LOCUS Exported 4465 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV vector with CaMKIIa promoter, EGFP, and W3SL(shorten expression cassette with relatively high expression to maximize expression of larger genes) . ACCESSION . VERSION . KEYWORDS pAAV-CW3SL-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4465) AUTHORS Choi JH, Yu NK, Baek GC, Bakes J, Seo D, Nam HJ, Baek SH, Lim CS, Lee YS, Kaang BK TITLE Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons. JOURNAL Mol Brain. 2014 Mar 11;7:17. doi: 10.1186/1756-6606-7-17. PUBMED 24618276 REFERENCE 2 (bases 1 to 4465) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4465 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" regulatory 550..559 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 556..1275 /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 556..1272 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(601..622) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(862..881) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1209..1230 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1348..1368) /label=WPRE-R /note="WPRE, reverse primer" repeat_region 1728..1868 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 1728..1857 /label=AAV2 ITR rep_origin 1943..2398 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2030..2049) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2240..2261 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2415..2434) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2534..2556 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(2594..2612) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2680..2784 /gene="bla" /label=AmpR promoter CDS 2785..3645 /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(3003..3022) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3816..4404 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4305..4324 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggccgcag atgctgcaca cttgtggact aagtttgttc gcatcccctt 181 ctccaacccc ctcagtacat caccctgggg gaacagggtc cacttgctcc tgggcccaca 241 cagtcctgca gtattgtgta tataaggcca gggcaaagag gagcaggttt taaagtgaaa 301 ggcaggcagg tgttggggag gcagttaccg gggcaacggg aacagggcgt ttcggaggtg 361 gttgccatgg ggacctggat gctgacgaag gctcgcgagg ctgtgagcag ccacagtgcc 421 ctgctcagaa gccccaagct cgtcagtcaa gccggttctc cgtttgcact caggagcacg 481 ggcaggcgag tggcccctag ttctgggggc agcgtgaaaa gacggtatcg ataagcttga 541 ttcgagctag ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 601 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 661 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 721 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 781 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 841 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 901 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 961 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 1021 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 1081 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 1141 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 1201 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 1261 gagctgtaca agtaaatcga attccgctcg agataatcaa cctctggatt acaaaatttg 1321 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc 1381 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta 1441 taaatcctgg ttagttcttg ccacggcgga actcatcgcc gcctgccttg cccgctgctg 1501 gacaggggct cggctgttgg gcactgacaa ttccgtggtg tttatttgtg aaatttgtga 1561 tgctattgct ttatttgtaa ccatctagct ttatttgtga aatttgtgat gctattgctt 1621 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta 1681 tgtttcaggt tcagggggag atgtgggagg ttttttaaag cggccgcagg aacccctagt 1741 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa 1801 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg 1861 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 1921 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 1981 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct 2041 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 2101 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg 2161 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 2221 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc 2281 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat 2341 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta 2401 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 2461 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 2521 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 2581 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 2641 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 2701 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 2761 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 2821 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 2881 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 2941 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 3001 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 3061 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 3121 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 3181 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 3241 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 3301 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 3361 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 3421 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 3481 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 3541 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 3601 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 3661 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 3721 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 3781 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 3841 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 3901 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 3961 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 4021 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 4081 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 4141 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 4201 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 4261 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 4321 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 4381 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 4441 ttttgctggc cttttgctca catgt //