LOCUS       sequence-298652-        7664 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of OsTIR1 and mAID-mCherry protein in DT40 cells under 
            the control of CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7664)
  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 7664)
  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-CMV-CmCherry-mAID
FEATURES             Location/Qualifiers
     source          1..7664
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             546..2270
                     /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             2271..2324
                     /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(2751..2770)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     misc_feature    join(2834..3408,3409..3411,3412..3420)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3001..3018)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3228..3247
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3463..3666
                     /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             3667..4377
                     /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(3812..3830)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4080..4099
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4392..4513
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4429..4448)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4483..4502
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4520..4975)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4657..4678)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4869..4888
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5002..5106
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5108..5465
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5108..5299
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5131..5151)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5316..5451
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5500..6294
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5500..6294
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5554..5573)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6164..6183
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6482..6501)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6526..6573
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6902..7490
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7391..7410
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgctg
      541 ccaccatgac atactttcct gaagaggtcg tcgaacacat ttttagcttc ctgcctgcac
      601 agagagatag aaacacagtg agcctggtct gcaaagtgtg gtacgagatc gaacgcctga
      661 gccggagagg agtgttcgtc ggcaactgct atgctgtgag agcaggcagg gtcgccgcta
      721 ggtttccaaa tgtgcgcgca ctgaccgtca aggggaaacc ccacttcgcc gactttaacc
      781 tggtgccccc tgattgggga ggatacgccg gcccttggat cgaggcagcc gctcgcggct
      841 gtcatggact ggaggaactg cgcatgaagc gaatggtggt ctctgacgaa agtctggagc
      901 tgctggctcg gagcttccct aggtttcgcg cactggtgct gatttcttgc gaaggcttca
      961 gcaccgatgg actggcagcc gtggcctccc actgtaagct gctgcgggag ctggacctcc
     1021 aggagaatga agtggaggat agaggcccca gatggctgtc ttgcttccca gactcatgta
     1081 ccagcctggt gtccctgaac tttgcctgca tcaaaggcga agtgaatgct gggtccctgg
     1141 agcggctggt ctcaagaagc cccaacctga ggtctctgcg gctgaaccgg agcgtgagcg
     1201 tggacactct ggctaagatt ctgctgagaa cccctaacct ggaggatctg ggaaccggca
     1261 atctgacaga cgatttccag acagaatcct actttaaact gacttctgcc ctggagaagt
     1321 gtaaaatgct gaggagtctg tcaggattct gggatgcttc acccgtgtgc ctgagcttta
     1381 tctaccctct gtgtgcacag ctgacaggcc tgaacctgag ctatgcacca accctggacg
     1441 ccagtgatct gacaaagatg atctcacgct gcgtgaaact ccagcgactg tgggtgctgg
     1501 actgtatttc cgataagggg ctccaggtgg tcgccagctc ctgcaaggac ctccaggagc
     1561 tgagagtgtt cccatctgat ttttacgtgg ccggatatag tgctgtcact gaggaaggcc
     1621 tggtggcagt ctcactggga tgcccaaagc tgaacagcct gctgtatttc tgtcatcaga
     1681 tgactaatgc tgcactggtg accgtcgcca agaactgccc taatttcacc cgatttcggc
     1741 tgtgtattct ggaaccaggc aaacccgacg tggtcacatc ccagccactg gatgaagggt
     1801 ttggagctat cgtgagagag tgcaagggac tccagaggct gagcatttcc ggcctgctga
     1861 cagacaaagt gttcatgtac atcggcaagt atgctaagca gctggagatg ctgagcattg
     1921 catttgccgg agactccgat aagggcatga tgcacgtgat gaacgggtgt aagaatctgc
     1981 gaaaactgga aatccgggac agccctttcg gggatgccgc tctgctggga aactttgcca
     2041 gatacgagac aatgaggagc ctgtggatgt ctagttgcaa tgtgactctg aagggctgtc
     2101 aggtcctggc tagtaaaatg cctatgctga acgtggaagt cattaatgag cgggacgggt
     2161 ctaacgaaat ggaggaaaat catggcgacc tgccaaaggt ggagaaactg tatgtgtatc
     2221 ggaccaccgc aggggcaaga gatgatgctc ccaactttgt gaagattctg gagggccgcg
     2281 gcagcctgct gacctgcggc gacgtggagg agaaccccgg ccccatgaaa acatttaaca
     2341 tttctcaaca agatctagaa ttagtagaag tagcgacaga gaagattaca atgctttatg
     2401 aggataataa acatcatgtg ggagcggcaa ttcgtacgaa aacaggagaa atcatttcgg
     2461 cagtacatat tgaagcgtat ataggacgag taactgtttg tgcagaagcc attgcgattg
     2521 gtagtgcagt ttcgaatgga caaaaggatt ttgacacgat tgtagctgtt agacaccctt
     2581 attctgacga agtagataga agtattcgag tggtaagtcc ttgtggtatg tgtagggagt
     2641 tgatttcaga ctatgcacca gattgttttg tgttaataga aatgaatggc aagttagtca
     2701 aaactacgat tgaagaactc attccactca aatatacccg aaattaagga tctttttccc
     2761 tctgccaaaa attatgggga catcatgaag ccccttgagg tggaacgatc attcaagaga
     2821 tccccgggat ccgcccctct ccctcccccc cccctaacgt tactggccga agccgcttgg
     2881 aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca
     2941 atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc
     3001 ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag
     3061 cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg
     3121 gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac
     3181 aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa
     3241 gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc
     3301 tggggcctcg gtacacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc
     3361 ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc
     3421 atgtgcgata tcggagctgg tgcaggcgct ggagcgggtg ccaaggagaa gagtgcttgt
     3481 cctaaagatc cagccaaacc tccggccaag gcacaagttg tgggatggcc accggtgaga
     3541 tcataccgga agaacgtgat ggtttcctgc caaaaatcaa gcggtggccc ggaggcggcg
     3601 gcgttcgtga aggtatcaat ggacggagca ccgtacttga ggaaaatcga tttgaggatg
     3661 tataaaatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     3721 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     3781 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     3841 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     3901 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     3961 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     4021 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     4081 cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag
     4141 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     4201 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     4261 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     4321 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtgacaa
     4381 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4441 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4501 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     4561 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     4621 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     4681 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     4741 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     4801 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     4861 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     4921 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     4981 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5041 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5101 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     5161 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     5221 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     5281 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     5341 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     5401 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     5461 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     5521 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     5581 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     5641 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     5701 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     5761 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     5821 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     5881 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     5941 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     6001 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     6061 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     6121 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     6181 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     6241 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     6301 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     6361 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     6421 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     6481 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     6541 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     6601 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     6661 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     6721 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     6781 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6841 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6901 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6961 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7021 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     7081 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7141 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7201 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7261 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7321 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7381 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7441 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7501 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     7561 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa
     7621 tcaattacgg ggtcattagt tcatagccca tatatggagt tccg
//
