LOCUS       sequence-402557-        7155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SaCas9 expression in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7155)
  AUTHORS   Huang Q, Chen AT, Chan KY, Sorensen H, Barry AJ, Azari B, Zheng Q, 
            Beddow T, Zhao B, Tobey IG, Moncada-Reid C, Eid FE, Walkey CJ, 
            Ljungberg MC, Lagor WR, Heaney JD, Chan YA, Deverman BE
  TITLE     Targeting AAV vectors to the central nervous system by engineering 
            capsid-receptor interactions that enable crossing of the blood-brain
            barrier.
  JOURNAL   PLoS Biol. 2023 Jul 19;21(7):e3002112. doi: 
            10.1371/journal.pbio.3002112. eCollection 2023 Jul.
  PUBMED    37467291
REFERENCE   2  (bases 1 to 7155)
  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: "PLoS 
            Biol."; date: "2023-07-19"; volume: "21(7):e3002112. doi"; pages: " 
            10.1371/journal.pbio.3002112. eCollection 2023 Jul"
COMMENT     Sequence Label: AAV-GfABC1D-SaCas9-WPRE3-pA
FEATURES             Location/Qualifiers
     source          1..7155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      823..832
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             835..855
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             880..4035
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             4036..4083
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4154..4174)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4361..4409
                     /note="synthetic polyadenylation signal"
     repeat_region   4418..4558
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4418..4547
                     /label=AAV2 ITR
     rep_origin      4633..5088
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4720..4739)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4930..4951
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5105..5124)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5224..5246
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5284..5302)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5370..5474
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5475..5543,5544..6335)
                     /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      6506..7094
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6995..7014
                     /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 gcggcctcta gaaacatatc ctggtgtgga gtaggggacg ctgctctgac
      181 agaggctcgg gggcctgagc tggctctgtg agctggggag gaggcagaca gccaggcctt
      241 gtctgcaagc agacctggca gcattgggct ggccgccccc cagggcctcc tcttcatgcc
      301 cagtgaatga ctcaccttgg cacagacaca atgttcgggg tgggcacagt gcctgcttcc
      361 cgccgcaccc cagcccccct caaatgcctt ccgagaagcc cattgagcag ggggcttgca
      421 ttgcacccca gcctgacagc ctggcatctt gggataaaag cagcacagcc ccctaggggc
      481 tgcccttgct gtgtggcgcc accggcggtg gagaacaagg ctctattcag cctgtgccca
      541 ggaaagggga tcaggggatg cccaggcatg gacagtgggt ggcagggggg gagaggaggg
      601 ctgtctgctt cccagaagtc caaggacaca aatgggtgag gggagagctc tccccatagc
      661 tgggctgcgg cccaacccca ccccctcagg ctatgccagg gggtgttgcc aggggcaccc
      721 gggcatcgcc agtctagccc actccttcat aaagccctcg tatcccagga gcgagcagag
      781 ccagagcagg ttggagagga gacgcatcac ctctgctgct tggccaccat ggccccaaag
      841 aagaagcgga aggtcggtat ccacggagtc ccagcagcca agcggaacta catcctgggc
      901 ctggacatcg gcatcaccag cgtgggctac ggcatcatcg actacgagac acgggacgtg
      961 atcgatgccg gcgtgcggct gttcaaagag gccaacgtgg aaaacaacga gggcaggcgg
     1021 agcaagagag gcgccagaag gctgaagcgg cggaggcggc atagaatcca gagagtgaag
     1081 aagctgctgt tcgactacaa cctgctgacc gaccacagcg agctgagcgg catcaacccc
     1141 tacgaggcca gagtgaaggg cctgagccag aagctgagcg aggaagagtt ctctgccgcc
     1201 ctgctgcacc tggccaagag aagaggcgtg cacaacgtga acgaggtgga agaggacacc
     1261 ggcaacgagc tgtccaccaa agagcagatc agccggaaca gcaaggccct ggaagagaaa
     1321 tacgtggccg aactgcagct ggaacggctg aagaaagacg gcgaagtgcg gggcagcatc
     1381 aacagattca agaccagcga ctacgtgaaa gaagccaaac agctgctgaa ggtgcagaag
     1441 gcctaccacc agctggacca gagcttcatc gacacctaca tcgacctgct ggaaacccgg
     1501 cggacctact atgagggacc tggcgagggc agccccttcg gctggaagga catcaaagaa
     1561 tggtacgaga tgctgatggg ccactgcacc tacttccccg aggaactgcg gagcgtgaag
     1621 tacgcctaca acgccgacct gtacaacgcc ctgaacgacc tgaacaatct cgtgatcacc
     1681 agggacgaga acgagaagct ggaatattac gagaagttcc agatcatcga gaacgtgttc
     1741 aagcagaaga agaagcccac cctgaagcag atcgccaaag aaatcctcgt gaacgaagag
     1801 gatattaagg gctacagagt gaccagcacc ggcaagcccg agttcaccaa cctgaaggtg
     1861 taccacgaca tcaaggacat taccgcccgg aaagagatta ttgagaacgc cgagctgctg
     1921 gatcagattg ccaagatcct gaccatctac cagagcagcg aggacatcca ggaagaactg
     1981 accaatctga actccgagct gacccaggaa gagatcgagc agatctctaa tctgaagggc
     2041 tataccggca cccacaacct gagcctgaag gccatcaacc tgatcctgga cgagctgtgg
     2101 cacaccaacg acaaccagat cgctatcttc aaccggctga agctggtgcc caagaaggtg
     2161 gacctgtccc agcagaaaga gatccccacc accctggtgg acgacttcat cctgagcccc
     2221 gtcgtgaaga gaagcttcat ccagagcatc aaagtgatca acgccatcat caagaagtac
     2281 ggcctgccca acgacatcat tatcgagctg gcccgcgaga agaactccaa ggacgcccag
     2341 aaaatgatca acgagatgca gaagcggaac cggcagacca acgagcggat cgaggaaatc
