LOCUS       sequence-487019-        7295 bp    DNA     circular SYN 23-SEP-2025
DEFINITION  Negative control AAV vector for SaCas9-based CRISPR KO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7295)
  AUTHORS   Parra-Rivas LA, Sharma R, Rust TE, Bazick HO, Carlson-Stevermer J, 
            Zylka MJ, Ogawa Y, Roy S
  TITLE     Protocol for CRISPR-based manipulation and visualization of 
            endogenous alpha-synuclein in cultured mouse hippocampal neurons.
  JOURNAL   STAR Protoc. 2025 Jul 21;6(3):103945. doi: 
            10.1016/j.xpro.2025.103945.
  PUBMED    40700012
REFERENCE   2  (bases 1 to 7295)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 23, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "STAR 
            Protoc."; date: "2025-07-21"; volume: "6(3):103945. doi"; pages: " 
            10.1016/j.xpro.2025.103945"
COMMENT     Sequence Label: pLP110_AAV Scramble Control
FEATURES             Location/Qualifiers
     source          1..7295
                     /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        149..596
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      603..612
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             615..635
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             660..3815
                     /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             3816..3863
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3870..3950
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3978..3995)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3984..4191
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4198..4446
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4198..4218
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4369..4388
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4469..4544
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4558..4698
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4558..4687
                     /label=AAV2 ITR
     rep_origin      4773..5228
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4860..4879)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5070..5091
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5245..5264)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5364..5386
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5424..5442)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5510..5614
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5615..5683,5684..6475)
                     /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(5833..5852)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6646..7234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7135..7154
                     /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 gactcgagag tgcaagtggg ttttaggacc aggatgaggc
      181 ggggtggggg tgcctacctg acgaccgacc ccgacccact ggacaagcac ccaaccccca
      241 ttccccaaat tgcgcatccc ctatcagaga gggggagggg aaacaggatg cggcgaggcg
      301 cgtgcgcact gccagcttca gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc
      361 gccaccgccg cctcagcact gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc
      421 cccttcccgg ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg
      481 cgaggcgcga gatagggggg cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag
      541 cgctgcctca gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga gcgcagaccg
      601 gtgccaccat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagcca
      661 agcggaacta catcctgggc ctggacatcg gcatcaccag cgtgggctac ggcatcatcg
      721 actacgagac acgggacgtg atcgatgccg gcgtgcggct gttcaaagag gccaacgtgg
      781 aaaacaacga gggcaggcgg agcaagagag gcgccagaag gctgaagcgg cggaggcggc
      841 atagaatcca gagagtgaag aagctgctgt tcgactacaa cctgctgacc gaccacagcg
      901 agctgagcgg catcaacccc tacgaggcca gagtgaaggg cctgagccag aagctgagcg
      961 aggaagagtt ctctgccgcc ctgctgcacc tggccaagag aagaggcgtg cacaacgtga
     1021 acgaggtgga agaggacacc ggcaacgagc tgtccaccaa agagcagatc agccggaaca
     1081 gcaaggccct ggaagagaaa tacgtggccg aactgcagct ggaacggctg aagaaagacg
     1141 gcgaagtgcg gggcagcatc aacagattca agaccagcga ctacgtgaaa gaagccaaac
     1201 agctgctgaa ggtgcagaag gcctaccacc agctggacca gagcttcatc gacacctaca
     1261 tcgacctgct ggaaacccgg cggacctact atgagggacc tggcgagggc agccccttcg
     1321 gctggaagga catcaaagaa tggtacgaga tgctgatggg ccactgcacc tacttccccg
     1381 aggaactgcg gagcgtgaag tacgcctaca acgccgacct gtacaacgcc ctgaacgacc
     1441 tgaacaatct cgtgatcacc agggacgaga acgagaagct ggaatattac gagaagttcc
     1501 agatcatcga gaacgtgttc aagcagaaga agaagcccac cctgaagcag atcgccaaag
     1561 aaatcctcgt gaacgaagag gatattaagg gctacagagt gaccagcacc ggcaagcccg
     1621 agttcaccaa cctgaaggtg taccacgaca tcaaggacat taccgcccgg aaagagatta
     1681 ttgagaacgc cgagctgctg gatcagattg ccaagatcct gaccatctac cagagcagcg
     1741 aggacatcca ggaagaactg accaatctga actccgagct gacccaggaa gagatcgagc
     1801 agatctctaa tctgaagggc tataccggca cccacaacct gagcctgaag gccatcaacc
     1861 tgatcctgga cgagctgtgg cacaccaacg acaaccagat cgctatcttc aaccggctga
     1921 agctggtgcc caagaaggtg gacctgtccc agcagaaaga gatccccacc accctggtgg
     1981 acgacttcat cctgagcccc gtcgtgaaga gaagcttcat ccagagcatc aaagtgatca
     2041 acgccatcat caagaagtac ggcctgccca acgacatcat tatcgagctg gcccgcgaga
     2101 agaactccaa ggacgcccag aaaatgatca acgagatgca gaagcggaac cggcagacca
     2161 acgagcggat cgaggaaatc atccggacca ccggcaaaga gaacgccaag tacctgatcg
     2221 agaagatcaa gctgcacgac atgcaggaag gcaagtgcct gtacagcctg gaagccatcc
