LOCUS       sequence-272984-        7346 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 7346)
  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 7346)
  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..7346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             24..1748
                     /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             1749..1802
                     /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(2229..2248)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     misc_feature    join(2285..2859,2860..2862,2863..2871)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2452..2469)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2679..2698
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2884..3579
                     /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(3017..3035)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3285..3304
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3580..3783
                     /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    3834..3955
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3871..3890)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3925..3944
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4109..4697)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4189..4208)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4924..5715,5716..5784))
                     /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    5889..5937
                     /note="synthetic polyadenylation signal"
     misc_feature    5951..6042
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5991..6010
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        6137..7319
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6368..7310
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7267..7287
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 tcttgtccag aggcgctgcc accatgacat actttcctga agaggtcgtc gaacacattt
       61 ttagcttcct gcctgcacag agagatagaa acacagtgag cctggtctgc aaagtgtggt
      121 acgagatcga acgcctgagc cggagaggag tgttcgtcgg caactgctat gctgtgagag
      181 caggcagggt cgccgctagg tttccaaatg tgcgcgcact gaccgtcaag gggaaacccc
      241 acttcgccga ctttaacctg gtgccccctg attggggagg atacgccggc ccttggatcg
      301 aggcagccgc tcgcggctgt catggactgg aggaactgcg catgaagcga atggtggtct
      361 ctgacgaaag tctggagctg ctggctcgga gcttccctag gtttcgcgca ctggtgctga
      421 tttcttgcga aggcttcagc accgatggac tggcagccgt ggcctcccac tgtaagctgc
      481 tgcgggagct ggacctccag gagaatgaag tggaggatag aggccccaga tggctgtctt
      541 gcttcccaga ctcatgtacc agcctggtgt ccctgaactt tgcctgcatc aaaggcgaag
      601 tgaatgctgg gtccctggag cggctggtct caagaagccc caacctgagg tctctgcggc
      661 tgaaccggag cgtgagcgtg gacactctgg ctaagattct gctgagaacc cctaacctgg
      721 aggatctggg aaccggcaat ctgacagacg atttccagac agaatcctac tttaaactga
      781 cttctgccct ggagaagtgt aaaatgctga ggagtctgtc aggattctgg gatgcttcac
      841 ccgtgtgcct gagctttatc taccctctgt gtgcacagct gacaggcctg aacctgagct
      901 atgcaccaac cctggacgcc agtgatctga caaagatgat ctcacgctgc gtgaaactcc
      961 agcgactgtg ggtgctggac tgtatttccg ataaggggct ccaggtggtc gccagctcct
     1021 gcaaggacct ccaggagctg agagtgttcc catctgattt ttacgtggcc ggatatagtg
     1081 ctgtcactga ggaaggcctg gtggcagtct cactgggatg cccaaagctg aacagcctgc
     1141 tgtatttctg tcatcagatg actaatgctg cactggtgac cgtcgccaag aactgcccta
     1201 atttcacccg atttcggctg tgtattctgg aaccaggcaa acccgacgtg gtcacatccc
     1261 agccactgga tgaagggttt ggagctatcg tgagagagtg caagggactc cagaggctga
     1321 gcatttccgg cctgctgaca gacaaagtgt tcatgtacat cggcaagtat gctaagcagc
     1381 tggagatgct gagcattgca tttgccggag actccgataa gggcatgatg cacgtgatga
     1441 acgggtgtaa gaatctgcga aaactggaaa tccgggacag ccctttcggg gatgccgctc
     1501 tgctgggaaa ctttgccaga tacgagacaa tgaggagcct gtggatgtct agttgcaatg
     1561 tgactctgaa gggctgtcag gtcctggcta gtaaaatgcc tatgctgaac gtggaagtca
     1621 ttaatgagcg ggacgggtct aacgaaatgg aggaaaatca tggcgacctg ccaaaggtgg
     1681 agaaactgta tgtgtatcgg accaccgcag gggcaagaga tgatgctccc aactttgtga
     1741 agattctgga gggccgcggc agcctgctga cctgcggcga cgtggaggag aaccccggcc
     1801 ccatgaaaac atttaacatt tctcaacaag atctagaatt agtagaagta gcgacagaga
     1861 agattacaat gctttatgag gataataaac atcatgtggg agcggcaatt cgtacgaaaa
     1921 caggagaaat catttcggca gtacatattg aagcgtatat aggacgagta actgtttgtg
     1981 cagaagccat tgcgattggt agtgcagttt cgaatggaca aaaggatttt gacacgattg
     2041 tagctgttag acacccttat tctgacgaag tagatagaag tattcgagtg gtaagtcctt
     2101 gtggtatgtg tagggagttg atttcagact atgcaccaga ttgttttgtg ttaatagaaa
     2161 tgaatggcaa gttagtcaaa actacgattg aagaactcat tccactcaaa tatacccgaa
     2221 attaaggatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgaggga
     2281 tccgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     2341 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     2401 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     2461 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     2521 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     2581 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     2641 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     2701 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     2761 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     2821 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac catgtgcgat
