LOCUS       sequence-243573-        7729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis of Grin1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7729)
  AUTHORS   Hunker AC, Soden ME, Krayushkina D, Heymann G, Awatramani R, Zweifel
            LS
  TITLE     Conditional Single Vector CRISPR/SaCas9 Viruses for Efficient 
            Mutagenesis in the Adult Mouse Nervous System.
  JOURNAL   Cell Rep. 2020 Mar 24;30(12):4303-4316.e6. doi: 
            10.1016/j.celrep.2020.02.092.
  PUBMED    32209486
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"; doi: 
            "10.1016/j.celrep.2020.02"; journalName: "Cell Rep"; date: 
            "2020-03-24- 24"; volume: "30"; issue: "12"; pages: "4303-4316.e6"
COMMENT     Sequence Label: pAAV-FLEX-SaCas9-U6-sgGrin1
FEATURES             Location/Qualifiers
     source          1..7729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(241..274)
                     /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(325..358)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(370..450)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(457..504)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(505..3660)
                     /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(3685..3705)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    3737..3770
                     /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    3821..3854
                     /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(3879..3896)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3885..4092
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4099..4347
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4099..4119
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4270..4289
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4370..4445
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4459..4599
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4459..4588
                     /label=AAV2 ITR
     rep_origin      4674..5129
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4761..4780)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4971..4992
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5146..5165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5265..5287
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5325..5343)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5411..5515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5516..5584,5585..6376)
                     /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(5734..5753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6547..7135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7036..7055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7197..7326
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        7350..7729
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactaccg gtgctagcgt cgactccgga
      241 ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg attgtagctg
      301 ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac gaagttatgg
      361 cgcgccttaa gcgtaatctg gaacatcgta tgggtaagcg taatctggaa catcgtatgg
      421 gtaagcgtaa tctggaacat cgtatgggta ggatcccttt ttcttttttg cctggccggc
      481 ctttttcgtg gccgccggcc ttttgccctt tttgatgatc tgagggtgct tcttagattt
      541 cacttcatac aggttgccca gaatgtctgt gctgtacttc ttaatgctct gggtcttgga
      601 ggcgattgtc ttaatgatcc tggggggcct cttgtcgttc atgttttcca ggtactcgcg
      661 gtaggtgatg tcgatcatgt tcacttcgat ccggttcagc aggtcgttgt tcacgccgat
      721 cactctatac agctcgccgt tgatcttgat cagatcgttg ttgtagaagg aggcgataaa
      781 ctcggcctgg ttgctgatct tcttcagctt cttagcttcc tcatagcact tgctattcac
      841 ttcgtagtag ttttcttttt tgatcacatc cagattcttc acggtcacga acttgtacac
      901 gccattgtcc aggtacacgt cgaatctgta gggcttcagg gacagcttca cgaccttgtt
      961 tctgctgttg gggtagtcgt cggtgatgtc cagatgggcg ttcagtttgt tgccgtaata
     1021 cttaatcttc ttgatcacgg ggccgttgtc ctttttggag tacttggtca ggtagttccc
     1081 ggtttcctcg tagtacttgt acaggggatt cttctcgtcg ccgtactgtt ccataatcag
     1141 cttcagtttc tggtaggtct gggggtcgtg gtggtacatc agcagctttt cggggctctt
     1201 gttgatcagc tttttcagct tgtcattgtc cttgtcgtac aggccgttca gattgttcac
     1261 gatcagggtg ttgcccttgt cgtccttccg ggtggagtac agggtgtcgt taatcagctc
     1321 tctattaggc ttcttgtcca cccggtggct gtacttgtag tccttgaagt ccttaatgtg
     1381 cttgatctgg tggggggtga tgaagatctc tttgtactcc tgctcggttt cgatctcggg
     1441 catgctctcg gcctgctttt cctcgaacat ctggttttcc atcacttttt tggccttgtc
     1501 cagtttcttc cactctttga agatgaaatc ggcgttggca atgatcaggg cgtcctcggc
     1561 gtggtgcttg taccccttgt tccgctcttt cttaaacttc cacttccgcc gcagaaagct
     1621 ggtgaagccg ccattgatgg acttcacttt cacgtccagg ttgttcactc tgaagtagct
     1681 ccgcagcagg ttcatcaggc ctctggtggc gtatctggta tccaccaggt tccggttgat
     1741 gaagtctttc tgcacggaga acctgttgat gtcccgttct tccagcagat actctttctt
     1801 ggtcttgctg attctgccct tgcccttggc cagattcagg atgtgcttct tgaaggtttc
     1861 gtagctgatc ttgctgtcgc tgctgctcag gtactggaat ggggtccggt tgcccttctt
     1921 gctgttttct tcctgcttca cgagcacctt gttgttgaag ctgttgtcga aggacacgct
     1981 tctggggatg atgtggtcca cctcatagtt gaaggggttg ttcagcagat cttccagagg
     2041 gatggcttcc aggctgtaca ggcacttgcc ttcctgcatg tcgtgcagct tgatcttctc
     2101 gatcaggtac ttggcgttct ctttgccggt ggtccggatg atttcctcga tccgctcgtt
     2161 ggtctgccgg ttccgcttct gcatctcgtt gatcattttc tgggcgtcct tggagttctt
     2221 ctcgcgggcc agctcgataa tgatgtcgtt gggcaggccg tacttcttga tgatggcgtt
     2281 gatcactttg atgctctgga tgaagcttct cttcacgacg gggctcagga tgaagtcgtc
     2341 caccagggtg gtggggatct ctttctgctg ggacaggtcc accttcttgg gcaccagctt
     2401 cagccggttg aagatagcga tctggttgtc gttggtgtgc cacagctcgt ccaggatcag
