LOCUS       sequence-434415-        7729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis of Cnr1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7729)
  AUTHORS   Gunduz-Cinar O, Castillo LI, Xia M, Van Leer E, Brockway ET, Pollack
            GA, Yasmin F, Bukalo O, Limoges A, Oreizi-Esfahani S, Kondev V, 
            Baldi R, Dong A, Harvey-White J, Cinar R, Kunos G, Li Y, Zweifel LS,
            Patel S, Holmes A
  TITLE     A cortico-amygdala neural substrate for endocannabinoid modulation 
            of fear extinction.
  JOURNAL   Neuron. 2023 Jul 19:S0896-6273(23)00482-8. doi: 
            10.1016/j.neuron.2023.06.023.
  PUBMED    37480845
REFERENCE   2  (bases 1 to 7729)
  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: "Neuron. 
            2023 Jul 19:S0896-6273(23)00482-8. doi: 
            10.1016/j.neuron.2023.06.023."
COMMENT     Sequence Label: pAAV-CMV-FLEX-SaCas9-U6-sgCnr1
FEATURES             Location/Qualifiers
     source          1..7729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        23..402
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        403..606
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     557..577
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(643..676)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(727..760)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(772..852)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(859..906)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(907..4062)
                     /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             complement(4087..4107)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4139..4172
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    4223..4256
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4281..4298)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4287..4494
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4501..4749
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4501..4521
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4672..4691
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4772..4847
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4861..5001
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4861..4990
                     /label=AAV2 ITR
     rep_origin      5076..5531
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5163..5182)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5373..5394
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5548..5567)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5667..5689
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5727..5745)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5813..5917
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5918..5986,5987..6778)
                     /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(6136..6155)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6949..7537
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7438..7457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7599..7728
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 cggcctctag actcgaggcg ttgacattga ttattgacta gttattaata gtaatcaatt
       61 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      121 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      181 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      241 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      301 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      361 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      421 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      481 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      541 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      601 agagctctct ggctaactac cggtgctagc gtcgactccg gaataacttc gtataggata
      661 ctttatacga agttatgcag aatggtagct ggattgtagc tgctattagc aatatgaaac
      721 ctcttaataa cttcgtatag catacattat acgaagttat ggcgcgcctt aagcgtaatc
      781 tggaacatcg tatgggtaag cgtaatctgg aacatcgtat gggtaagcgt aatctggaac
      841 atcgtatggg taggatccct ttttcttttt tgcctggccg gcctttttcg tggccgccgg
      901 ccttttgccc tttttgatga tctgagggtg cttcttagat ttcacttcat acaggttgcc
      961 cagaatgtct gtgctgtact tcttaatgct ctgggtcttg gaggcgattg tcttaatgat
     1021 cctggggggc ctcttgtcgt tcatgttttc caggtactcg cggtaggtga tgtcgatcat
     1081 gttcacttcg atccggttca gcaggtcgtt gttcacgccg atcactctat acagctcgcc
     1141 gttgatcttg atcagatcgt tgttgtagaa ggaggcgata aactcggcct ggttgctgat
     1201 cttcttcagc ttcttagctt cctcatagca cttgctattc acttcgtagt agttttcttt
     1261 tttgatcaca tccagattct tcacggtcac gaacttgtac acgccattgt ccaggtacac
     1321 gtcgaatctg tagggcttca gggacagctt cacgaccttg tttctgctgt tggggtagtc
     1381 gtcggtgatg tccagatggg cgttcagttt gttgccgtaa tacttaatct tcttgatcac
     1441 ggggccgttg tcctttttgg agtacttggt caggtagttc ccggtttcct cgtagtactt
     1501 gtacagggga ttcttctcgt cgccgtactg ttccataatc agcttcagtt tctggtaggt
     1561 ctgggggtcg tggtggtaca tcagcagctt ttcggggctc ttgttgatca gctttttcag
     1621 cttgtcattg tccttgtcgt acaggccgtt cagattgttc acgatcaggg tgttgccctt
     1681 gtcgtccttc cgggtggagt acagggtgtc gttaatcagc tctctattag gcttcttgtc
     1741 cacccggtgg ctgtacttgt agtccttgaa gtccttaatg tgcttgatct ggtggggggt
     1801 gatgaagatc tctttgtact cctgctcggt ttcgatctcg ggcatgctct cggcctgctt
     1861 ttcctcgaac atctggtttt ccatcacttt tttggccttg tccagtttct tccactcttt
     1921 gaagatgaaa tcggcgttgg caatgatcag ggcgtcctcg gcgtggtgct tgtacccctt
     1981 gttccgctct ttcttaaact tccacttccg ccgcagaaag ctggtgaagc cgccattgat
     2041 ggacttcact ttcacgtcca ggttgttcac tctgaagtag ctccgcagca ggttcatcag
     2101 gcctctggtg gcgtatctgg tatccaccag gttccggttg atgaagtctt tctgcacgga
     2161 gaacctgttg atgtcccgtt cttccagcag atactctttc ttggtcttgc tgattctgcc
     2221 cttgcccttg gccagattca ggatgtgctt cttgaaggtt tcgtagctga tcttgctgtc
     2281 gctgctgctc aggtactgga atggggtccg gttgcccttc ttgctgtttt cttcctgctt