     2881 aacatggtga gcaagggcga ggcagtgatc aaggagttca tgcggttcaa ggtgcacatg
     2941 gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     3001 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttctcctgg
     3061 gacatcctgt cccctcagtt catgtacggc tccagggcct tcatcaagca ccccgccgac
     3121 atccccgact actataagca gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     3181 ttcgaggacg gcggcgccgt gaccgtgacc caggacacct ccctggagga cggcaccctg
     3241 atctacaagg tgaagctccg cggcaccaac ttccctcctg acggccccgt aatgcagaag
     3301 aagacaatgg gctgggaagc gtccaccgag cggttgtacc ccgaggacgg cgtgctgaag
     3361 ggcgacatta agatggccct gcgcctgaag gacggcggcc gctacctggc ggacttcaag
     3421 accacctaca aggccaagaa gcccgtgcag atgcccggcg cctacaacgt cgaccgcaag
     3481 ttggacatca cctcccacaa cgaggactac accgtggtgg aacagtacga acgctccgag
     3541 ggccgccact ccaccggcgg catggacgag ctgtacaaga aggagaagag tgcttgtcct
     3601 aaagatccag ccaaacctcc ggccaaggca caagttgtgg gatggccacc ggtgagatca
     3661 taccggaaga acgtgatggt ttcctgccaa aaatcaagcg gtggcccgga ggcggcggcg
     3721 ttcgtgaagg tatcaatgga cggagcaccg tacttgagga aaatcgattt gaggatgtat
     3781 aaaggagctg gtgcaggcgc tggagcgggt gccgatatcc aattgttgtt gttaacttgt
     3841 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3901 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttagcgct
     3961 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4021 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4081 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4141 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4201 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4261 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4321 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4381 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4441 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4501 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4561 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4621 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4681 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4741 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4801 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4861 ctgacagccc ctgcgcaacg tggcaggaag cgcgcgctgg caaatgctaa accactgcag
     4921 tggttaccag tgcttgatca gtgaggcacc gatctcagcg atctgcctat ttcgttcgtc
     4981 catagtggcc tgactccccg tcgtgtagat cactacgatt cgtgagggct taccatcagg
     5041 ccccagcgca gcaatgatgc cgcgagagcc gcgttcaccg gcccccgatt tgtcagcaat
     5101 gaaccagcca gcagggaggg ccgagcgaag aagtggtcct gctactttgt ccgcctccat
     5161 ccagtctatg agctgctgtc gtgatgctag agtaagaagt tcgccagtga gtagtttccg
     5221 aagagttgtg gccattgcta ctggcatcgt ggtatcacgc tcgtcgttcg gtatggcttc
     5281 gttcaactct ggttcccagc ggtcaagccg ggtcacatga tcacccatat tatgaagaaa
     5341 tgcagtcagc tccttagggc ctccgatcgt tgtcagaagt aagttggccg cggtgttgtc
     5401 gctcatggta atggcagcac tacacaattc tcttaccgtc atgccatccg taagatgctt
     5461 ttccgtgacc ggcgagtact caaccaagtc gttttgtgag tagtgtatac ggcgaccaag
     5521 ctgctcttgc ccggcgtcta tacgggacaa caccgcgcca catagcagta ctttgaaagt
     5581 gctcatcatc gggaatcgtt cttcggggcg gaaagactca aggatcttgc cgctattgag
     5641 atccagttcg atatagccca ctcttgcacc cagttgatct tcagcatctt ttactttcac
     5701 cagcgtttcg gggtgtgcaa aaacaggcaa gcaaaatgcc gcaaagaagg gaatgagtgc
     5761 gacacgaaaa tgttggatgc tcatactcgt cctttttcaa tattattgaa gcatttatca
     5821 gggttactag tacgtctctc aaggataagt aagtaatatt aaggtacggg aggtattgga
     5881 caggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat
     5941 cgatagtact aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat
     6001 aggctgtccc cagtgcaagt gcaggtgcca gaacatttct ctggcctaac taagctttgc
     6061 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct
     6121 tgaaaggagt gcgtgaggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
     6181 ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg
     6241 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
     6301 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
     6361 aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc
     6421 ttgcgtgcct tgaattactt ccacgcccct ggctgcagta cgtgattctt gatcccgagc
     6481 ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc
     6541 gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc
     6601 ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg
     6661 ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg
     6721 gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt
     6781 cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg
     6841 gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa
     6901 ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg
     6961 cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac
     7021 aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg
     7081 cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg
     7141 gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc
     7201 cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt
     7261 tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgaa
     7321 ttgcggaagg ccgtcaaggc cacgtg
//
