LOCUS       sequence-298650-        7572 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of OsTIR1 and mScarlet-mAID protein in mammalian cells 
            under the control of EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7572)
  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 7572)
  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-EF1a-NmScarlet-mAID
FEATURES             Location/Qualifiers
     source          1..7572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1230..2954
                     /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             2955..3008
                     /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(3435..3454)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     misc_feature    join(3491..4065,4066..4068,4069..4077)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3658..3675)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3885..3904
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4090..4785
                     /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(4223..4241)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4491..4510
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4786..4989
                     /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    5040..5161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5077..5096)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5131..5150
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5315..5903)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5395..5414)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6130..6921,6922..6990))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     polyA_signal    7095..7143
                     /note="synthetic polyadenylation signal"
     misc_feature    7157..7248
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     7197..7216
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        7268..7478
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        7290..7486
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7337..7472
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgaa ttgcggaagg ccgtcaaggc
     1201 cacgtgtctt gtccagaggc gctgccacca tgacatactt tcctgaagag gtcgtcgaac
     1261 acatttttag cttcctgcct gcacagagag atagaaacac agtgagcctg gtctgcaaag
     1321 tgtggtacga gatcgaacgc ctgagccgga gaggagtgtt cgtcggcaac tgctatgctg
     1381 tgagagcagg cagggtcgcc gctaggtttc caaatgtgcg cgcactgacc gtcaagggga
     1441 aaccccactt cgccgacttt aacctggtgc cccctgattg gggaggatac gccggccctt
     1501 ggatcgaggc agccgctcgc ggctgtcatg gactggagga actgcgcatg aagcgaatgg
     1561 tggtctctga cgaaagtctg gagctgctgg ctcggagctt ccctaggttt cgcgcactgg
     1621 tgctgatttc ttgcgaaggc ttcagcaccg atggactggc agccgtggcc tcccactgta
     1681 agctgctgcg ggagctggac ctccaggaga atgaagtgga ggatagaggc cccagatggc
     1741 tgtcttgctt cccagactca tgtaccagcc tggtgtccct gaactttgcc tgcatcaaag
     1801 gcgaagtgaa tgctgggtcc ctggagcggc tggtctcaag aagccccaac ctgaggtctc
     1861 tgcggctgaa ccggagcgtg agcgtggaca ctctggctaa gattctgctg agaaccccta
     1921 acctggagga tctgggaacc ggcaatctga cagacgattt ccagacagaa tcctacttta
     1981 aactgacttc tgccctggag aagtgtaaaa tgctgaggag tctgtcagga ttctgggatg
     2041 cttcacccgt gtgcctgagc tttatctacc ctctgtgtgc acagctgaca ggcctgaacc
     2101 tgagctatgc accaaccctg gacgccagtg atctgacaaa gatgatctca cgctgcgtga
     2161 aactccagcg actgtgggtg ctggactgta tttccgataa ggggctccag gtggtcgcca
     2221 gctcctgcaa ggacctccag gagctgagag tgttcccatc tgatttttac gtggccggat
     2281 atagtgctgt cactgaggaa ggcctggtgg cagtctcact gggatgccca aagctgaaca
     2341 gcctgctgta tttctgtcat cagatgacta atgctgcact ggtgaccgtc gccaagaact
     2401 gccctaattt cacccgattt cggctgtgta ttctggaacc aggcaaaccc gacgtggtca
     2461 catcccagcc actggatgaa gggtttggag ctatcgtgag agagtgcaag ggactccaga
     2521 ggctgagcat ttccggcctg ctgacagaca aagtgttcat gtacatcggc aagtatgcta
     2581 agcagctgga gatgctgagc attgcatttg ccggagactc cgataagggc atgatgcacg
     2641 tgatgaacgg gtgtaagaat ctgcgaaaac tggaaatccg ggacagccct ttcggggatg
     2701 ccgctctgct gggaaacttt gccagatacg agacaatgag gagcctgtgg atgtctagtt
     2761 gcaatgtgac tctgaagggc tgtcaggtcc tggctagtaa aatgcctatg ctgaacgtgg
     2821 aagtcattaa tgagcgggac gggtctaacg aaatggagga aaatcatggc gacctgccaa
     2881 aggtggagaa actgtatgtg tatcggacca ccgcaggggc aagagatgat gctcccaact
     2941 ttgtgaagat tctggagggc cgcggcagcc tgctgacctg cggcgacgtg gaggagaacc
     3001 ccggccccat gaaaacattt aacatttctc aacaagatct agaattagta gaagtagcga
     3061 cagagaagat tacaatgctt tatgaggata ataaacatca tgtgggagcg gcaattcgta
     3121 cgaaaacagg agaaatcatt tcggcagtac atattgaagc gtatatagga cgagtaactg
     3181 tttgtgcaga agccattgcg attggtagtg cagtttcgaa tggacaaaag gattttgaca
     3241 cgattgtagc tgttagacac ccttattctg acgaagtaga tagaagtatt cgagtggtaa
     3301 gtccttgtgg tatgtgtagg gagttgattt cagactatgc accagattgt tttgtgttaa
     3361 tagaaatgaa tggcaagtta gtcaaaacta cgattgaaga actcattcca ctcaaatata
     3421 cccgaaatta aggatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt
     3481 gagggatccg cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat
     3541 aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg
     3601 tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc
     3661 tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt
     3721 cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg
     3781 acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac
     3841 cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg
     3901 tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg
     3961 ggcctcggta cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc
     4021 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg
     4081 tgcgataaca tggtgagcaa gggcgaggca gtgatcaagg agttcatgcg gttcaaggtg
     4141 cacatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     4201 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     4261 tcctgggaca tcctgtcccc tcagttcatg tacggctcca gggccttcat caagcacccc
     4321 gccgacatcc ccgactacta taagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg
     4381 atgaacttcg aggacggcgg cgccgtgacc gtgacccagg acacctccct ggaggacggc
     4441 accctgatct acaaggtgaa gctccgcggc accaacttcc ctcctgacgg ccccgtaatg
     4501 cagaagaaga caatgggctg ggaagcgtcc accgagcggt tgtaccccga ggacggcgtg
     4561 ctgaagggcg acattaagat ggccctgcgc ctgaaggacg gcggccgcta cctggcggac
     4621 ttcaagacca cctacaaggc caagaagccc gtgcagatgc ccggcgccta caacgtcgac
     4681 cgcaagttgg acatcacctc ccacaacgag gactacaccg tggtggaaca gtacgaacgc
     4741 tccgagggcc gccactccac cggcggcatg gacgagctgt acaagaagga gaagagtgct
     4801 tgtcctaaag atccagccaa acctccggcc aaggcacaag ttgtgggatg gccaccggtg
     4861 agatcatacc ggaagaacgt gatggtttcc tgccaaaaat caagcggtgg cccggaggcg
     4921 gcggcgttcg tgaaggtatc aatggacgga gcaccgtact tgaggaaaat cgatttgagg
     4981 atgtataaag gagctggtgc aggcgctgga gcgggtgccg atatccaatt gttgttgtta
     5041 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     5101 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5161 agcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5221 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5281 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5341 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5401 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5461 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5521 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5581 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5641 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5701 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5761 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5821 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5881 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5941 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6001 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6061 cttggtctga cagcccctgc gcaacgtggc aggaagcgcg cgctggcaaa tgctaaacca
     6121 ctgcagtggt taccagtgct tgatcagtga ggcaccgatc tcagcgatct gcctatttcg
     6181 ttcgtccata gtggcctgac tccccgtcgt gtagatcact acgattcgtg agggcttacc
     6241 atcaggcccc agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc ccgatttgtc
     6301 agcaatgaac cagccagcag ggagggccga gcgaagaagt ggtcctgcta ctttgtccgc
     6361 ctccatccag tctatgagct gctgtcgtga tgctagagta agaagttcgc cagtgagtag
     6421 tttccgaaga gttgtggcca ttgctactgg catcgtggta tcacgctcgt cgttcggtat
     6481 ggcttcgttc aactctggtt cccagcggtc aagccgggtc acatgatcac ccatattatg
     6541 aagaaatgca gtcagctcct tagggcctcc gatcgttgtc agaagtaagt tggccgcggt
     6601 gttgtcgctc atggtaatgg cagcactaca caattctctt accgtcatgc catccgtaag
     6661 atgcttttcc gtgaccggcg agtactcaac caagtcgttt tgtgagtagt gtatacggcg
     6721 accaagctgc tcttgcccgg cgtctatacg ggacaacacc gcgccacata gcagtacttt
     6781 gaaagtgctc atcatcggga atcgttcttc ggggcggaaa gactcaagga tcttgccgct
     6841 attgagatcc agttcgatat agcccactct tgcacccagt tgatcttcag catcttttac
     6901 tttcaccagc gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa agaagggaat
     6961 gagtgcgaca cgaaaatgtt ggatgctcat actcgtcctt tttcaatatt attgaagcat
     7021 ttatcagggt tactagtacg tctctcaagg ataagtaagt aatattaagg tacgggaggt
     7081 attggacagg ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt
     7141 gtgaatcgat agtactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag
     7201 caaaataggc tgtccccagt gcaagtgcag gtgccagaac atttctctgg cctaaccagg
     7261 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     7321 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     7381 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     7441 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tttgcaaaga
     7501 tggataaagt tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa
     7561 ggagtgggaa tt
//
