LOCUS Exported 6990 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV-CRISPR mediated genome editing in glia / astrocytes. ACCESSION . VERSION . KEYWORDS pAAV-U6sgp53-U6sgbbSapI-GFAPCre SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6990) AUTHORS Chow RD, Guzman CD, Wang G, Schmidt F, Youngblood MW, Ye L, Errami Y, Dong MB, Martinez MA, Zhang S, Renauer P, Bilguvar K, Gunel M, Sharp PA, Zhang F, Platt RJ, Chen S TITLE AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma JOURNAL Nat Neurosci. 2017 Aug 14. doi: 10.1038/nn.4620. REFERENCE 2 (bases 1 to 6990) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6990 /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 145..385 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 414..489 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 503..743 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 770..845 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 3082..3102 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4129..4155 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" polyA_signal 4177..4225 /note="synthetic polyadenylation signal" repeat_region 4253..4393 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4253..4382 /label=AAV2 ITR rep_origin 4468..4923 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4555..4574) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4765..4786 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4940..4959) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5059..5081 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5119..5137) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5205..5309 /gene="bla" /label=AmpR promoter CDS 5310..6170 /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(5528..5547) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6341..6929 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6830..6849 /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 gcgtgagggc ctatttccca tgattccttc atatttgcat 181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata 241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa 301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg 361 gctttatata tcttgtggaa aggacgaaac accgtgtaat agctcctgca tgggttttag 421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg 481 agtcggtgct ttttttctag aagagggcct atttcccatg attccttcat atttgcatat 541 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt 601 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt 661 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc 721 tttatatatc ttgtggaaag gacgaaacac cggaagagcg agctcttctg ttttagagct 781 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcatcgagtc 841 ggtgcttttt tggtaccccc acctccctct ctgtgctggg actcacagag ggagacctca 901 ggaggcagtc tgtccatcac atgtccaaat gcagagcata ccctgggctg ggcgcagtgg 961 cgcacaactg taattccagc actttgggag gctgatgtgg aaggatcact tgagcccaga 1021 agttctagac cagcctgggc aacatggcaa gaccctatct ctacaaaaaa agttaaaaaa 1081 tcagccacgt gtggtgacac acacctgtag tcccagctat tcaggaggct gaggtgaggg 1141 gatcacttaa ggctgggagg ttgaggctgc agtgagtcgt ggttgcgcca ctgcactcca 1201 gcctgggcaa cagtgagacc ctgtctcaaa agacaaaaaa aaaaaaaaaa aaaaaaagaa 1261 catatcctgg tgtggagtag gggacgctgc tctgacagag gctcgggggc ctgagctggc 1321 tctgtgagct ggggaggagg cagacagcca ggccttgtct gcaagcagac ctggcagcat 1381 tgggctggcc gccccccagg gcctcctctt catgcccagt gaatgactca ccttggcaca 1441 gacacaatgt tcggggtggg cacagtgcct gcttcccgcc gcaccccagc ccccctcaaa 1501 tgccttccga gaagcccatt gagcaggggg cttgcattgc accccagcct gacagcctgg 1561 catcttggga taaaagcagc acagccccct aggggctgcc cttgctgtgt ggcgccaccg 1621 gcggtggaga acaaggctct attcagcctg tgcccaggaa aggggatcag gggatgccca 1681 ggcatggaca gtgggtggca gggggggaga ggagggctgt ctgcttccca gaagtccaag 1741 gacacaaatg ggtgagggga ctgggcaggg ttctgaccct gtgggaccag agtggagggc 1801 gtagatggac ctgaagtctc cagggacaac agggcccagg tctcaggctc ctagttgggc 1861 ccagtggctc cagcgtttcc aaacccatcc atccccagag gttcttccca tctctccagg 1921 ctgatgtgtg ggaactcgag gaaataaatc tccagtggga gacggagggg tggccaggga 1981 aacggggcgc tgcaggaata aagacgagcc agcacagcca gctcatgtgt aacggctttg 2041 tggagctgtc aaggcctggt ctctgggaga gaggcacagg gaggccagac aaggaagggg 2101 tgacctggag ggacagatcc aggggctaaa gtcctgataa ggcaagagag tgccggcccc 2161 ctcttgccct atcaggacct ccactgccac atagaggcca tgattgaccc ttagacaaag 2221 ggctggtgtc caatcccagc ccccagcccc agaactccag ggaatgaatg ggcagagagc 2281 aggaatgtgg gacatctgtg ttcaagggaa ggactccagg agtctgctgg gaatgaggcc 2341 tagtaggaaa tgaggtggcc cttgagggta cagaacaggt tcattcttcg ccaaattccc 2401 agcaccttgc aggcacttac agctgagtga gataatgcct gggttatgaa atcaaaaagt 2461 tggaaagcag gtcagaggtc atctggtaca gcccttcctt cccttttttt tttttttttt 2521 ttgtgagaca aggtctctct ctgttgccca ggctggagtg gcgcaaacac agctcactgc 2581 agcctcaacc tactgggctc aagcaatcct ccagcctcag cctcccaaag tgctgggatt 2641 acaagcatga gccaccccac tcagcccttt ccttcctttt taattgatgc ataataattg 2701 taagtattca tcatggtcca accaaccctt tcttgaccca ccttcctaga gagagggtcc 2761 tcttgcttca gcggtcaggg ccccagaccc atggtctggc tccaggtacc acctgcctca 2821 tgcaggagtt ggcgtgccca ggaagctctg cctctgggca cagtgacctc agtggggtga 2881 ggggagctct ccccatagct gggctgcggc ccaaccccac cccctcaggc tatgccaggg 2941 ggtgttgcca ggggcacccg ggcatcgcca gtctagccca ctccttcata aagccctcgc 3001 atcccaggag cgagcagagc cagagcaggt tggagaggag acgcatcacc tccgctgctc 3061 gccgggcgta cggccaccat gcccaagaag aagaggaagg tgtccaatct cctgactgtt 3121 caccagaacc tccctgcgct gccagtagat gccactagcg atgaggtcag gaaaaatctc 3181 atggatatgt ttagggatag acaggcgttt tctgaacaca cctggaaaat gctgcttagc 3241 gtgtgccgat cctgggcagc ctggtgtaag ctgaacaatc gcaaatggtt ccccgccgag 3301 ccggaggacg tgcgcgatta cctgctgtat ctccaggcaa gagggctggc tgtcaagact 3361 atccagcagc acttgggcca actgaatatg ctgcatcgac gcagcgggct cccccggcct 3421 agcgattcaa acgcagtctc ccttgttatg aggagaatta gaaaggaaaa cgtagatgcg 3481 ggtgagaggg ctaagcaggc tctcgctttt gagcggactg atttcgacca ggtcagatcc 3541 ctgatggaga acagcgatcg gtgccaggac atcaggaacc tcgcatttct gggaattgca 3601 tataacacac ttctgcgcat agctgagatc gcccggatca gagtgaaaga catcagtcga 3661 acggacggcg gccggatgct tattcatatt ggacgcacaa agacattggt cagcaccgct 3721 ggcgttgaaa aggccttgtc cctgggcgta acgaagctgg tggaaagatg gatctcagtg 3781 tccggcgtgg ctgacgaccc taataattac ttgttctgtc gagtgagaaa aaacggagtc 3841 gccgcgccct ctgccaccag ccaattgagt acacgggccc ttgaagggat ctttgaggca 3901 acccaccgac tcatatacgg agccaaggat gacagtggcc agaggtatct cgcctggtca 3961 ggtcattctg ctagggtggg ggccgcacga gacatggcgc gggcaggagt ctccatacca 4021 gagattatgc aagctggagg ttggacaaat gtgaacatcg ttatgaacta tatccgcaat 4081 cttgactctg aaaccggggc catggtgaga ctgctcgaag atggtgacta cccatacgat 4141 gttccagatt acgcttaaga attcgatatc aagcttaata aaagatcttt attttcatta 4201 gatctgtgtg ttggtttttt gtgtggtaac cacgtgcgga ccgagcggcc gcaggaaccc 4261 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 4321 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 4381 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 4441 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg 4501 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt 4561 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 4621 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 4681 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 4741 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact 4801 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa 4861 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 4921 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 4981 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 5041 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 5101 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 5161 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 5221 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 5281 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 5341 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 5401 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 5461 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 5521 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 5581 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 5641 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 5701 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 5761 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 5821 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 5881 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 5941 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 6001 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 6061 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 6121 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 6181 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 6241 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 6301 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 6361 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 6421 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 6481 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 6541 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 6601 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 6661 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 6721 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 6781 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 6841 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 6901 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 6961 tggccttttg ctggcctttt gctcacatgt //