LOCUS Exported 7601 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION OsTIR1(F74G)-P2A-mAID-EGFP-NES. ACCESSION . VERSION . KEYWORDS pAAV-hSyn-OsTIR1(F74G) 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) 11(1):5701 REFERENCE 2 (bases 1 to 7601) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7601 /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 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 2658..3374 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 2658..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 5926..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 //