LOCUS       sequence-243677-        7726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis of Gria3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7726)
  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 7726)
  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-FLEXfrt-SaCas9-U6-sgGria3
FEATURES             Location/Qualifiers
     source          1..7726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        24..403
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        404..607
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     558..578
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    650..683
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(755..835)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(842..889)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATRKAGQAKKKK"
     CDS             complement(890..4045)
                     /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(4070..4090)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4280..4297)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4286..4493
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4500..4748
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4500..4520
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4671..4690
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4770..4845
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4859..4999
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4859..4988
                     /label=AAV2 ITR
     rep_origin      5074..5529
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5161..5180)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5371..5392
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5546..5565)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5665..5687
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5725..5743)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5811..5915
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5916..5984,5985..6776)
                     /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(6134..6153)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6947..7535
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7436..7455
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7597..7726
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcggcctcta gactcgaggc gttgacattg attattgact agttattaat agtaatcaat
       61 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      121 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      181 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      241 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      301 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      361 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      421 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      481 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      541 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      601 cagagctctc tggctaacta ccggtgctag cgtcgacaga attggatccg aagttcctat
      661 tctctagaaa gtataggaac ttccaaggcc tgaagttcct attcttcaaa aggtatagga
      721 acttcgcggc cgcttgatat cttggcgcgc cttaagcgta atctggaaca tcgtatgggt
      781 aagcgtaatc tggaacatcg tatgggtaag cgtaatctgg aacatcgtat gggtaggatc
      841 cctttttctt ttttgcctgg ccggcctttc tcgtggccgc cggccttttg ccctttttga
      901 tgatctgagg gtgcttctta gatttcactt catacaggtt gcccagaatg tctgtgctgt
      961 acttcttaat gctctgggtc ttggaggcga ttgtcttaat gatcctgggg ggcctcttgt
     1021 cgttcatgtt ttccaggtac tcgcggtagg tgatgtcgat catgttcact tcgatccggt
     1081 tcagcaggtc gttgttcacg ccgatcactc tatacagctc gccgttgatc ttgatcagat
     1141 cgttgttgta gaaggaggcg ataaactcgg cctggttgct gatcttcttc agcttcttag
     1201 cttcctcata gcacttgcta ttcacttcgt agtagttttc ttttttgatc acatccagat
     1261 tcttcacggt cacgaacttg tacacgccat tgtccaggta cacgtcgaat ctgtagggct
     1321 tcagggacag cttcacgacc ttgtttctgc tgttggggta gtcgtcggtg atgtccagat
     1381 gggcgttcag tttgttgccg taatacttaa tcttcttgat cacggggccg ttgtcctttt
     1441 tggagtactt ggtcaggtag ttcccggttt cctcgtagta cttgtacagg ggattcttct
     1501 cgtcgccgta ctgttccata atcagcttca gtttctggta ggtctggggg tcgtggtggt
     1561 acatcagcag cttttcgggg ctcttgttga tcagcttttt cagcttgtca ttgtccttgt
     1621 cgtacaggcc gttcagattg ttcacgatca gggtgttgcc cttgtcgtcc ttccgggtgg
     1681 agtacagggt gtcgttaatc agctctctat taggcttctt gtccacccgg tggctgtact
     1741 tgtagtcctt gaagtcctta atgtgcttga tctggtgggg ggtgatgaag atctctttgt
     1801 actcctgctc ggtttcgatc tcgggcatgc tctcggcctg cttttcctcg aacatctggt
     1861 tttccatcac ttttttggcc ttgtccagtt tcttccactc tttgaagatg aaatcggcgt
     1921 tggcaatgat cagggcgtcc tcggcgtggt gcttgtaccc cttgttccgc tctttcttaa
     1981 acttccactt ccgccgcaga aagctggtga agccgccatt gatggacttc actttcacgt
     2041 ccaggttgtt cactctgaag tagctccgca gcaggttcat caggcctctg gtggcgtatc
     2101 tggtatccac caggttccgg ttgatgaagt ctttctgcac ggagaacctg ttgatgtccc
     2161 gttcttccag cagatactct ttcttggtct tgctgattct gcccttgccc ttggccagat
     2221 tcaggatgtg cttcttgaag gtttcgtagc tgatcttgct gtcgctgctg ctcaggtact
     2281 ggaatggggt ccggttgccc ttcttgctgt tttcttcctg cttcacgagc accttgttgt
     2341 tgaagctgtt gtcgaaggac acgcttctgg ggatgatgtg gtccacctca tagttgaagg
     2401 ggttgttcag cagatcttcc agagggatgg cttccaggct gtacaggcac ttgccttcct
     2461 gcatgtcgtg cagcttgatc ttctcgatca ggtacttggc gttctctttg ccggtggtcc
     2521 ggatgatttc ctcgatccgc tcgttggtct gccggttccg cttctgcatc tcgttgatca
     2581 ttttctgggc gtccttggag ttcttctcgc gggccagctc gataatgatg tcgttgggca
