LOCUS       Exported                6989 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV-CRISPR library for pool mutagenesis of top tumor suppressor 
            genes.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-U6sgp53-mTSG-GFAPCre
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6989)
  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 6989)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6989
                     /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        772..847
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             902..922
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_RNA        2951..3026
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             3081..3101
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4128..4154
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4176..4224
                     /note="synthetic polyadenylation signal"
     repeat_region   4252..4392
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4252..4381
                     /label=AAV2 ITR
     rep_origin      4467..4922
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4554..4573)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4764..4785
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4939..4958)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5058..5080
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5118..5136)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5204..5308
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5309..6169
                     /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(5527..5546)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6340..6928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6829..6848
                     /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 cgggtgtgga ggtgtctgat ggttttagag
      781 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      841 tcggtgcttt tttggtaccc ccacctccct ctctgtgctc gccgggcgta cggccaccat
      901 gcccaagaag aagaggaagg tgtccaatct cctgactgtt caccagaacc tcgcagtggc
      961 gcacaactgt aattccagca ctttgggagg ctgatgtgga aggatcactt gagcccagaa
     1021 gttctagacc agcctgggca acatggcaag accctatctc tacaaaaaaa gttaaaaaat
     1081 cagccacgtg tggtgacaca cacctgtagt cccagctatt caggaggctg aggtgagggg
     1141 atcacttaag gctgggaggt tgaggctgca gtgagtcgtg gttgcgccac tgcactccag
     1201 cctgggcaac agtgagaccc tgtctmaaaa gacaaaaaaa aaaaaaaaaa aaaaaagaac
     1261 atatcctggt gtggagtagg ggacgctgct ctgacagagg ctcgggggcc tgagctggct
     1321 ctgtgagctg gggaggaggc agacagccag gccttgtctg caagcagacc tggcagcatt
     1381 gggctggccg ccccccaggg cctcctcttc atgcccagtg aatgactcac cttggcacag
     1441 acacaatgtt cggggtgggc acagtgcctg cttcccgccg caccccagcc cccctcaaat
     1501 gccttccgag aagcccattg agcagggggc ttgcattgca ccccagcctg acagcctggc
     1561 atcttgggat aaaagcagca cagcccccta ggggctgccc ttgctgtgtg gcgccaccgg
     1621 cggtggagaa caaggctcta ttcagcctgt gcccaggaaa ggggatcagg ggatgcccag
     1681 gcatggacag tgggtggcag ggggggagag gagggctgtc tgcttcccag aagtccaagg
     1741 acacaaatgg gtgaggggac tgggcagggt tctgaccctg tgggaccaga gtggagggcg
     1801 tagatggacc tgaagtctcc agggacaaca gggcccaggt ctcaggctcc tagttgggcc
     1861 cagtggctcc agcgtttcca aacccatcca tccccagagg ttcttcccat ctctccaggc
     1921 tgatgtgtgg gaactcgagg aaataaatct ccagtgggag acggaggggt ggccagggaa
     1981 acggggcgct gcaggaataa agacgagcca gcacagccag ctcatgtgta acggctttgt
     2041 ggagctgtca aggcctggtc tctgggagag aggcacaggg aggccagaca aggaaggggt
     2101 gacctggagg gacagatcca ggggctaaag tcctgataag gcaagagagt gccggccccc
     2161 tcttgcccta tcaggacctc cactgccaca tagaggccat gattgaccct tagacaaagg
     2221 gctggtgtcc aatcccagcc cccagcccca gaactccagg gaatgaatgg gcagagagca
     2281 ggaatgtggg acatctgtgt tcaagggaag gactccagga gtctgctggg aatgaggcct
     2341 agtaggaaat gaggtggccc ttgagggtac agaacaggtt cattcttcgc caaattccca
     2401 gcaccttgca ggcacttaca gctgagtgag ataatgcctg ggttatgaaa tcaaaaagtt
     2461 ggaaagcagg tcagaggtca tctggtacag cccttccttc cctttttttt tttttttttt
     2521 tttgagacaa ggtctctctc tgttgcccag gctggagtgg cgcaaacaca gctcactgca
     2581 gcctcaacct actgggctca agcaatcctc carcctcagc ctcccaaagt gctgggatta
     2641 caagcatgag ccaccccact cagccctttc cttccttttt aattgatgca taataattgt
     2701 aagtattcat catggtccaa ccaacccttt cttgacccac cttcctagag agagggtcct
     2761 cttgcttcag cggtcagggc cccagaccca tggtctggct ccaggtacca cctgcctcat
     2821 gcaggagttg gcgtgcccag gaagctctgc ctctgggcac agtgacctca gtggggtgag
