LOCUS Exported 7062 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION A CMV driven de-catalyzed SaCas9 fused to VP64 domain for increased transcription in targeted region. ACCESSION . VERSION . KEYWORDS pJEP303-pAAV-CMV-dSaCas9-VP64-pA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7062) AUTHORS Kumar N, Stanford W, Aradhana CS, Abraham ND, Dao TJ, Thaseen S, Sairavi A, Gonzalez CU, Ploski JE. TITLE The Development of an AAV-Based CRISPR SaCas9 Genome Editing System That Can Be Delivered to Neurons in vivo and Regulated via Doxycycline and Cre-Recombinase. JOURNAL Front. Mol. Neurosci. (2018) 13 REFERENCE 2 (bases 1 to 7062) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7062 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" enhancer 154..457 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 458..661 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 612..632 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 658..682 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 748..757 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 760..780 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3961..4008 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 4039..4188 /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" primer_bind complement(4267..4286) /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 4325..4465 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4325..4454 /label=AAV2 ITR rep_origin 4540..4995 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4627..4646) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4837..4858 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5012..5031) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5131..5153 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5191..5209) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5277..5381 /gene="bla" /label=AmpR promoter CDS 5382..6242 /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(5600..5619) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6413..7001 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6902..6921 /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 gcggccgcac gcgtggagtt ccgcgttaca taacttacgg taaatggccc 181 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 241 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 301 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 361 cagtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 421 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 481 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 541 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 601 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 661 tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag 721 aagacaccgg aattcggtac caccggtgcc accatggccc caaagaagaa gcggaaggtc 781 ggtatccacg gagtcccagc agccaagcgg aactacatcc tgggcctggc catcggcatc 841 accagcgtgg gctacggcat catcgactac gagacacggg acgtgatcga tgccggcgtg 901 cggctgttca aagaggccaa cgtggaaaac aacgagggca ggcggagcaa gagaggcgcc 961 agaaggctga agcggcggag gcggcataga atccagagag tgaagaagct gctgttcgac 1021 tacaacctgc tgaccgacca cagcgagctg agcggcatca acccctacga ggccagagtg 1081 aagggcctga gccagaagct gagcgaggaa gagttctctg ccgccctgct gcacctggcc 1141 aagagaagag gcgtgcacaa cgtgaacgag gtggaagagg acaccggcaa cgagctgtcc 1201 accaaagagc agatcagccg gaacagcaag gccctggaag agaaatacgt ggccgaactg 1261 cagctggaac ggctgaagaa agacggcgaa gtgcggggca gcatcaacag attcaagacc 1321 agcgactacg tgaaagaagc caaacagctg ctgaaggtgc agaaggccta ccaccagctg 1381 gaccagagct tcatcgacac ctacatcgac ctgctggaaa cccggcggac ctactatgag 1441 ggacctggcg agggcagccc cttcggctgg aaggacatca aagaatggta cgagatgctg 1501 atgggccact gcacctactt ccccgaggaa ctgcggagcg tgaagtacgc ctacaacgcc 1561 gacctgtaca acgccctgaa cgacctgaac aatctcgtga tcaccaggga cgagaacgag 1621 aagctggaat attacgagaa gttccagatc atcgagaacg tgttcaagca gaagaagaag 1681 cccaccctga agcagatcgc caaagaaatc ctcgtgaacg aagaggatat taagggctac 1741 agagtgacca gcaccggcaa gcccgagttc accaacctga aggtgtacca cgacatcaag 1801 gacattaccg cccggaaaga gattattgag aacgccgagc tgctggatca gattgccaag 1861 atcctgacca tctaccagag cagcgaggac atccaggaag aactgaccaa tctgaactcc 1921 gagctgaccc aggaagagat cgagcagatc tctaatctga agggctatac cggcacccac 1981 aacctgagcc tgaaggccat caacctgatc ctggacgagc tgtggcacac caacgacaac 2041 cagatcgcta tcttcaaccg gctgaagctg gtgcccaaga aggtggacct gtcccagcag 2101 aaagagatcc ccaccaccct ggtggacgac ttcatcctga gccccgtcgt gaagagaagc 2161 ttcatccaga gcatcaaagt gatcaacgcc atcatcaaga agtacggcct gcccaacgac 2221 atcattatcg agctggcccg cgagaagaac tccaaggacg cccagaaaat gatcaacgag 2281 atgcagaagc ggaaccggca gaccaacgag cggatcgagg aaatcatccg gaccaccggc 2341 aaagagaacg ccaagtacct gatcgagaag atcaagctgc acgacatgca ggaaggcaag 2401 tgcctgtaca gcctggaagc catccctctg gaagatctgc tgaacaaccc cttcaactat 2461 gaggtggacc acatcatccc cagaagcgtg tccttcgaca acagcttcaa