LOCUS       sequence-344152-        7447 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A single vector containing Cas9 from Staphylococcus aureus (SaCas9) 
            and its sgRNA targeting Grpr.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7447)
  AUTHORS   Melzer S, Newmark ER, Mizuno GO, Hyun M, Philson AC, Quiroli E, 
            Righetti B, Gregory MR, Huang KW, Levasseur J, Tian L, Sabatini BL
  TITLE     Bombesin-like peptide recruits disinhibitory cortical circuits and 
            enhances fear memories.
  JOURNAL   Cell. 2021 Oct 28;184(22):5622-5634.e25. doi: 
            10.1016/j.cell.2021.09.013. Epub 2021 Oct 4.
  PUBMED    34610277
REFERENCE   2  (bases 1 to 7447)
  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"; doi: 
            "10.1016/j.cell.2021.09.013"; journalName: "Cell"; date: 
            "2021-10-28- 28"; volume: "184"; issue: "22"; pages: "5622-5634.e25"
COMMENT     Sequence Label: 
            pX601-AAV-CMV::NLS-saCas9-NLS-3xHA-bGHpa;U6::BsaI-sgRNA-Grpr1
FEATURES             Location/Qualifiers
     source          1..7447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   69..198
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        222..601
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        602..805
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     756..776
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      824..833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             836..856
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             881..4036
                     /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             4037..4084
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4091..4171
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4199..4216)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4205..4412
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4419..4667
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4419..4439
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4590..4609
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4689..4764
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4778..4918
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4778..4907
                     /label=AAV2 ITR
     rep_origin      4993..5448
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5080..5099)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5290..5311
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5465..5484)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5584..5606
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5644..5662)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5730..5834
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5835..5903,5904..6695)
                     /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(6053..6072)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6866..7447,1..7)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7355..7374
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
       61 tcacatgtcc tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg
      121 cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact
      181 ccatcactag gggttcctgc ggcctctaga ctcgaggcgt tgacattgat tattgactag
      241 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      301 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      361 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      421 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      481 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      541 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      601 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      661 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      721 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      781 gtgggaggtc tatataagca gagctctctg gctaactacc ggtgccacca tggccccaaa
      841 gaagaagcgg aaggtcggta tccacggagt cccagcagcc aagcggaact acatcctggg
      901 cctggacatc ggcatcacca gcgtgggcta cggcatcatc gactacgaga cacgggacgt
      961 gatcgatgcc ggcgtgcggc tgttcaaaga ggccaacgtg gaaaacaacg agggcaggcg
     1021 gagcaagaga ggcgccagaa ggctgaagcg gcggaggcgg catagaatcc agagagtgaa
     1081 gaagctgctg ttcgactaca acctgctgac cgaccacagc gagctgagcg gcatcaaccc
     1141 ctacgaggcc agagtgaagg gcctgagcca gaagctgagc gaggaagagt tctctgccgc
     1201 cctgctgcac ctggccaaga gaagaggcgt gcacaacgtg aacgaggtgg aagaggacac
     1261 cggcaacgag ctgtccacca aagagcagat cagccggaac agcaaggccc tggaagagaa
     1321 atacgtggcc gaactgcagc tggaacggct gaagaaagac ggcgaagtgc ggggcagcat
     1381 caacagattc aagaccagcg actacgtgaa agaagccaaa cagctgctga aggtgcagaa
     1441 ggcctaccac cagctggacc agagcttcat cgacacctac atcgacctgc tggaaacccg
     1501 gcggacctac tatgagggac ctggcgaggg cagccccttc ggctggaagg acatcaaaga
     1561 atggtacgag atgctgatgg gccactgcac ctacttcccc gaggaactgc ggagcgtgaa
     1621 gtacgcctac aacgccgacc tgtacaacgc cctgaacgac ctgaacaatc tcgtgatcac
     1681 cagggacgag aacgagaagc tggaatatta cgagaagttc cagatcatcg agaacgtgtt
     1741 caagcagaag aagaagccca ccctgaagca gatcgccaaa gaaatcctcg tgaacgaaga
     1801 ggatattaag ggctacagag tgaccagcac cggcaagccc gagttcacca acctgaaggt
     1861 gtaccacgac atcaaggaca ttaccgcccg gaaagagatt attgagaacg ccgagctgct
     1921 ggatcagatt gccaagatcc tgaccatcta ccagagcagc gaggacatcc aggaagaact
     1981 gaccaatctg aactccgagc tgacccagga agagatcgag cagatctcta atctgaaggg
     2041 ctataccggc acccacaacc tgagcctgaa ggccatcaac ctgatcctgg acgagctgtg
     2101 gcacaccaac gacaaccaga tcgctatctt caaccggctg aagctggtgc ccaagaaggt
     2161 ggacctgtcc cagcagaaag agatccccac caccctggtg gacgacttca tcctgagccc
     2221 cgtcgtgaag agaagcttca tccagagcat caaagtgatc aacgccatca tcaagaagta
     2281 cggcctgccc aacgacatca ttatcgagct ggcccgcgag aagaactcca aggacgccca
     2341 gaaaatgatc aacgagatgc agaagcggaa ccggcagacc aacgagcgga tcgaggaaat
     2401 catccggacc accggcaaag agaacgccaa gtacctgatc gagaagatca agctgcacga
     2461 catgcaggaa ggcaagtgcc tgtacagcct ggaagccatc cctctggaag atctgctgaa
     2521 caaccccttc aactatgagg tggaccacat catccccaga agcgtgtcct tcgacaacag
     2581 cttcaacaac aaggtgctcg tgaagcagga agaaaacagc aagaagggca accggacccc
     2641 attccagtac ctgagcagca gcgacagcaa gatcagctac gaaaccttca agaagcacat
     2701 cctgaatctg gccaagggca agggcagaat cagcaagacc aagaaagagt atctgctgga
     2761 agaacgggac atcaacaggt tctccgtgca gaaagacttc atcaaccgga acctggtgga
     2821 taccagatac gccaccagag gcctgatgaa cctgctgcgg agctacttca gagtgaacaa
     2881 cctggacgtg aaagtgaagt ccatcaatgg cggcttcacc agctttctgc ggcggaagtg
     2941 gaagtttaag aaagagcgga acaaggggta caagcaccac gccgaggacg ccctgatcat
     3001 tgccaacgcc gatttcatct tcaaagagtg gaagaaactg gacaaggcca aaaaagtgat
     3061 ggaaaaccag atgttcgagg aaaagcaggc cgagagcatg cccgagatcg aaaccgagca
     3121 ggagtacaaa gagatcttca tcacccccca ccagatcaag cacattaagg acttcaagga
     3181 ctacaagtac agccaccggg tggacaagaa gcctaataga gagctgatta acgacaccct
     3241 gtactccacc cggaaggacg acaagggcaa caccctgatc gtgaacaatc tgaacggcct
     3301 gtacgacaag gacaatgaca agctgaaaaa gctgatcaac aagagccccg aaaagctgct
     3361 gatgtaccac cacgaccccc agacctacca gaaactgaag ctgattatgg aacagtacgg
     3421 cgacgagaag aatcccctgt acaagtacta cgaggaaacc gggaactacc tgaccaagta
     3481 ctccaaaaag gacaacggcc ccgtgatcaa gaagattaag tattacggca acaaactgaa
     3541 cgcccatctg gacatcaccg acgactaccc caacagcaga aacaaggtcg tgaagctgtc
     3601 cctgaagccc tacagattcg acgtgtacct ggacaatggc gtgtacaagt tcgtgaccgt
     3661 gaagaatctg gatgtgatca aaaaagaaaa ctactacgaa gtgaatagca agtgctatga
     3721 ggaagctaag aagctgaaga agatcagcaa ccaggccgag tttatcgcct ccttctacaa
     3781 caacgatctg atcaagatca acggcgagct gtatagagtg atcggcgtga acaacgacct
     3841 gctgaaccgg atcgaagtga acatgatcga catcacctac cgcgagtacc tggaaaacat
     3901 gaacgacaag aggcccccca ggatcattaa gacaatcgcc tccaagaccc agagcattaa
     3961 gaagtacagc acagacattc tgggcaacct gtatgaagtg aaatctaaga agcaccctca
     4021 gatcatcaaa aagggcaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa
     4081 aaagggatcc tacccatacg atgttccaga ttacgcttac ccatacgatg ttccagatta
     4141 cgcttaccca tacgatgttc cagattacgc ttaagaattc ctagagctcg ctgatcagcc
     4201 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     4261 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     4321 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     4381 gattgggaag agaatagcag gcatgctggg gaggtaccga gggcctattt cccatgattc
     4441 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     4501 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     4561 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     4621 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgat gataagccca
     4681 taaactgcgt tttagtactc tggaaacaga atctactaaa acaaggcaaa atgccgtgtt
     4741 tatctcgtca acttgttggc gagatttttg cggccgcagg aacccctagt gatggagttg
     4801 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4861 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4921 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4981 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     5041 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     5101 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5161 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     5221 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5281 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     5341 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     5401 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     5461 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     5521 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5581 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5641 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5701 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5761 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5821 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5881 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5941 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     6001 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     6061 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     6121 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     6181 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     6241 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6301 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6361 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6421 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6481 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6541 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6601 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6661 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6721 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6781 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6841 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6901 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6961 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     7021 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     7081 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     7141 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     7201 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     7261 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7321 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7381 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7441 ggagcct
//
