaggta cgggaggtat tggacaggcc gcaataaaat atctttattt tcattacatc
     7141 tgtgtgttgg ttttttgtgt gaatcgatag tactaacata cgctctccat caaaacaaaa
     7201 cgaaacaaaa caaactagca aaataggctg tccccagtgc aagtgcaggt gccagaacat
     7261 ttctctggcc taaccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     7321 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     7381 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     7441 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     7501 caaaaagctt tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg
     7561 accttctagg tcttgaaagg agtgggaatt
//
