LOCUS       sequence-433357-        7341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector plasmid expressing Cas9 from Staphylococcus aureus 
            (SaCas9) under the human synapsin (SYN) promoter and its sgRNA under
            the U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7341)
  TITLE     Adeno-associated viral vector-mediated delivery of SaCas9 in neurons
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7341)
  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: 
            "Unpublished"
COMMENT     Sequence Label: AAV-SYN-NLS-SaCas9-NLS-3xHA-bGHpA-U6-BsaI-sgRNA
FEATURES             Location/Qualifiers
     source          1..7341
                     /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        170..617
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      651..660
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             663..683
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             708..3863
                     /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             3864..3911
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3918..3998
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4026..4043)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4032..4239
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4246..4494
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4246..4266
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4417..4436
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4515..4590
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4604..4744
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4604..4733
                     /label=AAV2 ITR
     rep_origin      4819..5274
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4906..4925)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5116..5137
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5291..5310)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5410..5432
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5470..5488)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5556..5660
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5661..5729,5730..6521)
                     /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(5879..5898)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6692..7280
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7181..7200
                     /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 gactctagct gcagagggac ctgcgtatga gtgcaagtgg
      181 gttttaggac caggatgagg cggggtgggg gtgcctacct gacgaccgac cccgacccac
      241 tggacaagca cccaaccccc attccccaaa ttgcgcatcc cctatcagag agggggaggg
      301 gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc agcaccgcgg acagtgcctt
      361 cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac tgaaggcgcg ctgacgtcac
      421 tcgccggtcc cccgcaaact ccccttcccg gccaccttgg tcgcgtccgc gccgccgccg
      481 gcccagccgg accgcaccac gcgaggcgcg agataggggg gcacgggcgc gaccatctgc
      541 gctgcggcgc cggcgactca gcgctgcctc agtctgcggt gggcagcgga ggagtcgtgt
      601 cgtgcctgag agcgcagtcg aattcaagct gctagcaagg atccaccggt gccaccatgg
      661 ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccaag cggaactaca
      721 tcctgggcct ggacatcggc atcaccagcg tgggctacgg catcatcgac tacgagacac
      781 gggacgtgat cgatgccggc gtgcggctgt tcaaagaggc caacgtggaa aacaacgagg
      841 gcaggcggag caagagaggc gccagaaggc tgaagcggcg gaggcggcat agaatccaga
      901 gagtgaagaa gctgctgttc gactacaacc tgctgaccga ccacagcgag ctgagcggca
      961 tcaaccccta cgaggccaga gtgaagggcc tgagccagaa gctgagcgag gaagagttct
     1021 ctgccgccct gctgcacctg gccaagagaa gaggcgtgca caacgtgaac gaggtggaag
     1081 aggacaccgg caacgagctg tccaccaaag agcagatcag ccggaacagc aaggccctgg
     1141 aagagaaata cgtggccgaa ctgcagctgg aacggctgaa gaaagacggc gaagtgcggg
     1201 gcagcatcaa cagattcaag accagcgact acgtgaaaga agccaaacag ctgctgaagg
     1261 tgcagaaggc ctaccaccag ctggaccaga gcttcatcga cacctacatc gacctgctgg
     1321 aaacccggcg gacctactat gagggacctg gcgagggcag ccccttcggc tggaaggaca
     1381 tcaaagaatg gtacgagatg ctgatgggcc actgcaccta cttccccgag gaactgcgga
     1441 gcgtgaagta cgcctacaac gccgacctgt acaacgccct gaacgacctg aacaatctcg
     1501 tgatcaccag ggacgagaac gagaagctgg aatattacga gaagttccag atcatcgaga
     1561 acgtgttcaa gcagaagaag aagcccaccc tgaagcagat cgccaaagaa atcctcgtga
     1621 acgaagagga tattaagggc tacagagtga ccagcaccgg caagcccgag ttcaccaacc
     1681 tgaaggtgta ccacgacatc aaggacatta ccgcccggaa agagattatt gagaacgccg
     1741 agctgctgga tcagattgcc aagatcctga ccatctacca gagcagcgag gacatccagg
     1801 aagaactgac caatctgaac tccgagctga cccaggaaga gatcgagcag atctctaatc
     1861 tgaagggcta taccggcacc cacaacctga gcctgaaggc catcaacctg atcctggacg
     1921 agctgtggca caccaacgac aaccagatcg ctatcttcaa ccggctgaag ctggtgccca
     1981 agaaggtgga cctgtcccag cagaaagaga tccccaccac cctggtggac gacttcatcc
     2041 tgagccccgt cgtgaagaga agcttcatcc agagcatcaa agtgatcaac gccatcatca
     2101 agaagtacgg cctgcccaac gacatcatta tcgagctggc ccgcgagaag aactccaagg
     2161 acgcccagaa aatgatcaac gagatgcaga agcggaaccg gcagaccaac gagcggatcg
     2221 aggaaatcat ccggaccacc ggcaaagaga acgccaagta cctgatcgag aagatcaagc
     2281 tgcacgacat gcaggaaggc aagtgcctgt acagcctgga agccatccct ctggaagatc
     2341 tgctgaacaa ccccttcaac tatgaggtgg accacatcat ccccagaagc gtgtccttcg