     2341 cacgagcacc ttgttgttga agctgttgtc gaaggacacg cttctgggga tgatgtggtc
     2401 cacctcatag ttgaaggggt tgttcagcag atcttccaga gggatggctt ccaggctgta
     2461 caggcacttg ccttcctgca tgtcgtgcag cttgatcttc tcgatcaggt acttggcgtt
     2521 ctctttgccg gtggtccgga tgatttcctc gatccgctcg ttggtctgcc ggttccgctt
     2581 ctgcatctcg ttgatcattt tctgggcgtc cttggagttc ttctcgcggg ccagctcgat
     2641 aatgatgtcg ttgggcaggc cgtacttctt gatgatggcg ttgatcactt tgatgctctg
     2701 gatgaagctt ctcttcacga cggggctcag gatgaagtcg tccaccaggg tggtggggat
     2761 ctctttctgc tgggacaggt ccaccttctt gggcaccagc ttcagccggt tgaagatagc
     2821 gatctggttg tcgttggtgt gccacagctc gtccaggatc aggttgatgg ccttcaggct
     2881 caggttgtgg gtgccggtat agcccttcag attagagatc tgctcgatct cttcctgggt
     2941 cagctcggag ttcagattgg tcagttcttc ctggatgtcc tcgctgctct ggtagatggt
     3001 caggatcttg gcaatctgat ccagcagctc ggcgttctca ataatctctt tccgggcggt
     3061 aatgtccttg atgtcgtggt acaccttcag gttggtgaac tcgggcttgc cggtgctggt
     3121 cactctgtag cccttaatat cctcttcgtt cacgaggatt tctttggcga tctgcttcag
     3181 ggtgggcttc ttcttctgct tgaacacgtt ctcgatgatc tggaacttct cgtaatattc
     3241 cagcttctcg ttctcgtccc tggtgatcac gagattgttc aggtcgttca gggcgttgta
     3301 caggtcggcg ttgtaggcgt acttcacgct ccgcagttcc tcggggaagt aggtgcagtg
     3361 gcccatcagc atctcgtacc attctttgat gtccttccag ccgaaggggc tgccctcgcc
     3421 aggtccctca tagtaggtcc gccgggtttc cagcaggtcg atgtaggtgt cgatgaagct
     3481 ctggtccagc tggtggtagg ccttctgcac cttcagcagc tgtttggctt ctttcacgta
     3541 gtcgctggtc ttgaatctgt tgatgctgcc ccgcacttcg ccgtctttct tcagccgttc
     3601 cagctgcagt tcggccacgt atttctcttc cagggccttg ctgttccggc tgatctgctc
     3661 tttggtggac agctcgttgc cggtgtcctc ttccacctcg ttcacgttgt gcacgcctct
     3721 tctcttggcc aggtgcagca gggcggcaga gaactcttcc tcgctcagct tctggctcag
     3781 gcccttcact ctggcctcgt aggggttgat gccgctcagc tcgctgtggt cggtcagcag
     3841 gttgtagtcg aacagcagct tcttcactct ctggattcta tgccgcctcc gccgcttcag
     3901 ccttctggcg cctctcttgc tccgcctgcc ctcgttgttt tccacgttgg cctctttgaa
     3961 cagccgcacg ccggcatcga tcacgtcccg tgtctcgtag tcgatgatgc cgtagcccac
     4021 gctggtgatg ccgatgtcca ggcccaggat gtagttccgc ttggctgctg ggactccgtg
     4081 gataccgacc ttccgcttct tctttggggc catgcggccg ctgcagaccg gtgctagcat
     4141 aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat tatcactgtt
     4201 attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga agttatgaat
     4261 tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     4321 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     4381 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     4441 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggaggtacc
     4501 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
     4561 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
     4621 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
     4681 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
     4741 cgaaacaccg aaggcctgca tcggagactg cgttttagta ctctggaaac agaatctact
     4801 aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt ttgcggccgc
     4861 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     4921 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     4981 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     5041 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     5101 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     5161 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5221 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5281 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5341 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5401 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     5461 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5521 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5581 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5641 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5701 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5761 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     5821 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5881 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     5941 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6001 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6061 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6121 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6181 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6241 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6301 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6361 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6421 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6481 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6541 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6601 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
     6661 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6721 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6781 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6841 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6901 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6961 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7021 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7081 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7141 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7201 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7261 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7321 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7381 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7441 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7501 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7561 acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg
     7621 ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
     7681 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctg
//
