LOCUS       sequence-311521-        7628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of OsTIR1F74A 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 7628)
  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 7628)
  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: pAIDFA-CMV-NmScarlet-mAID
FEATURES             Location/Qualifiers
     source          1..7628
                     /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             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(2807..3381,3382..3384,3385..3393)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2974..2991)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3201..3220
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3406..4101
                     /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(3539..3557)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3807..3826
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4102..4305
                     /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    4356..4477
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4393..4412)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4447..4466
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4484..4939)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4621..4642)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4833..4852
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4966..5070
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5072..5429
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5072..5263
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5095..5115)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5280..5415
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5464..6258
                     /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             5464..6258
                     /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(5518..5537)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6128..6147
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6446..6465)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6490..6537
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6866..7454
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7355..7374
                     /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 ccacgccgcc 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 gatccgcccc tctccctccc
     2821 ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata
     2881 tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg
     2941 tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt
     3001 tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag
     3061 cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc
     3121 cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga
     3181 tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg
     3241 cccagaaggt accccattgt atgggatctg atctggggcc tcggtacaca tgctttacat
     3301 gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc
     3361 tttgaaaaac acgatgataa tatggccaca accatgtgcg ataacatggt gagcaagggc
     3421 gaggcagtga tcaaggagtt catgcggttc aaggtgcaca tggagggctc catgaacggc
     3481 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     3541 aagctgaagg tgaccaaggg tggccccctg cccttctcct gggacatcct gtcccctcag
     3601 ttcatgtacg gctccagggc cttcatcaag caccccgccg acatccccga ctactataag
     3661 cagtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgcc
     3721 gtgaccgtga cccaggacac ctccctggag gacggcaccc tgatctacaa ggtgaagctc
     3781 cgcggcacca acttccctcc tgacggcccc gtaatgcaga agaagacaat gggctgggaa
     3841 gcgtccaccg agcggttgta ccccgaggac ggcgtgctga agggcgacat taagatggcc
     3901 ctgcgcctga aggacggcgg ccgctacctg gcggacttca agaccaccta caaggccaag
     3961 aagcccgtgc agatgcccgg cgcctacaac gtcgaccgca agttggacat cacctcccac
     4021 aacgaggact acaccgtggt ggaacagtac gaacgctccg agggccgcca ctccaccggc
     4081 ggcatggacg agctgtacaa gaaggagaag agtgcttgtc ctaaagatcc agccaaacct
     4141 ccggccaagg cacaagttgt gggatggcca ccggtgagat cataccggaa gaacgtgatg
     4201 gtttcctgcc aaaaatcaag cggtggcccg gaggcggcgg cgttcgtgaa ggtatcaatg
     4261 gacggagcac cgtacttgag gaaaatcgat ttgaggatgt ataaaggagc tggtgcaggc
     4321 gctggagcgg gtgccgatat ccaattgttg ttgttaactt gtttattgca gcttataatg
     4381 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4441 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat
     4501 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     4561 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     4621 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     4681 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     4741 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     4801 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     4861 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     4921 gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4981 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5041 atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg
     5101 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     5161 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     5221 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     5281 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     5341 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     5401 gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt
     5461 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     5521 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     5581 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     5641 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     5701 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     5761 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     5821 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     5881 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     5941 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc
     6001 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     6061 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     6121 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     6181 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     6241 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     6301 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     6361 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     6421 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     6481 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata
     6541 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     6601 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     6661 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata
     6721 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6781 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6841 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6901 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6961 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     7021 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     7081 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     7141 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     7201 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     7261 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7321 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7381 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7441 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7501 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     7561 ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     7621 gagttccg
//
