LOCUS       sequence-272991-        7358 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 7358)
  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 7358)
  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..7358
                     /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..3591
                     /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(3029..3047)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3297..3316
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3592..3795
                     /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    3846..3967
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3883..3902)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3937..3956
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4121..4709)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4201..4220)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4936..5727,5728..5796))
                     /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    5901..5949
                     /note="synthetic polyadenylation signal"
     misc_feature    5963..6054
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6003..6022
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        6149..7331
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6380..7322
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7279..7299
                     /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 ggaggataac atggccatca tcaaggagtt catgcgcttc
     2941 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     3001 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
     3061 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag
     3121 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     3181 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     3241 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     3301 gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac
     3361 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac
     3421 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac
     3481 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     3541 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gaaggagaag
     3601 agtgcttgtc ctaaagatcc agccaaacct ccggccaagg cacaagttgt gggatggcca
     3661 ccggtgagat cataccggaa gaacgtgatg gtttcctgcc aaaaatcaag cggtggcccg
     3721 gaggcggcgg cgttcgtgaa ggtatcaatg gacggagcac cgtacttgag gaaaatcgat
     3781 ttgaggatgt ataaaggagc tggtgcaggc gctggagcgg gtgccgatat ccaattgttg
     3841 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3901 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3961 tatcttagcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4021 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4081 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4141 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4201 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4261 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4321 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4381 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4441 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4501 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4561 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4621 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4681 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4741 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4801 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4861 agtaaacttg gtctgacagc ccctgcgcaa cgtggcagga agcgcgcgct ggcaaatgct
     4921 aaaccactgc agtggttacc agtgcttgat cagtgaggca ccgatctcag cgatctgcct
     4981 atttcgttcg tccatagtgg cctgactccc cgtcgtgtag atcactacga ttcgtgaggg
     5041 cttaccatca ggccccagcg cagcaatgat gccgcgagag ccgcgttcac cggcccccga
     5101 tttgtcagca atgaaccagc cagcagggag ggccgagcga agaagtggtc ctgctacttt
     5161 gtccgcctcc atccagtcta tgagctgctg tcgtgatgct agagtaagaa gttcgccagt
     5221 gagtagtttc cgaagagttg tggccattgc tactggcatc gtggtatcac gctcgtcgtt
     5281 cggtatggct tcgttcaact ctggttccca gcggtcaagc cgggtcacat gatcacccat
     5341 attatgaaga aatgcagtca gctccttagg gcctccgatc gttgtcagaa gtaagttggc
     5401 cgcggtgttg tcgctcatgg taatggcagc actacacaat tctcttaccg tcatgccatc
     5461 cgtaagatgc ttttccgtga ccggcgagta ctcaaccaag tcgttttgtg agtagtgtat
     5521 acggcgacca agctgctctt gcccggcgtc tatacgggac aacaccgcgc cacatagcag
     5581 tactttgaaa gtgctcatca tcgggaatcg ttcttcgggg cggaaagact caaggatctt
     5641 gccgctattg agatccagtt cgatatagcc cactcttgca cccagttgat cttcagcatc
     5701 ttttactttc accagcgttt cggggtgtgc aaaaacaggc aagcaaaatg ccgcaaagaa
     5761 gggaatgagt gcgacacgaa aatgttggat gctcatactc gtcctttttc aatattattg
     5821 aagcatttat cagggttact agtacgtctc tcaaggataa gtaagtaata ttaaggtacg
     5881 ggaggtattg gacaggccgc aataaaatat ctttattttc attacatctg tgtgttggtt
     5941 ttttgtgtga atcgatagta ctaacatacg ctctccatca aaacaaaacg aaacaaaaca
     6001 aactagcaaa ataggctgtc cccagtgcaa gtgcaggtgc cagaacattt ctctggccta
     6061 actaagcttt gcaaagatgg ataaagtttt aaacagagag gaatctttgc agctaatgga
     6121 ccttctaggt cttgaaagga gtgcgtgagg ctccggtgcc cgtcagtggg cagagcgcac
     6181 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag
     6241 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     6301 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     6361 gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac
     6421 gggttatggc ccttgcgtgc cttgaattac ttccacgccc ctggctgcag tacgtgattc
     6481 ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag
     6541 ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa
     6601 tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt
     6661 tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag
     6721 atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc
     6781 agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt
     6841 agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc
     6901 cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc
     6961 ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg
     7021 agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca
     7081 cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt
     7141 ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga
     7201 ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt
     7261 ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc
     7321 aggtgtcgtg aattgcggaa ggccgtcaag gccacgtg
//
