LOCUS       sequence-272989-        7367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of OsTIR1 and GFP-mAID 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 7367)
  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 7367)
  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-NGFP-mAID
FEATURES             Location/Qualifiers
     source          1..7367
                     /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             join(2884..2886,2887..2889,2890..3600)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2884..2886,2887..2889,2890..3600)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2929..2950)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3190..3209)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3537..3558
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3601..3804
                     /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"
     polyA_signal    3855..3976
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3892..3911)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3946..3965
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4130..4718)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4210..4229)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4945..5736,5737..5805))
                     /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    5910..5958
                     /note="synthetic polyadenylation signal"
     misc_feature    5972..6063
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6012..6031
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        6158..7340
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6389..7331
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7288..7308
                     /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 aacatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2941 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     3001 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     3061 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     3121 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     3181 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     3241 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     3301 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     3361 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     3421 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     3481 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     3541 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3601 aaggagaaga gtgcttgtcc taaagatcca gccaaacctc cggccaaggc acaagttgtg
     3661 ggatggccac cggtgagatc ataccggaag aacgtgatgg tttcctgcca aaaatcaagc
     3721 ggtggcccgg aggcggcggc gttcgtgaag gtatcaatgg acggagcacc gtacttgagg
     3781 aaaatcgatt tgaggatgta taaaggagct ggtgcaggcg ctggagcggg tgccgatatc
     3841 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3901 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3961 tcatcaatgt atcttagcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4021 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4081 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4141 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4201 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4261 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4321 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4381 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4441 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4501 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4561 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4621 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4681 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4741 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4801 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4861 gtatatatga gtaaacttgg tctgacagcc cctgcgcaac gtggcaggaa gcgcgcgctg
     4921 gcaaatgcta aaccactgca gtggttacca gtgcttgatc agtgaggcac cgatctcagc
     4981 gatctgccta tttcgttcgt ccatagtggc ctgactcccc gtcgtgtaga tcactacgat
     5041 tcgtgagggc ttaccatcag gccccagcgc agcaatgatg ccgcgagagc cgcgttcacc
     5101 ggcccccgat ttgtcagcaa tgaaccagcc agcagggagg gccgagcgaa gaagtggtcc
     5161 tgctactttg tccgcctcca tccagtctat gagctgctgt cgtgatgcta gagtaagaag
     5221 ttcgccagtg agtagtttcc gaagagttgt ggccattgct actggcatcg tggtatcacg
     5281 ctcgtcgttc ggtatggctt cgttcaactc tggttcccag cggtcaagcc gggtcacatg
     5341 atcacccata ttatgaagaa atgcagtcag ctccttaggg cctccgatcg ttgtcagaag
     5401 taagttggcc gcggtgttgt cgctcatggt aatggcagca ctacacaatt ctcttaccgt
     5461 catgccatcc gtaagatgct tttccgtgac cggcgagtac tcaaccaagt cgttttgtga
     5521 gtagtgtata cggcgaccaa gctgctcttg cccggcgtct atacgggaca acaccgcgcc
     5581 acatagcagt actttgaaag tgctcatcat cgggaatcgt tcttcggggc ggaaagactc
     5641 aaggatcttg ccgctattga gatccagttc gatatagccc actcttgcac ccagttgatc
     5701 ttcagcatct tttactttca ccagcgtttc ggggtgtgca aaaacaggca agcaaaatgc
     5761 cgcaaagaag ggaatgagtg cgacacgaaa atgttggatg ctcatactcg tcctttttca
     5821 atattattga agcatttatc agggttacta gtacgtctct caaggataag taagtaatat
     5881 taaggtacgg gaggtattgg acaggccgca ataaaatatc tttattttca ttacatctgt
     5941 gtgttggttt tttgtgtgaa tcgatagtac taacatacgc tctccatcaa aacaaaacga
     6001 aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag tgcaggtgcc agaacatttc
     6061 tctggcctaa ctaagctttg caaagatgga taaagtttta aacagagagg aatctttgca
     6121 gctaatggac cttctaggtc ttgaaaggag tgcgtgaggc tccggtgccc gtcagtgggc
     6181 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
     6241 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     6301 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     6361 tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg
     6421 cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacgcccc tggctgcagt
     6481 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
     6541 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
     6601 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
     6661 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
     6721 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
     6781 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
     6841 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
     6901 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
     6961 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
     7021 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
     7081 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga
     7141 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
     7201 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
     7261 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
     7321 ttccatttca ggtgtcgtga attgcggaag gccgtcaagg ccacgtg
//
