LOCUS Exported 7211 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Vector D encodes pAAV-pMecp2-dSaCas9-VP160-spA-pU6-sgRNA (BsaI) transgenes for AAV packaging and expression of CRISPR activator in neurons. ACCESSION . VERSION . KEYWORDS pAAV-pMecp2-dSaCas9-VP160-pU6-sgRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7211) 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 7211) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7211 /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 3660..4043 /codon_start=1 /product="10 tandem repeats of the minimal activation domain of herpes simplex virus VP16 (Cheng et al., 2013)" /label=VP160 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS DALDDFDLDMLGSDALDDFDLDML" polyA_signal 4062..4110 /note="synthetic polyadenylation signal" promoter 4116..4356 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 4116..4136 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 4287..4306 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4385..4460 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" repeat_region 4474..4614 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4474..4603 /label=AAV2 ITR rep_origin 4689..5144 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4776..4795) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4986..5007 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5161..5180) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5280..5302 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5340..5358) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5426..5530 /gene="bla" /label=AmpR promoter CDS 5531..6391 /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(5749..5768) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6562..7150 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7051..7070 /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 gggcgcgccg 3661 acgcgctgga cgatttcgat ctcgacatgc tgggttctga tgccctcgat gactttgacc 3721 tggatatgtt gggaagcgac gcattggatg actttgatct ggacatgctc ggctccgatg 3781 ctctggacga tttcgatctc gatatgttag ggtcagacgc actggatgat ttcgaccttg 3841 atatgttggg aagcgatgcc cttgatgatt tcgacctgga catgctcggc agcgacgccc 3901 tggacgattt cgatctggac atgctggggt ccgatgcctt ggatgatttt gacttggata 3961 tgctggggag tgatgccctg gacgactttg acctggacat gctgggctcc gatgcgctcg 4021 atgacttcga tttggatatg ttgtatatcg attaagaatt caataaaaga tctttatttt 4081 cattagatct gtgtgttggt tttttgtgtg gtaccgaggg cctatttccc atgattcctt 4141 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa 4201 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg 4261 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt 4321 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagac cacggcaggt 4381 ctcagtttta gtactctgga aacagaatct actaaaacaa ggcaaaatgc cgtgtttatc 4441 tcgtcaactt gttggcgaga tttttgcggc cgcaggaacc cctagtgatg gagttggcca 4501 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 4561 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc 4621 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca 4681 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 4741 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt 4801 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 4861 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg 4921 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 4981 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt 5041 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca 5101 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag 5161 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga 5221 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 5281 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg 5341 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc 5401 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 5461 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 5521 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 5581 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 5641 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 5701 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 5761 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 5821 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 5881 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 5941 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 6001 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 6061 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 6121 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 6181 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 6241 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 6301 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 6361 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 6421 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 6481 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 6541 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 6601 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 6661 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 6721 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 6781 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 6841 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 6901 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 6961 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 7021 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 7081 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 7141 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 7201 tgctcacatg t //