LOCUS       sequence-296425-        7601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  OsTIR1(F74G)-P2A-mAID-EGFP-NES.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7601)
  AUTHORS   Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano 
            R, Nakaoka H, Fukui K, Gamo K, Tominari Y, Takeuchi H, Saga Y, 
            Hayashi KI, Kanemaki MT
  TITLE     The auxin-inducible degron 2 technology provides sharp degradation 
            control in yeast, mammalian cells, and mice.
  JOURNAL   Nat Commun. 2020 Nov 11;11(1):5701. doi: 10.1038/s41467-020-19532-z.
  PUBMED    33177522
REFERENCE   2  (bases 1 to 7601)
  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: "Nat 
            Commun."; date: "2020-11-11"; volume: "11(1):5701. doi"; pages: " 
            10.1038/s41467-020-19532-z"
COMMENT     Sequence Label: pAAV-hSyn-OsTIR1(F74G)
FEATURES             Location/Qualifiers
     source          1..7601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   6..135
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        180..627
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             645..2369
                     /codon_start=1
                     /product="rice transport inhibitor response 1 protein, 
                     mutated for use in the AID2 system of inducible degradation
                     (Yesbolatova et al., 2020)"
                     /label=OsTIR1(F74G)
                     /note="F-box protein that mediates auxin-dependent 
                     interaction of a ubiquitin ligase with an auxin-inducible 
                     degron (Tan et al., 2007)"
                     /translation="MTYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIERLSRRGVFV
                     GNCYAVRAGRVAARFPNVRALTVKGKPHGADFNLVPPDWGGYAGPWIEAAARGCHGLEE
                     LRMKRMVVSDESLELLARSFPRFRALVLISCEGFSTDGLAAVASHCKLLRELDLQENEV
                     EDRGPRWLSCFPDSCTSLVSLNFACIKGEVNAGSLERLVSRSPNLRSLRLNRSVSVDTL
                     AKILLRTPNLEDLGTGNLTDDFQTESYFKLTSALEKCKMLRSLSGFWDASPVCLSFIYP
                     LCAQLTGLNLSYAPTLDASDLTKMISRCVKLQRLWVLDCISDKGLQVVASSCKDLQELR
                     VFPSDFYVAGYSAVTEEGLVAVSLGCPKLNSLLYFCHQMTNAALVTVAKNCPNFTRFRL
                     CILEPGKPDVVTSQPLDEGFGAIVRECKGLQRLSISGLLTDKVFMYIGKYAKQLEMLSI
                     AFAGDSDKGMMHVMNGCKNLRKLEIRDSPFGDAALLGNFARYETMRSLWMSSCNVTLKG
                     CQVLASKMPMLNVEVINERDGSNEMEENHGDLPKVEKLYVYRTTAGARDDAPNFVKIL"
     CDS             2385..2441
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2448..2651
                     /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"
     CDS             join(2658..2660,2661..2663,2664..3374)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2658..2660,2661..2663,2664..3374)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2703..2724)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2964..2983)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3311..3332
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3411..3437
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     polyA_signal    3588..3709
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3625..3644)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3679..3698
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    3733..4321
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3786..3806)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4204..4215)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4353..4829
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4552..4571)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4869..5009
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4869..4998
                     /label=AAV2 ITR
     rep_origin      5084..5539
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5171..5190)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5381..5402
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5556..5575)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5675..5697
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5735..5753)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5821..5925
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5926..5994,5995..6786)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6144..6163)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6957..7545
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7446..7465
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg cgtcgggcga
       61 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
      121 tcactagggg ttcctgcggc cgcacgcgtg tgtctagact gcagagggcc ctgcgtatga
      181 gtgcaagtgg gttttaggac caggatgagg cggggtgggg gtgcctacct gacgaccgac
      241 cccgacccac tggacaagca cccaaccccc attccccaaa ttgcgcatcc cctatcagag
      301 agggggaggg gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc agcaccgcgg
      361 acagtgcctt cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac tgaaggcgcg
      421 ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg gccaccttgg tcgcgtccgc
      481 gccgccgccg gcccagccgg accgcaccac gcgaggcgcg agataggggg gcacgggcgc
      541 gaccatctgc gctgcggcgc cggcgactca gcgctgcctc agtctgcggt gggcagcgga
      601 ggagtcgtgt cgtgcctgag agcgcagtcg agaggatcgc caccatgaca tactttcctg
      661 aagaggtcgt cgaacacatt tttagcttcc tgcctgcaca gagagataga aacacagtga
      721 gcctggtctg caaagtgtgg tacgagatcg aacgcctgag ccggagagga gtgttcgtcg
      781 gcaactgcta tgctgtgaga gcaggcaggg tcgccgctag gtttccaaat gtgcgcgcac
      841 tgaccgtcaa ggggaaaccc cacggcgccg actttaacct ggtgccccct gattggggag
      901 gatacgccgg cccttggatc gaggcagccg ctcgcggctg tcatggactg gaggaactgc
      961 gcatgaagcg aatggtggtc tctgacgaaa gtctggagct gctggctcgg agcttcccta
     1021 ggtttcgcgc actggtgctg atttcttgcg aaggcttcag caccgatgga ctggcagccg
     1081 tggcctccca ctgtaagctg ctgcgggagc tggacctcca ggagaatgaa gtggaggata
     1141 gaggccccag atggctgtct tgcttcccag actcatgtac cagcctggtg tccctgaact
     1201 ttgcctgcat caaaggcgaa gtgaatgctg ggtccctgga gcggctggtc tcaagaagcc
     1261 ccaacctgag gtctctgcgg ctgaaccgga gcgtgagcgt ggacactctg gctaagattc
     1321 tgctgagaac ccctaacctg gaggatctgg gaaccggcaa tctgacagac gatttccaga
     1381 cagaatccta ctttaaactg acttctgccc tggagaagtg taaaatgctg aggagtctgt
     1441 caggattctg ggatgcttca cccgtgtgcc tgagctttat ctaccctctg tgtgcacagc
     1501 tgacaggcct gaacctgagc tatgcaccaa ccctggacgc cagtgatctg acaaagatga
     1561 tctcacgctg cgtgaaactc cagcgactgt gggtgctgga ctgtatttcc gataaggggc