     2281 ctctggaaga tctgctgaac aaccccttca actatgaggt ggaccacatc atccccagaa
     2341 gcgtgtcctt cgacaacagc ttcaacaaca aggtgctcgt gaagcaggaa gaaaacagca
     2401 agaagggcaa ccggacccca ttccagtacc tgagcagcag cgacagcaag atcagctacg
     2461 aaaccttcaa gaagcacatc ctgaatctgg ccaagggcaa gggcagaatc agcaagacca
     2521 agaaagagta tctgctggaa gaacgggaca tcaacaggtt ctccgtgcag aaagacttca
     2581 tcaaccggaa cctggtggat accagatacg ccaccagagg cctgatgaac ctgctgcgga
     2641 gctacttcag agtgaacaac ctggacgtga aagtgaagtc catcaatggc ggcttcacca
     2701 gctttctgcg gcggaagtgg aagtttaaga aagagcggaa caaggggtac aagcaccacg
     2761 ccgaggacgc cctgatcatt gccaacgccg atttcatctt caaagagtgg aagaaactgg
     2821 acaaggccaa aaaagtgatg gaaaaccaga tgttcgagga aaagcaggcc gagagcatgc
     2881 ccgagatcga aaccgagcag gagtacaaag agatcttcat caccccccac cagatcaagc
     2941 acattaagga cttcaaggac tacaagtaca gccaccgggt ggacaagaag cctaatagag
     3001 agctgattaa cgacaccctg tactccaccc ggaaggacga caagggcaac accctgatcg
     3061 tgaacaatct gaacggcctg tacgacaagg acaatgacaa gctgaaaaag ctgatcaaca
     3121 agagccccga aaagctgctg atgtaccacc acgaccccca gacctaccag aaactgaagc
     3181 tgattatgga acagtacggc gacgagaaga atcccctgta caagtactac gaggaaaccg
     3241 ggaactacct gaccaagtac tccaaaaagg acaacggccc cgtgatcaag aagattaagt
     3301 attacggcaa caaactgaac gcccatctgg acatcaccga cgactacccc aacagcagaa
     3361 acaaggtcgt gaagctgtcc ctgaagccct acagattcga cgtgtacctg gacaatggcg
     3421 tgtacaagtt cgtgaccgtg aagaatctgg atgtgatcaa aaaagaaaac tactacgaag
     3481 tgaatagcaa gtgctatgag gaagctaaga agctgaagaa gatcagcaac caggccgagt
     3541 ttatcgcctc cttctacaac aacgatctga tcaagatcaa cggcgagctg tatagagtga
     3601 tcggcgtgaa caacgacctg ctgaaccgga tcgaagtgaa catgatcgac atcacctacc
     3661 gcgagtacct ggaaaacatg aacgacaaga ggccccccag gatcattaag acaatcgcct
     3721 ccaagaccca gagcattaag aagtacagca cagacattct gggcaacctg tatgaagtga
     3781 aatctaagaa gcaccctcag atcatcaaaa agggcaaaag gccggcggcc acgaaaaagg
     3841 ccggccaggc aaaaaagaaa aagggatcct acccatacga tgttccagat tacgcttacc
     3901 catacgatgt tccagattac gcttacccat acgatgttcc agattacgct taagaattcc
     3961 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     4021 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4081 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4141 gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg aggtaccgag
     4201 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     4261 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     4321 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     4381 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     4441 aacaccgacg gaggctaagc gtcgcaaggt tttagtactc tggaaacaga atctactaaa
     4501 acaaggcaaa atgccgtgtt tatctcgtca acttgttggc gagatttttg cggccgcagg
     4561 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     4621 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     4681 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4741 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     4801 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     4861 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4921 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4981 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5041 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5101 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     5161 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     5221 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5281 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5341 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5401 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     5461 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5521 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5581 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5641 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5701 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5761 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5821 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5881 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5941 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6001 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6061 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6121 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6181 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6241 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6301 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     6361 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6421 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6481 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6541 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6601 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6661 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6721 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6781 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6841 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6901 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6961 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     7021 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     7081 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     7141 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     7201 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7261 gttcctggcc ttttgctggc cttttgctca catgt
//
