LOCUS       Exported                7447 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  OsTIR1(WT)-P2A-mAID-EGFP-NES.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-hSyn-OsTIR1(WT)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7447)
  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) 11(1):5701
REFERENCE   2  (bases 1 to 7447)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7447
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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             653..2377
                     /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             2393..2449
                     /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             2456..2659
                     /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             2666..3382
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2666..3382
                     /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(2711..2732)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2972..2991)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3319..3340
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3419..3445
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     misc_feature    3579..4167
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3632..3652)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4050..4061)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4199..4675
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4398..4417)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4715..4855
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4715..4844
                     /label=AAV2 ITR
     rep_origin      4930..5385
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5017..5036)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5227..5248
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5402..5421)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5521..5543
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5581..5599)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5667..5771
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5772..6632
                     /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(5990..6009)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6803..7391
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7292..7311
                     /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 agaaggtacc ggatccgcca ccatgacata
      661 ctttcctgaa gaggtcgtcg aacacatttt tagcttcctg cctgcacaga gagatagaaa
      721 cacagtgagc ctggtctgca aagtgtggta cgagatcgaa cgcctgagcc ggagaggagt
      781 gttcgtcggc aactgctatg ctgtgagagc aggcagggtc gccgctaggt ttccaaatgt
      841 gcgcgcactg accgtcaagg ggaaacccca cttcgccgac tttaacctgg tgccccctga
      901 ttggggagga tacgccggcc cttggatcga ggcagccgct cgcggctgtc atggactgga
      961 ggaactgcgc atgaagcgaa tggtggtctc tgacgaaagt ctggagctgc tggctcggag
     1021 cttccctagg tttcgcgcac tggtgctgat ttcttgcgaa ggcttcagca ccgatggact
     1081 ggcagccgtg gcctcccact gtaagctgct gcgggagctg gacctccagg agaatgaagt
     1141 ggaggataga ggccccagat ggctgtcttg cttcccagac tcatgtacca gcctggtgtc
     1201 cctgaacttt gcctgcatca aaggcgaagt gaatgctggg tccctggagc ggctggtctc
     1261 aagaagcccc aacctgaggt ctctgcggct gaaccggagc gtgagcgtgg acactctggc
     1321 taagattctg ctgagaaccc ctaacctgga ggatctggga accggcaatc tgacagacga
     1381 tttccagaca gaatcctact ttaaactgac ttctgccctg gagaagtgta aaatgctgag
     1441 gagtctgtca ggattctggg atgcttcacc cgtgtgcctg agctttatct accctctgtg
     1501 tgcacagctg acaggcctga acctgagcta tgcaccaacc ctggacgcca gtgatctgac
     1561 aaagatgatc tcacgctgcg tgaaactcca gcgactgtgg gtgctggact gtatttccga
     1621 taaggggctc caggtggtcg ccagctcctg caaggacctc caggagctga gagtgttccc
     1681 atctgatttt tacgtggccg gatatagtgc tgtcactgag gaaggcctgg tggcagtctc
     1741 actgggatgc ccaaagctga acagcctgct gtatttctgt catcagatga ctaatgctgc
     1801 actggtgacc gtcgccaaga actgccctaa tttcacccga tttcggctgt gtattctgga
     1861 accaggcaaa cccgacgtgg tcacatccca gccactggat gaagggtttg gagctatcgt
     1921 gagagagtgc aagggactcc agaggctgag catttccggc ctgctgacag acaaagtgtt
     1981 catgtacatc ggcaagtatg ctaagcagct ggagatgctg agcattgcat ttgccggaga
     2041 ctccgataag ggcatgatgc acgtgatgaa cgggtgtaag aatctgcgaa aactggaaat
     2101 ccgggacagc cctttcgggg atgccgctct gctgggaaac tttgccagat acgagacaat
     2161 gaggagcctg tggatgtcta gttgcaatgt gactctgaag ggctgtcagg tcctggctag
     2221 taaaatgcct atgctgaacg tggaagtcat taatgagcgg gacgggtcta acgaaatgga
     2281 ggaaaatcat ggcgacctgc caaaggtgga gaaactgtat gtgtatcgga ccaccgcagg
     2341 ggcaagagat gatgctccca actttgtgaa gattctgaga tcaggaagcg gagctactaa
     2401 cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggaccta gatctaagga
     2461 gaagagtgct tgtcctaaag atccagccaa acctccggcc aaggcacaag ttgtgggatg
     2521 gccaccggtg agatcatacc ggaagaacgt gatggtttcc tgccaaaaat caagcggtgg
     2581 cccggaggcg gcggcgttcg tgaaggtatc aatggacgga gcaccgtact tgaggaaaat
     2641 cgatttgagg atgtataaag ctagcatggt gagcaagggc gaggagctgt tcaccggggt
     2701 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2761 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2821 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     2881 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2941 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     3001 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     3061 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     3121 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     3181 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     3241 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     3301 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3361 cggcatggac gagctgtaca agggtgcagg cgcccaagct tcgaattctg cagtcgacct
     3421 gccccccctg gagcgcctga ccctgatcga cggtaccgcg ggcccgggat ccaccggatc
     3481 tagataactg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     3541 cctcccacac ctcccaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     3601 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     3661 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3721 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     3781 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3841 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3901 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3961 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc
     4021 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     4081 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     4141 ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg
     4201 gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt
     4261 gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt
     4321 ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac
     4381 ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct
     4441 cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt
     4501 gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg
     4561 tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg
     4621 gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat
     4681 tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     4741 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     4801 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     4861 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     4921 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4981 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     5041 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5101 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     5161 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5221 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     5281 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     5341 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     5401 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     5461 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     5521 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     5581 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5641 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5701 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5761 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5821 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5881 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5941 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     6001 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     6061 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     6121 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6181 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6241 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6301 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6361 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6421 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6481 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6541 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6601 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6661 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6721 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6781 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6841 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6901 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6961 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     7021 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     7081 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     7141 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     7201 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7261 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7321 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7381 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7441 ttgctca
//