     1621 tccaggtggt cgccagctcc tgcaaggacc tccaggagct gagagtgttc ccatctgatt
     1681 tttacgtggc cggatatagt gctgtcactg aggaaggcct ggtggcagtc tcactgggat
     1741 gcccaaagct gaacagcctg ctgtatttct gtcatcagat gactaatgct gcactggtga
     1801 ccgtcgccaa gaactgccct aatttcaccc gatttcggct gtgtattctg gaaccaggca
     1861 aacccgacgt ggtcacatcc cagccactgg atgaagggtt tggagctatc gtgagagagt
     1921 gcaagggact ccagaggctg agcatttccg gcctgctgac agacaaagtg ttcatgtaca
     1981 tcggcaagta tgctaagcag ctggagatgc tgagcattgc atttgccgga gactccgata
     2041 agggcatgat gcacgtgatg aacgggtgta agaatctgcg aaaactggaa atccgggaca
     2101 gccctttcgg ggatgccgct ctgctgggaa actttgccag atacgagaca atgaggagcc
     2161 tgtggatgtc tagttgcaat gtgactctga agggctgtca ggtcctggct agtaaaatgc
     2221 ctatgctgaa cgtggaagtc attaatgagc gggacgggtc taacgaaatg gaggaaaatc
     2281 atggcgacct gccaaaggtg gagaaactgt atgtgtatcg gaccaccgca ggggcaagag
     2341 atgatgctcc caactttgtg aagattctga gatcaggaag cggagctact aacttcagcc
     2401 tgctgaagca ggctggagac gtggaggaga accctggacc tagatctaag gagaagagtg
     2461 cttgtcctaa agatccagcc aaacctccgg ccaaggcaca agttgtggga tggccaccgg
     2521 tgagatcata ccggaagaac gtgatggttt cctgccaaaa atcaagcggt ggcccggagg
     2581 cggcggcgtt cgtgaaggta tcaatggacg gagcaccgta cttgaggaaa atcgatttga
     2641 ggatgtataa agctagcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     2701 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     2761 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     2821 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     2881 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     2941 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     3001 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     3061 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     3121 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     3181 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     3241 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     3301 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     3361 acgagctgta caagggtgca ggcgcccaag cttcgaattc tgcagtcgac ctgccccccc
     3421 tggagcgcct gaccctgatc gacggtaccg cgggcccggg atccaccgga tctagataac
     3481 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     3541 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     3601 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3661 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa attcgatatc
     3721 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     3781 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     3841 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     3901 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     3961 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     4021 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     4081 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     4141 ccttggctgc tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     4201 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     4261 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     4321 catcgatacc gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag
     4381 tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat
     4441 taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg
     4501 tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg
     4561 aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc
     4621 aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc
     4681 tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct
     4741 ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg
     4801 cgtgaaccac tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga
     4861 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4921 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4981 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     5041 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     5101 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5161 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5221 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5281 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5341 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5401 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     5461 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5521 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5581 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5641 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5701 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     5761 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5821 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5881 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5941 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     6001 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     6061 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     6121 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     6181 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6241 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6301 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6361 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6421 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6481 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6541 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6601 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6661 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6721 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6781 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6841 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6901 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6961 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7021 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7081 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     7141 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7201 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7261 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7321 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7381 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7441 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7501 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7561 gcctttttac ggttcctggc cttttgctgg ccttttgctc a
//
