LOCUS Exported 5825 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV vector including gRNA expression cassette and mEGFP knock-in donor targeting mouse camk2a. ACCESSION . VERSION . KEYWORDS pAAV-HDR-mEGFP-camk2a SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5825) AUTHORS Nishiyama J, Mikuni T, Yasuda R TITLE Virus-Mediated Genome Editing via Homology-Directed Repair in Mitotic and Postmitotic Cells in Mammalian Brain. JOURNAL Neuron. 2017 Oct 18. pii: S0896-6273(17)30933-9. doi: 10.1016/j.neuron.2017.10.004. PUBMED 29056297 REFERENCE 2 (bases 1 to 5825) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5825 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 149..389 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 149..169 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 320..339 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 419..494 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 1441..2160 /codon_start=1 /product="enhanced GFP with monomerizing A206K mutation (Zacharias et al., 2002)" /label=mEGFP /note="mammalian codon-optimized" /translation="MSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVL LEFVTAAGITLGMDELYK" primer_bind complement(1489..1510) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1750..1769) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2097..2118 /label=EGFP-C /note="EGFP, forward primer" repeat_region 3088..3228 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3088..3217 /label=AAV2 ITR rep_origin 3303..3758 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3390..3409) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3600..3621 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3775..3794) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3894..3916 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3954..3972) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4040..4144 /gene="bla" /label=AmpR promoter CDS 4145..5005 /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(4363..4382) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5176..5764 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5665..5684 /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 gcggcctcta gaaagcttga gggcctattt cccatgattc cttcatattt 181 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa 241 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt 301 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt 361 cttggcttta tatatcttgt ggaaaggacg aaacaccgct gcctgcccag tgccaggagt 421 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg 481 caccgagtcg gtgctttttt gaattcgctg tggcagaggc ctcaggaggg gccctgctgc 541 tcagtggtga cagatagggg tgagaaagca gacagagtca ttccgtcagc attctgggtc 601 tgtttggtac ttcttctcac gataaggtgg cggtgtgata tgcacaatgg ctaaaaagca 661 gggagagctg gaaagaaaca aggacagaga cagaggccaa gtcaaccaga ccaattccca 721 gaggaagcaa agaaaccatt acagagacta caagggggaa gggaaggaga gatgaattag 781 cttcccctgt aaaccttaga acccagctgt tgccagggca acggggcaat acctgtctct 841 tcagaggaga tgaagttgcc agggtaacta catcctgtct ttctcaagga ccatcccaga 901 atgtggcacc cactagccgt taccatagca actgcctctt tgccccactt aatcccatcc 961 cgtctgttaa aagggcccta tagttggagg tgggggaggt aggaagagcg atgatcactt 1021 gtggactaag tttgttcgca tccccttctc caaccccctc agtacatcac cctgggggaa 1081 cagggtccac ttgctcctgg gcccacacag tcctgcagta ttgtgtatat aaggccaggg 1141 caaagaggag caggttttaa agtgaaaggc aggcaggtgt tggggaggca gttaccgggg 1201 caacgggaac agggcgtttc ggaggtggtt gccatgggga cctggatgct gacgaaggct 1261 cgcgaggctg tgagcagcca cagtgccctg ctcagaagcc ccaagctcgt cagtcaagcc 1321 ggttctccgt ttgcactcag gagcacgggc aggcgagtgg cccctagttc tgggggcagc 1381 gcttcagcat cccagcccta gttcccagcc taaagcctcg cctgcctgcc cagtgccagg 1441 atgtccgtga gcaagggcga ggagctcttc accggggtgg tgcccatcct ggtcgagctg 1501 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 1561 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 1621 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 1681 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 1741 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 1801 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 1861 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag 1921 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc 1981 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 2041 cactacctga gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg 2101 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 2161 gctaccatca cctgcacccg attcacagaa gagtaccagc tctttgagga actgggaaag 2221 tgagtcacac ctcagggagt cctagagagc atggggatca gtctccttgg gcagggggag 2281 ggggcgtgtg cctatgtggc cagagctgtc ccgtgcatgc agagggaggt gtgcagcatg 2341 atttggaagc actgtgtatg catagggtgt ccagagttag gcgctgacac atttatgagt 2401 ccagtcagcc tggtgtgtgt gtgtgtgtgt gtgtgtgtga tggaaaggaa acagattggt 2461 ggtgtatgca cacacctatg catataaata cacatgacct gggttcacaa agatatctag 2521 aaacttgggg tttgaaaggg attttcccac ttggctggat ctgagctgga atctattttt 2581 gtagttgtac ttagaaacct ccaatgacaa agaaagtttg ccaccccctg acctggagat 2641 cttgtcttta gacactgtgc agagtggcta gctttctcct agcacatccc ctccggcttc 2701 cccagatacc ttccgggagc aacagcttca gtacagaagg agttaatata aagcaatcac 2761 agtgttggga ggaggggaag gacagtcagc cagccggggg gtgccactgt ggcactgact 2821 atacaaaaca actgtggtcc tttggcaaag ttgcccatcg tgttatatct caggaggctg 2881 agaggagaaa ggttccctcc agaagagcag caggctacag atataagaga aggtttctag 2941 atgttcccag ctaggttatg gggtcctggg ttggatcagg agttggggga gcagtttggc 3001 agagtgcccc acggatgcat acgtgctcct accagtcctg tttggttcct gctgtagcgc 3061 agtaacatga gacgtcgacg cggccgcagg aacccctagt gatggagttg gccactccct 3121 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct 3181 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc 3241 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata 3301 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 3361 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 3421 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 3481 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg 3541 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 3601 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt 3661 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt 3721 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat 3781 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc 3841 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 3901 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt 3961 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg 4021 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 4081 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 4141 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 4201 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 4261 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 4321 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 4381 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 4441 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 4501 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 4561 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 4621 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 4681 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 4741 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 4801 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 4861 aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 4921 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 4981 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 5041 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 5101 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 5161 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 5221 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 5281 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 5341 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 5401 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 5461 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 5521 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 5581 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 5641 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 5701 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 5761 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 5821 catgt //