LOCUS       sequence-298653-        7667 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 7667)
  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 7667)
  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..7667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             242..1966
                     /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             1967..2020
                     /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(2447..2466)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     misc_feature    join(2530..3104,3105..3107,3108..3116)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2697..2714)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2924..2943
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3129..3836
                     /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(3274..3292)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3542..3561
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3837..4040
                     /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    4091..4212
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4128..4147)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4182..4201
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4219..4674)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4356..4377)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4568..4587
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4701..4805
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4807..5164
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4807..4998
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4830..4850)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5015..5150
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5199..5993
                     /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             5199..5993
                     /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(5253..5272)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5863..5882
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6181..6200)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6225..6272
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6601..7189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7090..7109
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7364..7667
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctgccac
      241 catgacatac tttcctgaag aggtcgtcga acacattttt agcttcctgc ctgcacagag
      301 agatagaaac acagtgagcc tggtctgcaa agtgtggtac gagatcgaac gcctgagccg
      361 gagaggagtg ttcgtcggca actgctatgc tgtgagagca ggcagggtcg ccgctaggtt
      421 tccaaatgtg cgcgcactga ccgtcaaggg gaaaccccac ttcgccgact ttaacctggt
      481 gccccctgat tggggaggat acgccggccc ttggatcgag gcagccgctc gcggctgtca
      541 tggactggag gaactgcgca tgaagcgaat ggtggtctct gacgaaagtc tggagctgct
      601 ggctcggagc ttccctaggt ttcgcgcact ggtgctgatt tcttgcgaag gcttcagcac
      661 cgatggactg gcagccgtgg cctcccactg taagctgctg cgggagctgg acctccagga
      721 gaatgaagtg gaggatagag gccccagatg gctgtcttgc ttcccagact catgtaccag
      781 cctggtgtcc ctgaactttg cctgcatcaa aggcgaagtg aatgctgggt ccctggagcg
      841 gctggtctca agaagcccca acctgaggtc tctgcggctg aaccggagcg tgagcgtgga
      901 cactctggct aagattctgc tgagaacccc taacctggag gatctgggaa ccggcaatct
      961 gacagacgat ttccagacag aatcctactt taaactgact tctgccctgg agaagtgtaa
     1021 aatgctgagg agtctgtcag gattctggga tgcttcaccc gtgtgcctga gctttatcta
     1081 ccctctgtgt gcacagctga caggcctgaa cctgagctat gcaccaaccc tggacgccag
     1141 tgatctgaca aagatgatct cacgctgcgt gaaactccag cgactgtggg tgctggactg
     1201 tatttccgat aaggggctcc aggtggtcgc cagctcctgc aaggacctcc aggagctgag
     1261 agtgttccca tctgattttt acgtggccgg atatagtgct gtcactgagg aaggcctggt
     1321 ggcagtctca ctgggatgcc caaagctgaa cagcctgctg tatttctgtc atcagatgac
     1381 taatgctgca ctggtgaccg tcgccaagaa ctgccctaat ttcacccgat ttcggctgtg
     1441 tattctggaa ccaggcaaac ccgacgtggt cacatcccag ccactggatg aagggtttgg
     1501 agctatcgtg agagagtgca agggactcca gaggctgagc atttccggcc tgctgacaga
     1561 caaagtgttc atgtacatcg gcaagtatgc taagcagctg gagatgctga gcattgcatt
     1621 tgccggagac tccgataagg gcatgatgca cgtgatgaac gggtgtaaga atctgcgaaa
     1681 actggaaatc cgggacagcc ctttcgggga tgccgctctg ctgggaaact ttgccagata
     1741 cgagacaatg aggagcctgt ggatgtctag ttgcaatgtg actctgaagg gctgtcaggt
     1801 cctggctagt aaaatgccta tgctgaacgt ggaagtcatt aatgagcggg acgggtctaa
     1861 cgaaatggag gaaaatcatg gcgacctgcc aaaggtggag aaactgtatg tgtatcggac
     1921 caccgcaggg gcaagagatg atgctcccaa ctttgtgaag attctggagg gccgcggcag
     1981 cctgctgacc tgcggcgacg tggaggagaa ccccggcccc atgaaaacat ttaacatttc
     2041 tcaacaagat ctagaattag tagaagtagc gacagagaag attacaatgc tttatgagga
     2101 taataaacat catgtgggag cggcaattcg tacgaaaaca ggagaaatca tttcggcagt
     2161 acatattgaa gcgtatatag gacgagtaac tgtttgtgca gaagccattg cgattggtag
     2221 tgcagtttcg aatggacaaa aggattttga cacgattgta gctgttagac acccttattc
     2281 tgacgaagta gatagaagta ttcgagtggt aagtccttgt ggtatgtgta gggagttgat
     2341 ttcagactat gcaccagatt gttttgtgtt aatagaaatg aatggcaagt tagtcaaaac
     2401 tacgattgaa gaactcattc cactcaaata tacccgaaat taaggatctt tttccctctg
     2461 ccaaaaatta tggggacatc atgaagcccc ttgaggtgga acgatcattc aagagatccc
     2521 cgggatccgc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata
     2581 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt
     2641 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct
     2701 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc
     2761 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga
     2821 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc
     2881 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt
     2941 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg
     3001 gcctcggtac acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc
     3061 gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccatgt
     3121 gcgataacat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     3181 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     3241 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     3301 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     3361 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     3421 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     3481 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     3541 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     3601 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     3661 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     3721 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     3781 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagaagg
     3841 agaagagtgc ttgtcctaaa gatccagcca aacctccggc caaggcacaa gttgtgggat
     3901 ggccaccggt gagatcatac cggaagaacg tgatggtttc ctgccaaaaa tcaagcggtg
     3961 gcccggaggc ggcggcgttc gtgaaggtat caatggacgg agcaccgtac ttgaggaaaa
     4021 tcgatttgag gatgtataaa ggagctggtg caggcgctgg agcgggtgcc gatatccaat
     4081 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4141 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4201 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     4261 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     4321 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     4381 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     4441 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     4501 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     4561 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     4621 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     4681 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4741 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4801 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     4861 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     4921 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4981 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     5041 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     5101 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     5161 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     5221 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     5281 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     5341 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     5401 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     5461 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     5521 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     5581 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     5641 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     5701 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     5761 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     5821 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     5881 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     5941 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     6001 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     6061 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     6121 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     6181 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     6241 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     6301 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     6361 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     6421 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     6481 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6541 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6601 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6661 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6721 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6781 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6841 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6901 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6961 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     7021 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     7081 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     7141 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     7201 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     7261 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
     7321 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     7381 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     7441 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     7501 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     7561 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     7621 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatg
//