     2881 gggagctctc cccatagcyt ttatatatct tgtggaaagg acgaaacacc gggtgtggag
     2941 gtgtctgatg gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     3001 ttgaaaaagt ggcaccgagt cggtgctttt ttggtacccc cacctccctc tctgtgctcg
     3061 ccgggcgtac ggccaccatg cccaagaaga agaggaaggt gtccaatctc ctgactgttc
     3121 accagaacct ccctgcgctg ccagtagatg ccactagcga tgaggtcagg aaaaatctca
     3181 tggatatgtt tagggataga caggcgtttt ctgaacacac ctggaaaatg ctgcttagcg
     3241 tgtgccgatc ctgggcagcc tggtgtaagc tgaacaatcg caaatggttc cccgccgagc
     3301 cggaggacgt gcgcgattac ctgctgtatc tccaggcaag agggctggct gtcaagacta
     3361 tccagcagca cttgggccaa ctgaatatgc tgcatcgacg cagcgggctc ccccggccta
     3421 gcgattcaaa cgcagtctcc cttgttatga ggagaattag aaaggaaaac gtagatgcgg
     3481 gtgagagggc taagcaggct ctcgcttttg agcggactga tttcgaccag gtcagatccc
     3541 tgatggagaa cagcgatcgg tgccaggaca tcaggaacct cgcatttctg ggaattgcat
     3601 ataacacact tctgcgcata gctgagatcg cccggatcag agtgaaagac atcagtcgaa
     3661 cggacggcgg ccggatgctt attcatattg gacgcacaaa gacattggtc agcaccgctg
     3721 gcgttgaaaa ggccttgtcc ctgggcgtaa cgaagctggt ggaaagatgg atctcagtgt
     3781 ccggcgtggc tgacgaccct aataattact tgttctgtcg agtgagaaaa aacggagtcg
     3841 ccgcgccctc tgccaccagc caattgagta cacgggccct tgaagggatc tttgaggcaa
     3901 cccaccgact catatacgga gccaaggatg acagtggcca gaggtatctc gcctggtcag
     3961 gtcattctgc tagggtgggg gccgcacgag acatggcgcg ggcaggagtc tccataccag
     4021 agattatgca agctggaggt tggacaaatg tgaacatcgt tatgaactat atccgcaatc
     4081 ttgactctga aaccggggcc atggtgagac tgctcgaaga tggtgactac ccatacgatg
     4141 ttccagatta cgcttaagaa ttcgatatca agcttaataa aagatcttta ttttcattag
     4201 atctgtgtgt tggttttttg tgtggtaacc acgtgcggac cgagcggccg caggaacccc
     4261 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     4321 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     4381 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4441 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     4501 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     4561 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4621 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4681 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4741 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     4801 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     4861 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     4921 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     4981 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5041 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5101 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     5161 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5221 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5281 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5341 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5401 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5461 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5521 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5581 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5641 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5701 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5761 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5821 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     5881 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     5941 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6001 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6061 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6121 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6181 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6241 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6301 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6361 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6421 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6481 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6541 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6601 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6661 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6721 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6781 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6841 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6901 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6961 ggccttttgc tggccttttg ctcacatgt
//
