LOCUS Exported 7034 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Vector E encodes pAAV-pMecp2-dSaCas9-KRAB-spA-pU6-sgRNA (BsaI) transgenes for AAV packaging and expression of CRISPR interference in neurons. ACCESSION . VERSION . KEYWORDS pAAV-pMecp2-dSaCas9-KRAB-pU6-sgRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7034) AUTHORS Lau CH, Ho JW, Lo PK, Tin C TITLE Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of Engineered AAV1 Expressing CRISPRa. JOURNAL Mol Ther Nucleic Acids. 2019 Jun 7;16:637-649. doi: 10.1016/j.omtn.2019.04.015. Epub 2019 Apr 23. PUBMED 31108320 REFERENCE 2 (bases 1 to 7034) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7034 /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..377 /label=Mecp2 promoter /note="promoter for selective expression in neurons (Adachi et al., 2005; Gray et al., 2011)" regulatory 384..393 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 396..416 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3597..3644 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 3681..3875 /codon_start=1 /product="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" /label=KRAB /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEPWLV" polyA_signal 3885..3933 /note="synthetic polyadenylation signal" promoter 3939..4179 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 3939..3959 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 4110..4129 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4208..4283 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" repeat_region 4297..4437 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4297..4426 /label=AAV2 ITR rep_origin 4512..4967 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4599..4618) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4809..4830 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4984..5003) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5103..5125 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5163..5181) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5249..5353 /gene="bla" /label=AmpR promoter CDS 5354..6214 /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(5572..5591) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6385..6973 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6874..6893 /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 gaaagcttag ctgaatgggg tccgcctctt ttccctgcct 181 aaacagacag gaactcctgc caattgaggg cgtcaccgct aaggctccgc cccagcctgg 241 gctccacaac caatgaaggg taatctcgac aaagagcaag gggtggggcg cgggcgcgca 301 ggtgcagcag cacacaggct ggtcgggagg gcggggcgcg acgtctgccg tgcggggtcc 361 cggcatcggt tgcgcgcacc ggtgccacca tggccccaaa gaagaagcgg aaggtcggta 421 tccacggagt cccagcagcc aagcggaact acatcctggg cctggccatc ggcatcacca 481 gcgtgggcta cggcatcatc gactacgaga cacgggacgt gatcgatgcc ggcgtgcggc 541 tgttcaaaga ggccaacgtg gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa 601 ggctgaagcg gcggaggcgg catagaatcc agagagtgaa gaagctgctg ttcgactaca 661 acctgctgac cgaccacagc gagctgagcg gcatcaaccc ctacgaggcc agagtgaagg 721 gcctgagcca gaagctgagc gaggaagagt tctctgccgc cctgctgcac ctggccaaga 781 gaagaggcgt gcacaacgtg aacgaggtgg aagaggacac cggcaacgag ctgtccacca 841 aagagcagat cagccggaac agcaaggccc tggaagagaa atacgtggcc gaactgcagc 901 tggaacggct gaagaaagac ggcgaagtgc ggggcagcat caacagattc aagaccagcg 961 actacgtgaa agaagccaaa cagctgctga aggtgcagaa ggcctaccac cagctggacc 1021 agagcttcat cgacacctac atcgacctgc tggaaacccg gcggacctac tatgagggac 1081 ctggcgaggg cagccccttc ggctggaagg acatcaaaga atggtacgag atgctgatgg 1141 gccactgcac ctacttcccc gaggaactgc ggagcgtgaa gtacgcctac aacgccgacc 1201 tgtacaacgc cctgaacgac ctgaacaatc tcgtgatcac cagggacgag aacgagaagc 1261 tggaatatta cgagaagttc cagatcatcg agaacgtgtt caagcagaag aagaagccca 1321 ccctgaagca gatcgccaaa gaaatcctcg tgaacgaaga ggatattaag ggctacagag 1381 tgaccagcac cggcaagccc gagttcacca acctgaaggt gtaccacgac atcaaggaca 1441 ttaccgcccg gaaagagatt attgagaacg ccgagctgct ggatcagatt gccaagatcc 1501 tgaccatcta ccagagcagc gaggacatcc aggaagaact gaccaatctg aactccgagc 1561 tgacccagga agagatcgag cagatctcta atctgaaggg ctataccggc acccacaacc 1621 tgagcctgaa ggccatcaac ctgatcctgg acgagctgtg gcacaccaac gacaaccaga 1681 tcgctatctt caaccggctg aagctggtgc ccaagaaggt ggacctgtcc cagcagaaag 1741 agatccccac caccctggtg gacgacttca tcctgagccc cgtcgtgaag agaagcttca 1801 tccagagcat caaagtgatc aacgccatca tcaagaagta cggcctgccc aacgacatca 1861 ttatcgagct ggcccgcgag aagaactcca aggacgccca gaaaatgatc aacgagatgc 1921 agaagcggaa ccggcagacc aacgagcgga tcgaggaaat catccggacc accggcaaag 1981 agaacgccaa gtacctgatc gagaagatca agctgcacga catgcaggaa ggcaagtgcc 2041 tgtacagcct ggaagccatc cctctggaag atctgctgaa caaccccttc aactatgagg 2101 tggaccacat catccccaga agcgtgtcct tcgacaacag cttcaacaac aaggtgctcg 2161 tgaagcagga agaagccagc aagaagggca accggacccc attccagtac ctgagcagca 2221 gcgacagcaa gatcagctac gaaaccttca agaagcacat cctgaatctg gccaagggca 2281 agggcagaat cagcaagacc aagaaagagt atctgctgga agaacgggac atcaacaggt 2341 tctccgtgca gaaagacttc