LOCUS       sequence-272995-        7637 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 7637)
  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 7637)
  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..7637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             34..237
                     /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             238..948
                     /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(383..401)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     651..670
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    963..1084
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1000..1019)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1054..1073
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1091..1546)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1228..1249)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1440..1459
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1573..1677
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1679..2036
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1679..1870
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1702..1722)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1887..2022
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2071..2865
                     /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             2071..2865
                     /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(2125..2144)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2735..2754
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3053..3072)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3097..3144
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3473..4061
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3962..3981
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4236..4539
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4540..4743
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4694..4714
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4781..6505
                     /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             6506..6559
                     /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(6986..7005)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     misc_feature    join(7042..7616,7617..7619,7620..7628)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7209..7226)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7436..7455
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
ORIGIN
        1 atcggagctg gtgcaggcgc tggagcgggt gccaaggaga agagtgcttg tcctaaagat
       61 ccagccaaac ctccggccaa ggcacaagtt gtgggatggc caccggtgag atcataccgg
      121 aagaacgtga tggtttcctg ccaaaaatca agcggtggcc cggaggcggc ggcgttcgtg
      181 aaggtatcaa tggacggagc accgtacttg aggaaaatcg atttgaggat gtataaaatg
      241 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
      301 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
      361 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
      421 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
      481 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
      541 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
      601 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
      661 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
      721 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
      781 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
      841 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
      901 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtgaca attgttgttg
      961 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     1021 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     1081 cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     1141 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     1201 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     1261 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     1321 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     1381 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     1441 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     1501 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt
     1561 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1621 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct
     1681 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     1741 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     1801 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     1861 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     1921 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     1981 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc
     2041 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     2101 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     2161 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     2221 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
     2281 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     2341 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     2401 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     2461 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     2521 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     2581 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     2641 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     2701 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     2761 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     2821 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     2881 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     2941 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     3001 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac
     3061 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     3121 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     3181 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     3241 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     3301 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat
     3361 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3421 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3481 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3541 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3601 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3661 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3721 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3781 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3841 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3901 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3961 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4021 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4081 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4141 ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg
     4201 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     4261 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     4321 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     4381 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     4441 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     4501 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     4561 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     4621 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     4681 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     4741 gctggtttag tgaaccgtca gatccgctag cgctgccacc atgacatact ttcctgaaga
     4801 ggtcgtcgaa cacattttta gcttcctgcc tgcacagaga gatagaaaca cagtgagcct
     4861 ggtctgcaaa gtgtggtacg agatcgaacg cctgagccgg agaggagtgt tcgtcggcaa
     4921 ctgctatgct gtgagagcag gcagggtcgc cgctaggttt ccaaatgtgc gcgcactgac
     4981 cgtcaagggg aaaccccact tcgccgactt taacctggtg ccccctgatt ggggaggata
     5041 cgccggccct tggatcgagg cagccgctcg cggctgtcat ggactggagg aactgcgcat
     5101 gaagcgaatg gtggtctctg acgaaagtct ggagctgctg gctcggagct tccctaggtt
     5161 tcgcgcactg gtgctgattt cttgcgaagg cttcagcacc gatggactgg cagccgtggc
     5221 ctcccactgt aagctgctgc gggagctgga cctccaggag aatgaagtgg aggatagagg
     5281 ccccagatgg ctgtcttgct tcccagactc atgtaccagc ctggtgtccc tgaactttgc
     5341 ctgcatcaaa ggcgaagtga atgctgggtc cctggagcgg ctggtctcaa gaagccccaa
     5401 cctgaggtct ctgcggctga accggagcgt gagcgtggac actctggcta agattctgct
     5461 gagaacccct aacctggagg atctgggaac cggcaatctg acagacgatt tccagacaga
     5521 atcctacttt aaactgactt ctgccctgga gaagtgtaaa atgctgagga gtctgtcagg
     5581 attctgggat gcttcacccg tgtgcctgag ctttatctac cctctgtgtg cacagctgac
     5641 aggcctgaac ctgagctatg caccaaccct ggacgccagt gatctgacaa agatgatctc
     5701 acgctgcgtg aaactccagc gactgtgggt gctggactgt atttccgata aggggctcca
     5761 ggtggtcgcc agctcctgca aggacctcca ggagctgaga gtgttcccat ctgattttta
     5821 cgtggccgga tatagtgctg tcactgagga aggcctggtg gcagtctcac tgggatgccc
     5881 aaagctgaac agcctgctgt atttctgtca tcagatgact aatgctgcac tggtgaccgt
     5941 cgccaagaac tgccctaatt tcacccgatt tcggctgtgt attctggaac caggcaaacc
     6001 cgacgtggtc acatcccagc cactggatga agggtttgga gctatcgtga gagagtgcaa
     6061 gggactccag aggctgagca tttccggcct gctgacagac aaagtgttca tgtacatcgg
     6121 caagtatgct aagcagctgg agatgctgag cattgcattt gccggagact ccgataaggg
     6181 catgatgcac gtgatgaacg ggtgtaagaa tctgcgaaaa ctggaaatcc gggacagccc
     6241 tttcggggat gccgctctgc tgggaaactt tgccagatac gagacaatga ggagcctgtg
     6301 gatgtctagt tgcaatgtga ctctgaaggg ctgtcaggtc ctggctagta aaatgcctat
     6361 gctgaacgtg gaagtcatta atgagcggga cgggtctaac gaaatggagg aaaatcatgg
     6421 cgacctgcca aaggtggaga aactgtatgt gtatcggacc accgcagggg caagagatga
     6481 tgctcccaac tttgtgaaga ttctggaggg ccgcggcagc ctgctgacct gcggcgacgt
     6541 ggaggagaac cccggcccca tgaaaacatt taacatttct caacaagatc tagaattagt
     6601 agaagtagcg acagagaaga ttacaatgct ttatgaggat aataaacatc atgtgggagc
     6661 ggcaattcgt acgaaaacag gagaaatcat ttcggcagta catattgaag cgtatatagg
     6721 acgagtaact gtttgtgcag aagccattgc gattggtagt gcagtttcga atggacaaaa
     6781 ggattttgac acgattgtag ctgttagaca cccttattct gacgaagtag atagaagtat
     6841 tcgagtggta agtccttgtg gtatgtgtag ggagttgatt tcagactatg caccagattg
     6901 ttttgtgtta atagaaatga atggcaagtt agtcaaaact acgattgaag aactcattcc
     6961 actcaaatat acccgaaatt aaggatcttt ttccctctgc caaaaattat ggggacatca
     7021 tgaagcccct tgagggatcc gcccctctcc ctcccccccc cctaacgtta ctggccgaag
     7081 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc
     7141 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg
     7201 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
     7261 tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc
     7321 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa
     7381 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct
     7441 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg
     7501 atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg
     7561 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
     7621 ccacaaccat gtgcgat
//
