LOCUS Exported 7624 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses dSa Cas9 fused to optimized combination of truncated activation domains from SCP1 promoter with dual snRP1 poly adenylation signal, and Sa sgRNA for Actc1, vector allows for strong activation of mouse Actc1, can be packaged and delivered as AAV. ACCESSION . VERSION . KEYWORDS pAAV-SCP1-dSa VPR mini.-2X snRP-1 Actc1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7624) TITLE Church Lab CRISPR plasmids 2017 REFERENCE 2 (bases 1 to 7624) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7624 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(5..27) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 127..146 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin complement(163..618) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(300..321) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 512..531 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 693..833 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 704..833 /label=AAV2 ITR promoter 842..1082 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 842..862 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1013..1032 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 1112..1187 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" regulatory 1294..1303 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1306..1326 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4507..4554 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 4585..4734 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 4759..4779 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4939..5295 /codon_start=1 /product="transcriptional activation domain of human RelA, also known as p65 (O'Shea and Perkins, 2008)" /label=RelA (p65) AD /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD EDFSSIADMDFSALL" repeat_region 5579..5719 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5579..5708 /label=AAV2 ITR rep_origin complement(5781..6369) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5861..5880) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6540..7400) /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 7163..7182 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7401..7505) /gene="bla" /label=AmpR promoter primer_bind 7573..7591 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 61 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta 121 actatgcggc atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata 181 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg 241 aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc 301 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 361 ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt 421 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 481 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 541 aggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 601 cgccgctaca gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag 661 atgcgtaagg agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg ctcgctcgct 721 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt 781 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg 841 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga 901 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag 961 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca 1021 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg 1081 acgaaacacc ggccattctt ggagccaagg ggttttagta ctctggaaac agaatctact 1141 aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt ttttggccgc 1201 ctcgaggtac ttatataagg gggtgggggc gcgttcgtcc tcagtcgcga tcgaacactc 1261 gagccgagca gacgtgccta cggaccgacc ggtgccacca tggccccaaa gaagaagcgg 1321 aaggtcggta tccacggagt cccagcagcc aagcggaact acatcctggg cctggccatc 1381 ggcatcacca gcgtgggcta cggcatcatc gactacgaga cacgggacgt gatcgatgcc 1441 ggcgtgcggc tgttcaaaga ggccaacgtg gaaaacaacg agggcaggcg gagcaagaga 1501 ggcgccagaa ggctgaagcg gcggaggcgg catagaatcc agagagtgaa gaagctgctg 1561 ttcgactaca acctgctgac cgaccacagc gagctgagcg gcatcaaccc ctacgaggcc 1621 agagtgaagg gcctgagcca gaagctgagc gaggaagagt tctctgccgc cctgctgcac 1681 ctggccaaga gaagaggcgt gcacaacgtg aacgaggtgg aagaggacac cggcaacgag 1741 ctgtccacca aagagcagat cagccggaac agcaaggccc tggaagagaa atacgtggcc 1801 gaactgcagc tggaacggct gaagaaagac ggcgaagtgc ggggcagcat caacagattc 1861 aagaccagcg actacgtgaa agaagccaaa cagctgctga aggtgcagaa ggcctaccac 1921 cagctggacc agagcttcat cgacacctac atcgacctgc tggaaacccg gcggacctac 1981 tatgagggac ctggcgaggg cagccccttc ggctggaagg acatcaaaga atggtacgag 2041 atgctgatgg gccactgcac ctacttcccc gaggaactgc ggagcgtgaa gtacgcctac 2101 aacgccgacc tgtacaacgc cctgaacgac ctgaacaatc tcgtgatcac cagggacgag 2161 aacgagaagc tggaatatta cgagaagttc cagatcatcg agaacgtgtt caagcagaag 2221 aagaagccca ccctgaagca gatcgccaaa gaaatcctcg tgaacgaaga ggatattaag 2281 ggctacagag tgaccagcac cggcaagccc gagttcacca acctgaaggt gtaccacgac 2341 atcaaggaca ttaccgcccg gaaagagatt attgagaacg ccgagctgct ggatcagatt 2401 gccaagatcc tgaccatcta ccagagcagc gaggacatcc aggaagaact gaccaatctg 2461 aactccgagc tgacccagga agagatcgag cagatctcta atctgaaggg ctataccggc 2521 acccacaacc tgagcctgaa ggccatcaac ctgatcctgg acgagctgtg gcacaccaac 2581 gacaaccaga tcgctatctt caaccggctg aagctggtgc ccaagaaggt ggacctgtcc 2641 cagcagaaag agatccccac caccctggtg gacgacttca tcctgagccc cgtcgtgaag 2701 agaagcttca tccagagcat caaagtgatc aacgccatca tcaagaagta