     2401 acaacagctt caacaacaag gtgctcgtga agcaggaaga aaacagcaag aagggcaacc
     2461 ggaccccatt ccagtacctg agcagcagcg acagcaagat cagctacgaa accttcaaga
     2521 agcacatcct gaatctggcc aagggcaagg gcagaatcag caagaccaag aaagagtatc
     2581 tgctggaaga acgggacatc aacaggttct ccgtgcagaa agacttcatc aaccggaacc
     2641 tggtggatac cagatacgcc accagaggcc tgatgaacct gctgcggagc tacttcagag
     2701 tgaacaacct ggacgtgaaa gtgaagtcca tcaatggcgg cttcaccagc tttctgcggc
     2761 ggaagtggaa gtttaagaaa gagcggaaca aggggtacaa gcaccacgcc gaggacgccc
     2821 tgatcattgc caacgccgat ttcatcttca aagagtggaa gaaactggac aaggccaaaa
     2881 aagtgatgga aaaccagatg ttcgaggaaa agcaggccga gagcatgccc gagatcgaaa
     2941 ccgagcagga gtacaaagag atcttcatca ccccccacca gatcaagcac attaaggact
     3001 tcaaggacta caagtacagc caccgggtgg acaagaagcc taatagagag ctgattaacg
     3061 acaccctgta ctccacccgg aaggacgaca agggcaacac cctgatcgtg aacaatctga
     3121 acggcctgta cgacaaggac aatgacaagc tgaaaaagct gatcaacaag agccccgaaa
     3181 agctgctgat gtaccaccac gacccccaga cctaccagaa actgaagctg attatggaac
     3241 agtacggcga cgagaagaat cccctgtaca agtactacga ggaaaccggg aactacctga
     3301 ccaagtactc caaaaaggac aacggccccg tgatcaagaa gattaagtat tacggcaaca
     3361 aactgaacgc ccatctggac atcaccgacg actaccccaa cagcagaaac aaggtcgtga
     3421 agctgtccct gaagccctac agattcgacg tgtacctgga caatggcgtg tacaagttcg
     3481 tgaccgtgaa gaatctggat gtgatcaaaa aagaaaacta ctacgaagtg aatagcaagt
     3541 gctatgagga agctaagaag ctgaagaaga tcagcaacca ggccgagttt atcgcctcct
     3601 tctacaacaa cgatctgatc aagatcaacg gcgagctgta tagagtgatc ggcgtgaaca
     3661 acgacctgct gaaccggatc gaagtgaaca tgatcgacat cacctaccgc gagtacctgg
     3721 aaaacatgaa cgacaagagg ccccccagga tcattaagac aatcgcctcc aagacccaga
     3781 gcattaagaa gtacagcaca gacattctgg gcaacctgta tgaagtgaaa tctaagaagc
     3841 accctcagat catcaaaaag ggcaaaaggc cggcggccac gaaaaaggcc ggccaggcaa
     3901 aaaagaaaaa gggatcctac ccatacgatg ttccagatta cgcttaccca tacgatgttc
     3961 cagattacgc ttacccatac gatgttccag attacgctta agaattccta gagctcgctg
     4021 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     4081 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     4141 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     4201 gggggaggat tgggaagaga atagcaggca tgctggggag gtaccgaggg cctatttccc
     4261 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     4321 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     4381 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     4441 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagac
     4501 cacggcaggt ctcagtttta gtactctgga aacagaatct actaaaacaa ggcaaaatgc
     4561 cgtgtttatc tcgtcaactt gttggcgaga tttttgcggc cgcaggaacc cctagtgatg
     4621 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     4681 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     4741 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4801 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4861 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     4921 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4981 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     5041 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5101 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     5161 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     5221 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     5281 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5341 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5401 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5461 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5521 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5581 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5641 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5701 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5761 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5821 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5881 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5941 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6001 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6061 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6121 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6181 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6241 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6301 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6361 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6421 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6481 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6541 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6601 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6661 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6721 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6781 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6841 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6901 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6961 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7021 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7081 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7141 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7201 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7261 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7321 gctggccttt tgctcacatg t
//
