LOCUS       sequence-298651-        7655 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of OsTIR1 and mScarlet-mAID 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 7655)
  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 7655)
  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-NmScarlet-mAID
FEATURES             Location/Qualifiers
     source          1..7655
                     /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             3433..4128
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(3566..3584)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3834..3853
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4129..4332
                     /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    4383..4504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4420..4439)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4474..4493
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4511..4966)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4648..4669)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4860..4879
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4993..5097
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5099..5456
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5099..5290
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5122..5142)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5307..5442
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5491..6285
                     /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             5491..6285
                     /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(5545..5564)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6155..6174
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6473..6492)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6517..6564
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6893..7481
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7382..7401
                     /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 acatggtgag caagggcgag gcagtgatca aggagttcat gcggttcaag
     3481 gtgcacatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc
     3541 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     3601 ttctcctggg acatcctgtc ccctcagttc atgtacggct ccagggcctt catcaagcac
     3661 cccgccgaca tccccgacta ctataagcag tccttccccg agggcttcaa gtgggagcgc
     3721 gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc aggacacctc cctggaggac
     3781 ggcaccctga tctacaaggt gaagctccgc ggcaccaact tccctcctga cggccccgta
     3841 atgcagaaga agacaatggg ctgggaagcg tccaccgagc ggttgtaccc cgaggacggc
     3901 gtgctgaagg gcgacattaa gatggccctg cgcctgaagg acggcggccg ctacctggcg
     3961 gacttcaaga ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc ctacaacgtc
     4021 gaccgcaagt tggacatcac ctcccacaac gaggactaca ccgtggtgga acagtacgaa
     4081 cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaagaa ggagaagagt
     4141 gcttgtccta aagatccagc caaacctccg gccaaggcac aagttgtggg atggccaccg
     4201 gtgagatcat accggaagaa cgtgatggtt tcctgccaaa aatcaagcgg tggcccggag
     4261 gcggcggcgt tcgtgaaggt atcaatggac ggagcaccgt acttgaggaa aatcgatttg
     4321 aggatgtata aaggagctgg tgcaggcgct ggagcgggtg ccgatatcca attgttgttg
     4381 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4441 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4501 cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     4561 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     4621 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     4681 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     4741 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     4801 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     4861 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     4921 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt
     4981 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5041 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct
     5101 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     5161 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     5221 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     5281 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     5341 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     5401 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc
     5461 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     5521 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     5581 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     5641 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
     5701 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     5761 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     5821 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     5881 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     5941 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     6001 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     6061 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     6121 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     6181 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     6241 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     6301 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     6361 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     6421 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac
     6481 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     6541 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     6601 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     6661 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     6721 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat
     6781 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6841 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6901 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6961 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     7021 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     7081 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     7141 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     7201 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     7261 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     7321 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7381 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7441 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7501 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     7561 ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg
     7621 gggtcattag ttcatagccc atatatggag ttccg
//