     2461 gttgatggcc ttcaggctca ggttgtgggt gccggtatag cccttcagat tagagatctg
     2521 ctcgatctct tcctgggtca gctcggagtt cagattggtc agttcttcct ggatgtcctc
     2581 gctgctctgg tagatggtca ggatcttggc aatctgatcc agcagctcgg cgttctcaat
     2641 aatctctttc cgggcggtaa tgtccttgat gtcgtggtac accttcaggt tggtgaactc
     2701 gggcttgccg gtgctggtca ctctgtagcc cttaatatcc tcttcgttca cgaggatttc
     2761 tttggcgatc tgcttcaggg tgggcttctt cttctgcttg aacacgttct cgatgatctg
     2821 gaacttctcg taatattcca gcttctcgtt ctcgtccctg gtgatcacga gattgttcag
     2881 gtcgttcagg gcgttgtaca ggtcggcgtt gtaggcgtac ttcacgctcc gcagttcctc
     2941 ggggaagtag gtgcagtggc ccatcagcat ctcgtaccat tctttgatgt ccttccagcc
     3001 gaaggggctg ccctcgccag gtccctcata gtaggtccgc cgggtttcca gcaggtcgat
     3061 gtaggtgtcg atgaagctct ggtccagctg gtggtaggcc ttctgcacct tcagcagctg
     3121 tttggcttct ttcacgtagt cgctggtctt gaatctgttg atgctgcccc gcacttcgcc
     3181 gtctttcttc agccgttcca gctgcagttc ggccacgtat ttctcttcca gggccttgct
     3241 gttccggctg atctgctctt tggtggacag ctcgttgccg gtgtcctctt ccacctcgtt
     3301 cacgttgtgc acgcctcttc tcttggccag gtgcagcagg gcggcagaga actcttcctc
     3361 gctcagcttc tggctcaggc ccttcactct ggcctcgtag gggttgatgc cgctcagctc
     3421 gctgtggtcg gtcagcaggt tgtagtcgaa cagcagcttc ttcactctct ggattctatg
     3481 ccgcctccgc cgcttcagcc ttctggcgcc tctcttgctc cgcctgccct cgttgttttc
     3541 cacgttggcc tctttgaaca gccgcacgcc ggcatcgatc acgtcccgtg tctcgtagtc
     3601 gatgatgccg tagcccacgc tggtgatgcc gatgtccagg cccaggatgt agttccgctt
     3661 ggctgctggg actccgtgga taccgacctt ccgcttcttc tttggggcca tgcggccgct
     3721 gcagaccggt gctagcataa cttcgtataa agtatcctat acgaagttat ttgccttaac
     3781 ccagaaatta tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataatgtatg
     3841 ctatacgaag ttatgaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     3901 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3961 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     4021 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag
     4081 gcatgctggg gaggtaccga gggcctattt cccatgattc cttcatattt gcatatacga
     4141 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
     4201 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     4261 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     4321 tatatcttgt ggaaaggacg aaacaccgcc gcgccgatgt tgacaatctg ttttagtact
     4381 ctggaaacag aatctactaa aacaaggcaa aatgccgtgt ttatctcgtc aacttgttgg
     4441 cgagattttt gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     4501 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     4561 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     4621 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     4681 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4741 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4801 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4861 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     4921 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4981 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     5041 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5101 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     5161 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5221 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5281 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     5341 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     5401 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5461 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5521 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5581 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5641 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5701 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5761 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5821 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5881 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5941 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6001 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6061 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6121 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6181 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg
     6241 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6301 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6361 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6421 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6481 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6541 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6601 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6661 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6721 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6781 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6841 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6901 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6961 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7021 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7081 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7141 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtcctg
     7201 caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg acctttggtc
     7261 gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg
     7321 gttcctgcgg cctctagact cgaggcgttg acattgatta ttgactagtt attaatagta
     7381 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     7441 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     7501 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     7561 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     7621 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     7681 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatg
//