     2641 ggccgtactt cttgatgatg gcgttgatca ctttgatgct ctggatgaag cttctcttca
     2701 cgacggggct caggatgaag tcgtccacca gggtggtggg gatctctttc tgctgggaca
     2761 ggtccacctt cttgggcacc agcttcagcc ggttgaagat agcgatctgg ttgtcgttgg
     2821 tgtgccacag ctcgtccagg atcaggttga tggccttcag gctcaggttg tgggtgccgg
     2881 tatagccctt cagattagag atctgctcga tctcttcctg ggtcagctcg gagttcagat
     2941 tggtcagttc ttcctggatg tcctcgctgc tctggtagat ggtcaggatc ttggcaatct
     3001 gatccagcag ctcggcgttc tcaataatct ctttccgggc ggtaatgtcc ttgatgtcgt
     3061 ggtacacctt caggttggtg aactcgggct tgccggtgct ggtcactctg tagcccttaa
     3121 tatcctcttc gttcacgagg atttctttgg cgatctgctt cagggtgggc ttcttcttct
     3181 gcttgaacac gttctcgatg atctggaact tctcgtaata ttccagcttc tcgttctcgt
     3241 ccctggtgat cacgagattg ttcaggtcgt tcagggcgtt gtacaggtcg gcgttgtagg
     3301 cgtacttcac gctccgcagt tcctcgggga agtaggtgca gtggcccatc agcatctcgt
     3361 accattcttt gatgtccttc cagccgaagg ggctgccctc gccaggtccc tcatagtagg
     3421 tccgccgggt ttccagcagg tcgatgtagg tgtcgatgaa gctctggtcc agctggtggt
     3481 aggccttctg caccttcagc agctgtttgg cttctttcac gtagtcgctg gtcttgaatc
     3541 tgttgatgct gccccgcact tcgccgtctt tcttcagccg ttccagctgc agttcggcca
     3601 cgtatttctc ttccagggcc ttgctgttcc ggctgatctg ctctttggtg gacagctcgt
     3661 tgccggtgtc ctcttccacc tcgttcacgt tgtgcacgcc tcttctcttg gccaggtgca
     3721 gcagggcggc agagaactct tcctcgctca gcttctggct caggcccttc actctggcct
     3781 cgtaggggtt gatgccgctc agctcgctgt ggtcggtcag caggttgtag tcgaacagca
     3841 gcttcttcac tctctggatt ctatgccgcc tccgccgctt cagccttctg gcgcctctct
     3901 tgctccgcct gccctcgttg ttttccacgt tggcctcttt gaacagccgc acgccggcat
     3961 cgatcacgtc ccgtgtctcg tagtcgatga tgccgtagcc cacgctggtg atgccgatgt
     4021 ccaggcccag gatgtagttc cgcttggctg ctgggactcc gtggataccg accttccgct
     4081 tcttctttgg ggccatgcgg ccgctgcaga ccggtttaga tctttactag tttaagcttc
     4141 aattggaagt tcctattctt tctagagtat aggaacttcc tgcagtaggc tagcgaagtt
     4201 cctattcctt ttgaagtata ggaacttcgt cgaggggggg cccgagctta tcgatgaatt
     4261 cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     4321 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     4381 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     4441 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggaggtaccg
     4501 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     4561 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     4621 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     4681 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     4741 gaaacaccgt gttcattgac atctggtcag ttttagtact ctggaaacag aatctactaa
     4801 aacaaggcaa aatgccgtgt ttatctcgtc aacttgttgg cgagattttt gcggccgcag
     4861 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     4921 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     4981 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     5041 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     5101 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     5161 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     5221 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     5281 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5341 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     5401 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     5461 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     5521 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     5581 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5641 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5701 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     5761 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5821 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5881 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5941 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     6001 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     6061 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6121 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6181 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6241 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6301 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6361 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6421 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6481 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6541 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6601 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     6661 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6721 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6781 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6841 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6901 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6961 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     7021 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     7081 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     7141 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     7201 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     7261 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7321 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7381 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7441 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7501 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7561 ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct
     7621 cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     7681 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcct
//