     2401 atccggacca ccggcaaaga gaacgccaag tacctgatcg agaagatcaa gctgcacgac
     2461 atgcaggaag gcaagtgcct gtacagcctg gaagccatcc ctctggaaga tctgctgaac
     2521 aaccccttca actatgaggt ggaccacatc atccccagaa gcgtgtcctt cgacaacagc
     2581 ttcaacaaca aggtgctcgt gaagcaggaa gaaaacagca agaagggcaa ccggacccca
     2641 ttccagtacc tgagcagcag cgacagcaag atcagctacg aaaccttcaa gaagcacatc
     2701 ctgaatctgg ccaagggcaa gggcagaatc agcaagacca agaaagagta tctgctggaa
     2761 gaacgggaca tcaacaggtt ctccgtgcag aaagacttca tcaaccggaa cctggtggat
     2821 accagatacg ccaccagagg cctgatgaac ctgctgcgga gctacttcag agtgaacaac
     2881 ctggacgtga aagtgaagtc catcaatggc ggcttcacca gctttctgcg gcggaagtgg
     2941 aagtttaaga aagagcggaa caaggggtac aagcaccacg ccgaggacgc cctgatcatt
     3001 gccaacgccg atttcatctt caaagagtgg aagaaactgg acaaggccaa aaaagtgatg
     3061 gaaaaccaga tgttcgagga aaagcaggcc gagagcatgc ccgagatcga aaccgagcag
     3121 gagtacaaag agatcttcat caccccccac cagatcaagc acattaagga cttcaaggac
     3181 tacaagtaca gccaccgggt ggacaagaag cctaatagag agctgattaa cgacaccctg
     3241 tactccaccc ggaaggacga caagggcaac accctgatcg tgaacaatct gaacggcctg
     3301 tacgacaagg acaatgacaa gctgaaaaag ctgatcaaca agagccccga aaagctgctg
     3361 atgtaccacc acgaccccca gacctaccag aaactgaagc tgattatgga acagtacggc
     3421 gacgagaaga atcccctgta caagtactac gaggaaaccg ggaactacct gaccaagtac
     3481 tccaaaaagg acaacggccc cgtgatcaag aagattaagt attacggcaa caaactgaac
     3541 gcccatctgg acatcaccga cgactacccc aacagcagaa acaaggtcgt gaagctgtcc
     3601 ctgaagccct acagattcga cgtgtacctg gacaatggcg tgtacaagtt cgtgaccgtg
     3661 aagaatctgg atgtgatcaa aaaagaaaac tactacgaag tgaatagcaa gtgctatgag
     3721 gaagctaaga agctgaagaa gatcagcaac caggccgagt ttatcgcctc cttctacaac
     3781 aacgatctga tcaagatcaa cggcgagctg tatagagtga tcggcgtgaa caacgacctg
     3841 ctgaaccgga tcgaagtgaa catgatcgac atcacctacc gcgagtacct ggaaaacatg
     3901 aacgacaaga ggccccccag gatcattaag acaatcgcct ccaagaccca gagcattaag
     3961 aagtacagca cagacattct gggcaacctg tatgaagtga aatctaagaa gcaccctcag
     4021 atcatcaaaa agggcaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa
     4081 aagtcgagct agaattcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     4141 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     4201 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttag
     4261 ttcttgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     4321 tgttgggcac tgacaattcc gtggtgttct cgagagatct aataaaatat ctttattttc
     4381 attacatctg tgtgttggtt ttttgtgtgg cggccgcagg aacccctagt gatggagttg
     4441 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4501 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4561 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4621 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4681 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     4741 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4801 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4861 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4921 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     4981 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     5041 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     5101 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     5161 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5221 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5281 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5341 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5401 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5461 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5521 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5581 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5641 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5701 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5761 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5821 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5881 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5941 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6001 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6061 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6121 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6181 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6241 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6301 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6361 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6421 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6481 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6541 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6601 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6661 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6721 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6781 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6841 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6901 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6961 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7021 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7081 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7141 cttttgctca catgt
//
