LOCUS       sequence-299317-        7584 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of OsTIR1 and mCherry-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 7584)
  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 7584)
  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-NmCherry-mAID
FEATURES             Location/Qualifiers
     source          1..7584
                     /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..4797
                     /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(4235..4253)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4503..4522
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4798..5001
                     /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    5052..5173
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5089..5108)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5143..5162
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5327..5915)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5407..5426)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6142..6933,6934..7002))
                     /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    7107..7155
                     /note="synthetic polyadenylation signal"
     misc_feature    7169..7260
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     7209..7228
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        7280..7490
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        7302..7498
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7349..7484
                     /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 gggcgaggag gataacatgg ccatcatcaa ggagttcatg
     4141 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     4201 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     4261 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
     4321 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     4381 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     4441 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     4501 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     4561 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     4621 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     4681 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     4741 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagaag
     4801 gagaagagtg cttgtcctaa agatccagcc aaacctccgg ccaaggcaca agttgtggga
     4861 tggccaccgg tgagatcata ccggaagaac gtgatggttt cctgccaaaa atcaagcggt
     4921 ggcccggagg cggcggcgtt cgtgaaggta tcaatggacg gagcaccgta cttgaggaaa
     4981 atcgatttga ggatgtataa aggagctggt gcaggcgctg gagcgggtgc cgatatccaa
     5041 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     5101 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     5161 tcaatgtatc ttagcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5221 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5281 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5341 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5401 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5461 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5521 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5581 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5641 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5701 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5761 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5821 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5881 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5941 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6001 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6061 tatatgagta aacttggtct gacagcccct gcgcaacgtg gcaggaagcg cgcgctggca
     6121 aatgctaaac cactgcagtg gttaccagtg cttgatcagt gaggcaccga tctcagcgat
     6181 ctgcctattt cgttcgtcca tagtggcctg actccccgtc gtgtagatca ctacgattcg
     6241 tgagggctta ccatcaggcc ccagcgcagc aatgatgccg cgagagccgc gttcaccggc
     6301 ccccgatttg tcagcaatga accagccagc agggagggcc gagcgaagaa gtggtcctgc
     6361 tactttgtcc gcctccatcc agtctatgag ctgctgtcgt gatgctagag taagaagttc
     6421 gccagtgagt agtttccgaa gagttgtggc cattgctact ggcatcgtgg tatcacgctc
     6481 gtcgttcggt atggcttcgt tcaactctgg ttcccagcgg tcaagccggg tcacatgatc
     6541 acccatatta tgaagaaatg cagtcagctc cttagggcct ccgatcgttg tcagaagtaa
     6601 gttggccgcg gtgttgtcgc tcatggtaat ggcagcacta cacaattctc ttaccgtcat
     6661 gccatccgta agatgctttt ccgtgaccgg cgagtactca accaagtcgt tttgtgagta
     6721 gtgtatacgg cgaccaagct gctcttgccc ggcgtctata cgggacaaca ccgcgccaca
     6781 tagcagtact ttgaaagtgc tcatcatcgg gaatcgttct tcggggcgga aagactcaag
     6841 gatcttgccg ctattgagat ccagttcgat atagcccact cttgcaccca gttgatcttc
     6901 agcatctttt actttcacca gcgtttcggg gtgtgcaaaa acaggcaagc aaaatgccgc
     6961 aaagaaggga atgagtgcga cacgaaaatg ttggatgctc atactcgtcc tttttcaata
     7021 ttattgaagc atttatcagg gttactagta cgtctctcaa ggataagtaa gtaatattaa
     7081 ggtacgggag gtattggaca ggccgcaata aaatatcttt attttcatta catctgtgtg
     7141 ttggtttttt gtgtgaatcg atagtactaa catacgctct ccatcaaaac aaaacgaaac
     7201 aaaacaaact agcaaaatag gctgtcccca gtgcaagtgc aggtgccaga acatttctct
     7261 ggcctaacca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     7321 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     7381 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     7441 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     7501 gctttgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc
     7561 taggtcttga aaggagtggg aatt
//