caacaaggtg 2521 ctcgtgaagc aggaagaagc cagcaagaag ggcaaccgga ccccattcca gtacctgagc 2581 agcagcgaca gcaagatcag ctacgaaacc ttcaagaagc acatcctgaa tctggccaag 2641 ggcaagggca gaatcagcaa gaccaagaaa gagtatctgc tggaagaacg ggacatcaac 2701 aggttctccg tgcagaaaga cttcatcaac cggaacctgg tggataccag atacgccacc 2761 agaggcctga tgaacctgct gcggagctac ttcagagtga acaacctgga cgtgaaagtg 2821 aagtccatca atggcggctt caccagcttt ctgcggcgga agtggaagtt taagaaagag 2881 cggaacaagg ggtacaagca ccacgccgag gacgccctga tcattgccaa cgccgatttc 2941 atcttcaaag agtggaagaa actggacaag gccaaaaaag tgatggaaaa ccagatgttc 3001 gaggaaaagc aggccgagag catgcccgag atcgaaaccg agcaggagta caaagagatc 3061 ttcatcaccc cccaccagat caagcacatt aaggacttca aggactacaa gtacagccac 3121 cgggtggaca agaagcctaa tagagagctg attaacgaca ccctgtactc cacccggaag 3181 gacgacaagg gcaacaccct gatcgtgaac aatctgaacg gcctgtacga caaggacaat 3241 gacaagctga aaaagctgat caacaagagc cccgaaaagc tgctgatgta ccaccacgac 3301 ccccagacct accagaaact gaagctgatt atggaacagt acggcgacga gaagaatccc 3361 ctgtacaagt actacgagga aaccgggaac tacctgacca agtactccaa aaaggacaac 3421 ggccccgtga tcaagaagat taagtattac ggcaacaaac tgaacgccca tctggacatc 3481 accgacgact accccaacag cagaaacaag gtcgtgaagc tgtccctgaa gccctacaga 3541 ttcgacgtgt acctggacaa tggcgtgtac aagttcgtga ccgtgaagaa tctggatgtg 3601 atcaaaaaag aaaactacta cgaagtgaat agcaagtgct atgaggaagc taagaagctg 3661 aagaagatca gcaaccaggc cgagtttatc gcctccttct acaacaacga tctgatcaag 3721 atcaacggcg agctgtatag agtgatcggc gtgaacaacg acctgctgaa ccggatcgaa 3781 gtgaacatga tcgacatcac ctaccgcgag tacctggaaa acatgaacga caagaggccc 3841 cccaggatca ttaagacaat cgcctccaag acccagagca ttaagaagta cagcacagac 3901 attctgggca acctgtatga agtgaaatct aagaagcacc ctcagatcat caaaaagggc 3961 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaaggg atccgaggcc 4021 agcggttccg gacgggctga cgcattggac gattttgatc tggatatgct gggaagtgac 4081 gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc 4141 gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taactcaaga 4201 tgatctagac atatggatat ctgactagtc tcgagtttat tgcagcttat aatggttaca 4261 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg gaccgagcgg 4321 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact 4381 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc 4441 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg 4501 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat 4561 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag 4621 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 4681 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 4741 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt 4801 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 4861 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg 4921 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 4981 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 5041 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 5101 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 5161 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta 5221 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg 5281 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 5341 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 5401 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 5461 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 5521 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 5581 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 5641 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 5701 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 5761 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 5821 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 5881 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 5941 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 6001 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 6061 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 6121 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 6181 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 6241 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 6301 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 6361 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 6421 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 6481 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 6541 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 6601 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 6661 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 6721 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 6781 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 6841 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 6901 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 6961 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 7021 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt //