LOCUS       sequence-198067-        6990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-CRISPR mediated genome editing in glia / astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
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.
  PUBMED    28805815
REFERENCE   2  (bases 1 to 6990)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Neurosci. 2017 Aug 14. doi: 10.1038/nn.4620."
COMMENT     Sequence Label: pAAV-U6sgp53-U6sgbbSapI-GFAPCre
FEATURES             Location/Qualifiers
     source          1..6990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        503..751
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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             join(5310..5378,5379..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"
     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
//
