LOCUS Exported 6345 bp ds-DNA circular SYN 28-JUN-2022 DEFINITION AAV expression of GFP and human alpha-Synuclein with S129D mutation from hSyn promoter. ACCESSION . VERSION . KEYWORDS pAAV-hSyn-GFP-IRES-hSNCA (S129D) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6345) TITLE Postsynaptic synucleins mediate vesicular exocytosis of endocannabinoids JOURNAL Unpublished REFERENCE 2 (bases 1 to 6345) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 28, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6345 /organism="synthetic DNA construct" /mol_type="other DNA" regulatory 593..602 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 599..1318 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 599..1315 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(644..665) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(905..924) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1252..1273 /label=EGFP-C /note="EGFP, forward primer" misc_feature 1447..1909 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(1503..1520) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 1730..1749 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" regulatory 1944..1953 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" misc_feature 2403..2991 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2456..2476) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2874..2885) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3023..3499 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3222..3241) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 3539..3679 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3539..3668 /label=AAV2 ITR rep_origin 3754..4209 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3841..3860) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4051..4072 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4226..4245) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4345..4367 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4405..4423) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4491..4595 /gene="bla" /label=AmpR promoter CDS 4596..5456 /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(4814..4833) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5627..6215 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6116..6135 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 6277..61 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" ORIGIN 1 gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc 61 tgcggccgca cgcgttaact gcagagggcc ctgcgtatga gtgcaagtgg gttttaggac 121 caggatgagg cggggtgggg gtgcctacct gacgaccgac cccgacccac tggacaagca 181 cccaaccccc attccccaaa ttgcgcatcc cctatcagag agggggaggg gaaacaggat 241 gcggcgaggc gcgtgcgcac tgccagcttc agcaccgcgg acagtgcctt cgcccccgcc 301 tggcggcgcg cgccaccgcc gcctcagcac tgaaggcgcg ctgacgtcac tcgccggtcc 361 cccgcaaact ccccttcccg gccaccttgg tcgcgtccgc gccgccgccg gcccagccgg 421 accgcaccac gcgaggcgcg agataggggg gcacgggcgc gaccatctgc gctgcggcgc 481 cggcgactca gcgctgcctc agtctgcggt gggcagcgga ggagtcgtgt cgtgcctgag 541 agcgcgtccg gagctattag caatatgaaa cctcttaggc gcgccaccgg tcgccaccat 601 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg 661 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg 721 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct 781 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca 841 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt 901 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt 961 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa 1021 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg 1081 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga 1141 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta 1201 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct 1261 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag 1321 ccagtgtggt ggtacgtagg aattctccct cccccctaac gttactggcc gaagccgctt 1381 ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg 1441 caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc 1501 ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga 1561 agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc 1621 tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc 1681 acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc 1741 aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga 1801 tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg 1861 ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat ggtaccgcgg 1921 gcccgggatc ctctagagtc gacgccacca tggatgtatt catgaaagga ctttcaaagg 1981 ccaaggaggg agttgtggct gctgctgaga aaaccaaaca gggtgtggca gaagcagcag 2041 gaaagacaaa agagggtgtt ctctatgtag gctccaaaac caaggaggga gtggtgcatg 2101 gtgtggcaac agtggctgag aagaccaaag agcaagtgac aaatgttgga ggagcagtgg 2161 tgacgggtgt gacagcagta gcccagaaga cagtggaggg agcagggagc attgcagcag 2221 ccactggctt tgtcaaaaag gaccagttgg gcaagaatga agaaggagcc ccacaggaag 2281 gaattctgga agatatgcct gtggatcctg acaatgaggc ttatgaaatg cctgatgagg 2341 aagggtatca agactacgaa cctgaagcct aagctagcga attcgatatc aagcttatcg 2401 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg 2461 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc 2521 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt 2581 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca 2641 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc 2701 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc 2761 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc 2821 tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc 2881 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc 2941 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc 3001 gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct 3061 ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat 3121 cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag 3181 caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg 3241 gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct 3301 cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt 3361 tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta 3421 atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac 3481 tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag 3541 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc 3601 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga 3661 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt 3721 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg 3781 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg 3841 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 3901 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 3961 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 4021 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 4081 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt 4141 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 4201 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 4261 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 4321 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 4381 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 4441 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 4501 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 4561 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 4621 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 4681 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 4741 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 4801 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 4861 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 4921 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 4981 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 5041 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 5101 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 5161 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 5221 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 5281 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc 5341 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 5401 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 5461 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 5521 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 5581 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 5641 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 5701 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 5761 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 5821 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 5881 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 5941 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 6001 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 6061 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 6121 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 6181 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 6241 ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct 6301 cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccg //