LOCUS       sequence-343478-        7448 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A single vector containing Cas9 from Staphylococcus aureus (SaCas9) 
            and its sgRNA targeting lacZ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7448)
  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 7448)
  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-lacZ
FEATURES             Location/Qualifiers
     source          1..7448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     56..75
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   217..346
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        370..749
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        750..953
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     904..924
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      972..981
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             984..1004
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1029..4184
                     /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             4185..4232
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4239..4319
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4347..4364)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4353..4560
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4567..4815
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4567..4587
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4738..4757
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4838..4913
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4927..5067
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4927..5056
                     /label=AAV2 ITR
     rep_origin      5142..5597
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5229..5248)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5439..5460
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5614..5633)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5733..5755
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5793..5811)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5879..5983
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5984..6052,6053..6844)
                     /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(6202..6221)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7015..7448,1..155)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
       61 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      121 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      181 ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct
      241 cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
      301 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagact
      361 cgaggcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
      421 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      481 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      541 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      601 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      661 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      721 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      781 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      841 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      901 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
      961 taactaccgg tgccaccatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
     1021 cagcagccaa gcggaactac atcctgggcc tggacatcgg catcaccagc gtgggctacg
     1081 gcatcatcga ctacgagaca cgggacgtga tcgatgccgg cgtgcggctg ttcaaagagg
     1141 ccaacgtgga aaacaacgag ggcaggcgga gcaagagagg cgccagaagg ctgaagcggc
     1201 ggaggcggca tagaatccag agagtgaaga agctgctgtt cgactacaac ctgctgaccg
     1261 accacagcga gctgagcggc atcaacccct acgaggccag agtgaagggc ctgagccaga
     1321 agctgagcga ggaagagttc tctgccgccc tgctgcacct ggccaagaga agaggcgtgc
     1381 acaacgtgaa cgaggtggaa gaggacaccg gcaacgagct gtccaccaaa gagcagatca
     1441 gccggaacag caaggccctg gaagagaaat acgtggccga actgcagctg gaacggctga
     1501 agaaagacgg cgaagtgcgg ggcagcatca acagattcaa gaccagcgac tacgtgaaag
     1561 aagccaaaca gctgctgaag gtgcagaagg cctaccacca gctggaccag agcttcatcg
     1621 acacctacat cgacctgctg gaaacccggc ggacctacta tgagggacct ggcgagggca
     1681 gccccttcgg ctggaaggac atcaaagaat ggtacgagat gctgatgggc cactgcacct
     1741 acttccccga ggaactgcgg agcgtgaagt acgcctacaa cgccgacctg tacaacgccc
     1801 tgaacgacct gaacaatctc gtgatcacca gggacgagaa cgagaagctg gaatattacg
     1861 agaagttcca gatcatcgag aacgtgttca agcagaagaa gaagcccacc ctgaagcaga
     1921 tcgccaaaga aatcctcgtg aacgaagagg atattaaggg ctacagagtg accagcaccg
     1981 gcaagcccga gttcaccaac ctgaaggtgt accacgacat caaggacatt accgcccgga
     2041 aagagattat tgagaacgcc gagctgctgg atcagattgc caagatcctg accatctacc
     2101 agagcagcga ggacatccag gaagaactga ccaatctgaa ctccgagctg acccaggaag
     2161 agatcgagca gatctctaat ctgaagggct ataccggcac ccacaacctg agcctgaagg
     2221 ccatcaacct gatcctggac gagctgtggc acaccaacga caaccagatc gctatcttca
     2281 accggctgaa gctggtgccc aagaaggtgg acctgtccca gcagaaagag atccccacca
     2341 ccctggtgga cgacttcatc ctgagccccg tcgtgaagag aagcttcatc cagagcatca
     2401 aagtgatcaa cgccatcatc aagaagtacg gcctgcccaa cgacatcatt atcgagctgg
     2461 cccgcgagaa gaactccaag gacgcccaga aaatgatcaa cgagatgcag aagcggaacc
     2521 ggcagaccaa cgagcggatc gaggaaatca tccggaccac cggcaaagag aacgccaagt
     2581 acctgatcga gaagatcaag ctgcacgaca tgcaggaagg caagtgcctg tacagcctgg
     2641 aagccatccc tctggaagat ctgctgaaca accccttcaa ctatgaggtg gaccacatca
     2701 tccccagaag cgtgtccttc gacaacagct tcaacaacaa ggtgctcgtg aagcaggaag
     2761 aaaacagcaa gaagggcaac cggaccccat tccagtacct gagcagcagc gacagcaaga
     2821 tcagctacga aaccttcaag aagcacatcc tgaatctggc caagggcaag ggcagaatca
     2881 gcaagaccaa gaaagagtat ctgctggaag aacgggacat caacaggttc tccgtgcaga
     2941 aagacttcat caaccggaac ctggtggata ccagatacgc caccagaggc ctgatgaacc
     3001 tgctgcggag ctacttcaga gtgaacaacc tggacgtgaa agtgaagtcc atcaatggcg
     3061 gcttcaccag ctttctgcgg cggaagtgga agtttaagaa agagcggaac aaggggtaca
     3121 agcaccacgc cgaggacgcc ctgatcattg ccaacgccga tttcatcttc aaagagtgga
     3181 agaaactgga caaggccaaa aaagtgatgg aaaaccagat gttcgaggaa aagcaggccg
     3241 agagcatgcc cgagatcgaa accgagcagg agtacaaaga gatcttcatc accccccacc
     3301 agatcaagca cattaaggac ttcaaggact acaagtacag ccaccgggtg gacaagaagc
     3361 ctaatagaga gctgattaac gacaccctgt actccacccg gaaggacgac aagggcaaca
     3421 ccctgatcgt gaacaatctg aacggcctgt acgacaagga caatgacaag ctgaaaaagc
     3481 tgatcaacaa gagccccgaa aagctgctga tgtaccacca cgacccccag acctaccaga
     3541 aactgaagct gattatggaa cagtacggcg acgagaagaa tcccctgtac aagtactacg
     3601 aggaaaccgg gaactacctg accaagtact ccaaaaagga caacggcccc gtgatcaaga
     3661 agattaagta ttacggcaac aaactgaacg cccatctgga catcaccgac gactacccca
     3721 acagcagaaa caaggtcgtg aagctgtccc tgaagcccta cagattcgac gtgtacctgg
     3781 acaatggcgt gtacaagttc gtgaccgtga agaatctgga tgtgatcaaa aaagaaaact
     3841 actacgaagt gaatagcaag tgctatgagg aagctaagaa gctgaagaag atcagcaacc
     3901 aggccgagtt tatcgcctcc ttctacaaca acgatctgat caagatcaac ggcgagctgt
     3961 atagagtgat cggcgtgaac aacgacctgc tgaaccggat cgaagtgaac atgatcgaca
     4021 tcacctaccg cgagtacctg gaaaacatga acgacaagag gccccccagg atcattaaga
     4081 caatcgcctc caagacccag agcattaaga agtacagcac agacattctg ggcaacctgt
     4141 atgaagtgaa atctaagaag caccctcaga tcatcaaaaa gggcaaaagg ccggcggcca
     4201 cgaaaaaggc cggccaggca aaaaagaaaa agggatccta cccatacgat gttccagatt
     4261 acgcttaccc atacgatgtt ccagattacg cttacccata cgatgttcca gattacgctt
     4321 aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     4381 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     4441 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     4501 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga
     4561 ggtaccgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     4621 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     4681 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     4741 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     4801 aaaggacgaa acaccgcatc gcgtgggcgt attcgcagtt ttagtactct ggaaacagaa
     4861 tctactaaaa caaggcaaaa tgccgtgttt atctcgtcaa cttgttggcg agatttttgc
     4921 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4981 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     5041 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     5101 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     5161 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     5221 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5281 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5341 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5401 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5461 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     5521 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5581 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5641 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5701 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5761 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5821 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5881 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5941 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     6001 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     6061 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     6121 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6181 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     6241 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     6301 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6361 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6421 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6481 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6541 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6601 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6661 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     6721 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6781 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6841 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6901 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6961 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     7021 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7081 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7141 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     7201 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7261 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7321 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7381 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7441 ggcggaca
//
