LOCUS       sequence-272997-        7640 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of OsTIR1 and mCherry-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 7640)
  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 7640)
  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-NmCherry-mAID
FEATURES             Location/Qualifiers
     source          1..7640
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    10..131
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(47..66)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     101..120
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(138..593)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(275..296)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     487..506
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        620..724
                     /gene="bla"
                     /label=AmpR promoter
     promoter        726..1083
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        726..917
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(749..769)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      934..1069
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1118..1912
                     /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             1118..1912
                     /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(1172..1191)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1782..1801
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2100..2119)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2144..2191
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2520..3108
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3009..3028
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        3283..3586
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3587..3790
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3741..3761
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3828..5552
                     /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             5553..5606
                     /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(6033..6052)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     misc_feature    join(6089..6663,6664..6666,6667..6675)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6256..6273)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     6483..6502
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             6688..7395
                     /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(6833..6851)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7101..7120
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             7396..7599
                     /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"
ORIGIN
        1 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
       61 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
      121 aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
      181 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
      241 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
      301 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
      361 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
      421 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
      481 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
      541 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
      601 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
      661 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      721 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
      781 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
      841 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
      901 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
      961 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     1021 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     1081 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     1141 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     1201 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     1261 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     1321 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     1381 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     1441 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     1501 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     1561 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     1621 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     1681 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     1741 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     1801 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     1861 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     1921 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     1981 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     2041 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     2101 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     2161 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     2221 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     2281 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     2341 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     2401 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     2461 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2521 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2581 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2641 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     2701 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2761 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2821 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2881 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2941 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3001 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3061 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3121 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3181 ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc
     3241 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     3301 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     3361 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     3421 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     3481 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     3541 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     3601 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     3661 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     3721 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     3781 aagcagagct ggtttagtga accgtcagat ccgctagcgc tgccaccatg acatactttc
     3841 ctgaagaggt cgtcgaacac atttttagct tcctgcctgc acagagagat agaaacacag
     3901 tgagcctggt ctgcaaagtg tggtacgaga tcgaacgcct gagccggaga ggagtgttcg
     3961 tcggcaactg ctatgctgtg agagcaggca gggtcgccgc taggtttcca aatgtgcgcg
     4021 cactgaccgt caaggggaaa ccccacttcg ccgactttaa cctggtgccc cctgattggg
     4081 gaggatacgc cggcccttgg atcgaggcag ccgctcgcgg ctgtcatgga ctggaggaac
     4141 tgcgcatgaa gcgaatggtg gtctctgacg aaagtctgga gctgctggct cggagcttcc
     4201 ctaggtttcg cgcactggtg ctgatttctt gcgaaggctt cagcaccgat ggactggcag
     4261 ccgtggcctc ccactgtaag ctgctgcggg agctggacct ccaggagaat gaagtggagg
     4321 atagaggccc cagatggctg tcttgcttcc cagactcatg taccagcctg gtgtccctga
     4381 actttgcctg catcaaaggc gaagtgaatg ctgggtccct ggagcggctg gtctcaagaa
     4441 gccccaacct gaggtctctg cggctgaacc ggagcgtgag cgtggacact ctggctaaga
     4501 ttctgctgag aacccctaac ctggaggatc tgggaaccgg caatctgaca gacgatttcc
     4561 agacagaatc ctactttaaa ctgacttctg ccctggagaa gtgtaaaatg ctgaggagtc
     4621 tgtcaggatt ctgggatgct tcacccgtgt gcctgagctt tatctaccct ctgtgtgcac
     4681 agctgacagg cctgaacctg agctatgcac caaccctgga cgccagtgat ctgacaaaga
     4741 tgatctcacg ctgcgtgaaa ctccagcgac tgtgggtgct ggactgtatt tccgataagg
     4801 ggctccaggt ggtcgccagc tcctgcaagg acctccagga gctgagagtg ttcccatctg
     4861 atttttacgt ggccggatat agtgctgtca ctgaggaagg cctggtggca gtctcactgg
     4921 gatgcccaaa gctgaacagc ctgctgtatt tctgtcatca gatgactaat gctgcactgg
     4981 tgaccgtcgc caagaactgc cctaatttca cccgatttcg gctgtgtatt ctggaaccag
     5041 gcaaacccga cgtggtcaca tcccagccac tggatgaagg gtttggagct atcgtgagag
     5101 agtgcaaggg actccagagg ctgagcattt ccggcctgct gacagacaaa gtgttcatgt
     5161 acatcggcaa gtatgctaag cagctggaga tgctgagcat tgcatttgcc ggagactccg
     5221 ataagggcat gatgcacgtg atgaacgggt gtaagaatct gcgaaaactg gaaatccggg
     5281 acagcccttt cggggatgcc gctctgctgg gaaactttgc cagatacgag acaatgagga
     5341 gcctgtggat gtctagttgc aatgtgactc tgaagggctg tcaggtcctg gctagtaaaa
     5401 tgcctatgct gaacgtggaa gtcattaatg agcgggacgg gtctaacgaa atggaggaaa
     5461 atcatggcga cctgccaaag gtggagaaac tgtatgtgta tcggaccacc gcaggggcaa
     5521 gagatgatgc tcccaacttt gtgaagattc tggagggccg cggcagcctg ctgacctgcg
     5581 gcgacgtgga ggagaacccc ggccccatga aaacatttaa catttctcaa caagatctag
     5641 aattagtaga agtagcgaca gagaagatta caatgcttta tgaggataat aaacatcatg
     5701 tgggagcggc aattcgtacg aaaacaggag aaatcatttc ggcagtacat attgaagcgt
     5761 atataggacg agtaactgtt tgtgcagaag ccattgcgat tggtagtgca gtttcgaatg
     5821 gacaaaagga ttttgacacg attgtagctg ttagacaccc ttattctgac gaagtagata
     5881 gaagtattcg agtggtaagt ccttgtggta tgtgtaggga gttgatttca gactatgcac
     5941 cagattgttt tgtgttaata gaaatgaatg gcaagttagt caaaactacg attgaagaac
     6001 tcattccact caaatatacc cgaaattaag gatctttttc cctctgccaa aaattatggg
     6061 gacatcatga agccccttga gggatccgcc cctctccctc ccccccccct aacgttactg
     6121 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
     6181 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
     6241 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
     6301 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc
     6361 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac
     6421 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca
     6481 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt
     6541 gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa
     6601 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat
     6661 aatatggcca caaccatgtg cgataacatg gtgagcaagg gcgaggagga taacatggcc
     6721 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     6781 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     6841 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     6901 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     6961 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     7021 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     7081 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     7141 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     7201 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     7261 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     7321 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     7381 gacgagctgt acaagaagga gaagagtgct tgtcctaaag atccagccaa acctccggcc
     7441 aaggcacaag ttgtgggatg gccaccggtg agatcatacc ggaagaacgt gatggtttcc
     7501 tgccaaaaat caagcggtgg cccggaggcg gcggcgttcg tgaaggtatc aatggacgga
     7561 gcaccgtact tgaggaaaat cgatttgagg atgtataaag gagctggtgc aggcgctgga
     7621 gcgggtgccg atatccaatt
//