atcaaccgga acctggtgga taccagatac gccaccagag 2401 gcctgatgaa cctgctgcgg agctacttca gagtgaacaa cctggacgtg aaagtgaagt 2461 ccatcaatgg cggcttcacc agctttctgc ggcggaagtg gaagtttaag aaagagcgga 2521 acaaggggta caagcaccac gccgaggacg ccctgatcat tgccaacgcc gatttcatct 2581 tcaaagagtg gaagaaactg gacaaggcca aaaaagtgat ggaaaaccag atgttcgagg 2641 aaaagcaggc cgagagcatg cccgagatcg aaaccgagca ggagtacaaa gagatcttca 2701 tcacccccca ccagatcaag cacattaagg acttcaagga ctacaagtac agccaccggg 2761 tggacaagaa gcctaataga gagctgatta acgacaccct gtactccacc cggaaggacg 2821 acaagggcaa caccctgatc gtgaacaatc tgaacggcct gtacgacaag gacaatgaca 2881 agctgaaaaa gctgatcaac aagagccccg aaaagctgct gatgtaccac cacgaccccc 2941 agacctacca gaaactgaag ctgattatgg aacagtacgg cgacgagaag aatcccctgt 3001 acaagtacta cgaggaaacc gggaactacc tgaccaagta ctccaaaaag gacaacggcc 3061 ccgtgatcaa gaagattaag tattacggca acaaactgaa cgcccatctg gacatcaccg 3121 acgactaccc caacagcaga aacaaggtcg tgaagctgtc cctgaagccc tacagattcg 3181 acgtgtacct ggacaatggc gtgtacaagt tcgtgaccgt gaagaatctg gatgtgatca 3241 aaaaagaaaa ctactacgaa gtgaatagca agtgctatga ggaagctaag aagctgaaga 3301 agatcagcaa ccaggccgag tttatcgcct ccttctacaa caacgatctg atcaagatca 3361 acggcgagct gtatagagtg atcggcgtga acaacgacct gctgaaccgg atcgaagtga 3421 acatgatcga catcacctac cgcgagtacc tggaaaacat gaacgacaag aggcccccca 3481 ggatcattaa gacaatcgcc tccaagaccc agagcattaa gaagtacagc acagacattc 3541 tgggcaacct gtatgaagtg aaatctaaga agcaccctca gatcatcaaa aagggcaaaa 3601 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagggatcc atggacgcga 3661 aatcacttac ggcatggtcg agaacactgg ttacgttcaa ggacgtgttt gtggacttta 3721 cacgtgagga gtggaaattg ctggatactg cgcaacaaat tgtgtatcga aatgtcatgc 3781 ttgagaatta caagaacctc gtcagtctcg gataccagtt gacgaaaccg gatgtgatcc 3841 ttaggctcga aaagggggaa gaaccttggc tggtataaga attcaataaa agatctttat 3901 tttcattaga tctgtgtgtt ggttttttgt gtggtaccga gggcctattt cccatgattc 3961 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg 4021 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt 4081 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt 4141 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgga gaccacggca 4201 ggtctcagtt ttagtactct ggaaacagaa tctactaaaa caaggcaaaa tgccgtgttt 4261 atctcgtcaa cttgttggcg agatttttgc ggccgcagga acccctagtg atggagttgg 4321 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 4381 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc 4441 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa 4501 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 4561 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc 4621 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 4681 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc 4741 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 4801 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc 4861 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta 4921 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct 4981 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 5041 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 5101 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 5161 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac 5221 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 5281 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 5341 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 5401 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 5461 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 5521 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 5581 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 5641 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 5701 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 5761 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 5821 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 5881 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 5941 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 6001 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 6061 tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 6121 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 6181 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 6241 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 6301 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 6361 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 6421 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 6481 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt 6541 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 6601 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 6661 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 6721 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 6781 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 6841 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 6901 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 6961 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 7021 ttttgctcac atgt //