cggcctgccc 2761 aacgacatca ttatcgagct ggcccgcgag aagaactcca aggacgccca gaaaatgatc 2821 aacgagatgc agaagcggaa ccggcagacc aacgagcgga tcgaggaaat catccggacc 2881 accggcaaag agaacgccaa gtacctgatc gagaagatca agctgcacga catgcaggaa 2941 ggcaagtgcc tgtacagcct ggaagccatc cctctggaag atctgctgaa caaccccttc 3001 aactatgagg tggaccacat catccccaga agcgtgtcct tcgacaacag cttcaacaac 3061 aaggtgctcg tgaagcagga agaagccagc aagaagggca accggacccc attccagtac 3121 ctgagcagca gcgacagcaa gatcagctac gaaaccttca agaagcacat cctgaatctg 3181 gccaagggca agggcagaat cagcaagacc aagaaagagt atctgctgga agaacgggac 3241 atcaacaggt tctccgtgca gaaagacttc atcaaccgga acctggtgga taccagatac 3301 gccaccagag gcctgatgaa cctgctgcgg agctacttca gagtgaacaa cctggacgtg 3361 aaagtgaagt ccatcaatgg cggcttcacc agctttctgc ggcggaagtg gaagtttaag 3421 aaagagcgga acaaggggta caagcaccac gccgaggacg ccctgatcat tgccaacgcc 3481 gatttcatct tcaaagagtg gaagaaactg gacaaggcca aaaaagtgat ggaaaaccag 3541 atgttcgagg aaaagcaggc cgagagcatg cccgagatcg aaaccgagca ggagtacaaa 3601 gagatcttca tcacccccca ccagatcaag cacattaagg acttcaagga ctacaagtac 3661 agccaccggg tggacaagaa gcctaataga gagctgatta acgacaccct gtactccacc 3721 cggaaggacg acaagggcaa caccctgatc gtgaacaatc tgaacggcct gtacgacaag 3781 gacaatgaca agctgaaaaa gctgatcaac aagagccccg aaaagctgct gatgtaccac 3841 cacgaccccc agacctacca gaaactgaag ctgattatgg aacagtacgg cgacgagaag 3901 aatcccctgt acaagtacta cgaggaaacc gggaactacc tgaccaagta ctccaaaaag 3961 gacaacggcc ccgtgatcaa gaagattaag tattacggca acaaactgaa cgcccatctg 4021 gacatcaccg acgactaccc caacagcaga aacaaggtcg tgaagctgtc cctgaagccc 4081 tacagattcg acgtgtacct ggacaatggc gtgtacaagt tcgtgaccgt gaagaatctg 4141 gatgtgatca aaaaagaaaa ctactacgaa gtgaatagca agtgctatga ggaagctaag 4201 aagctgaaga agatcagcaa ccaggccgag tttatcgcct ccttctacaa caacgatctg 4261 atcaagatca acggcgagct gtatagagtg atcggcgtga acaacgacct gctgaaccgg 4321 atcgaagtga acatgatcga catcacctac cgcgagtacc tggaaaacat gaacgacaag 4381 aggcccccca ggatcattaa gacaatcgcc tccaagaccc agagcattaa gaagtacagc 4441 acagacattc tgggcaacct gtatgaagtg aaatctaaga agcaccctca gatcatcaaa 4501 aagggcaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagggatcc 4561 gaggccagcg gttccggacg ggctgacgca ttggacgatt ttgatctgga tatgctggga 4621 agtgacgccc tcgatgattt tgaccttgac atgcttggtt cggatgccct tgatgacttt 4681 gacctcgaca tgctcggcag tgacgccctt gatgatttcg acctggacat gctgattaac 4741 tctagaagtt ccggatctcc gaaaaagaaa cgcaaagttg gttcgggagg tggttcgggt 4801 ggctctggat cagtgctgcc tcaggctcct gctcctgcac cagctccagc catggtgtct 4861 gcactggctc aggcaccagc acccgtgcct gtgctggctc ctggacctcc acaggctgtg 4921 gctccaccag cccctaaacc tacacaggcc ggcgagggca cactgtctga agctctgctg 4981 cagctgcagt tcgacgacga ggatctggga gccctgctgg gaaacagcac cgatcctgcc 5041 gtgttcaccg acctggccag cgtggacaac agcgagttcc agcagctgct gaaccagggc 5101 atccctgtgg cccctcacac caccgagccc atgctgatgg aataccccga ggccatcacc 5161 cggctcgtga caggcgctca gaggcctcct gatccagctc ctgcccctct gggagcacca 5221 ggcctgccta atggactgct gtctggcgac gaggacttca gctctatcgc cgatatggat 5281 ttctcagcct tgctgtcagg cggtggtagt ggtgggagcg gtagtgacct ttcccatccg 5341 cccccaaggg gccatctgga tgagctgaca accacacttg agtccatgac cgaggatctg 5401 aacctggact cacccctgac cccggaattg aacgagattc tggatacctt cctgaacgac 5461 gagtgcctct tgcatgccat gcatatcagc acaggactgt ccatcttcga cacatctctg 5521 ttttaggaat tcaaataaaa tacgaaatga aataaaatac gaaatgcaat tgggccgcag 5581 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc 5641 gggcgaccaa aggtcgcccg acgcccgggc tttgcccgag cggcctcagt gagcgagcga 5701 gcgcgcagct gcctgcagga catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 5761 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 5821 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 5881 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 5941 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 6001 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 6061 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 6121 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 6181 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 6241 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 6301 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 6361 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 6421 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 6481 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 6541 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 6601 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 6661 agtgctgcaa tgataccgcg gcttccacgc tcaccggctc cagatttatc agcaataaac 6721 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 6781 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 6841 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 6901 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 6961 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 7021 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 7081 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 7141 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 7201 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 7261 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 7321 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 7381 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 7441 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 7501 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 7561 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac 7621 ggtg //