LOCUS       sequence-243680-        7729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis of Gria2.
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-sgGria2
FEATURES             Location/Qualifiers
     source          1..7729
                     /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    complement(644..677)
                     /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(728..761)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(773..853)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(860..907)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(908..4063)
                     /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(4088..4108)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4140..4173
                     /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    4224..4257
                     /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(4282..4299)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4288..4495
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4502..4750
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4502..4522
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4673..4692
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4773..4848
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4862..5002
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4862..4991
                     /label=AAV2 ITR
     rep_origin      5077..5532
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5164..5183)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5374..5395
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5549..5568)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5668..5690
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5728..5746)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5814..5918
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5919..5987,5988..6779)
                     /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(6137..6156)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6950..7538
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7439..7458
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7600..7729
                     /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 cgtcgactcc ggaataactt cgtataggat
      661 actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag caatatgaaa
      721 cctcttaata acttcgtata gcatacatta tacgaagtta tggcgcgcct taagcgtaat
      781 ctggaacatc gtatgggtaa gcgtaatctg gaacatcgta tgggtaagcg taatctggaa
      841 catcgtatgg gtaggatccc tttttctttt ttgcctggcc ggcctttttc gtggccgccg
      901 gccttttgcc ctttttgatg atctgagggt gcttcttaga tttcacttca tacaggttgc
      961 ccagaatgtc tgtgctgtac ttcttaatgc tctgggtctt ggaggcgatt gtcttaatga
     1021 tcctgggggg cctcttgtcg ttcatgtttt ccaggtactc gcggtaggtg atgtcgatca
     1081 tgttcacttc gatccggttc agcaggtcgt tgttcacgcc gatcactcta tacagctcgc
     1141 cgttgatctt gatcagatcg ttgttgtaga aggaggcgat aaactcggcc tggttgctga
     1201 tcttcttcag cttcttagct tcctcatagc acttgctatt cacttcgtag tagttttctt
     1261 ttttgatcac atccagattc ttcacggtca cgaacttgta cacgccattg tccaggtaca
     1321 cgtcgaatct gtagggcttc agggacagct tcacgacctt gtttctgctg ttggggtagt
     1381 cgtcggtgat gtccagatgg gcgttcagtt tgttgccgta atacttaatc ttcttgatca
     1441 cggggccgtt gtcctttttg gagtacttgg tcaggtagtt cccggtttcc tcgtagtact
     1501 tgtacagggg attcttctcg tcgccgtact gttccataat cagcttcagt ttctggtagg
     1561 tctgggggtc gtggtggtac atcagcagct tttcggggct cttgttgatc agctttttca
     1621 gcttgtcatt gtccttgtcg tacaggccgt tcagattgtt cacgatcagg gtgttgccct
     1681 tgtcgtcctt ccgggtggag tacagggtgt cgttaatcag ctctctatta ggcttcttgt
     1741 ccacccggtg gctgtacttg tagtccttga agtccttaat gtgcttgatc tggtgggggg
     1801 tgatgaagat ctctttgtac tcctgctcgg tttcgatctc gggcatgctc tcggcctgct
     1861 tttcctcgaa catctggttt tccatcactt ttttggcctt gtccagtttc ttccactctt
     1921 tgaagatgaa atcggcgttg gcaatgatca gggcgtcctc ggcgtggtgc ttgtacccct
     1981 tgttccgctc tttcttaaac ttccacttcc gccgcagaaa gctggtgaag ccgccattga
     2041 tggacttcac tttcacgtcc aggttgttca ctctgaagta gctccgcagc aggttcatca
     2101 ggcctctggt ggcgtatctg gtatccacca ggttccggtt gatgaagtct ttctgcacgg
     2161 agaacctgtt gatgtcccgt tcttccagca gatactcttt cttggtcttg ctgattctgc
     2221 ccttgccctt ggccagattc aggatgtgct tcttgaaggt ttcgtagctg atcttgctgt
     2281 cgctgctgct caggtactgg aatggggtcc ggttgccctt cttgctgttt tcttcctgct
     2341 tcacgagcac cttgttgttg aagctgttgt cgaaggacac gcttctgggg atgatgtggt
     2401 ccacctcata gttgaagggg ttgttcagca gatcttccag agggatggct tccaggctgt
     2461 acaggcactt gccttcctgc atgtcgtgca gcttgatctt ctcgatcagg tacttggcgt
     2521 tctctttgcc ggtggtccgg atgatttcct cgatccgctc gttggtctgc cggttccgct
     2581 tctgcatctc gttgatcatt ttctgggcgt ccttggagtt cttctcgcgg gccagctcga
     2641 taatgatgtc gttgggcagg ccgtacttct tgatgatggc gttgatcact ttgatgctct
     2701 ggatgaagct tctcttcacg acggggctca ggatgaagtc gtccaccagg gtggtgggga
     2761 tctctttctg ctgggacagg tccaccttct tgggcaccag cttcagccgg ttgaagatag
     2821 cgatctggtt gtcgttggtg tgccacagct cgtccaggat caggttgatg gccttcaggc
     2881 tcaggttgtg ggtgccggta tagcccttca gattagagat ctgctcgatc tcttcctggg
     2941 tcagctcgga gttcagattg gtcagttctt cctggatgtc ctcgctgctc tggtagatgg
     3001 tcaggatctt ggcaatctga tccagcagct cggcgttctc aataatctct ttccgggcgg
     3061 taatgtcctt gatgtcgtgg tacaccttca ggttggtgaa ctcgggcttg ccggtgctgg
     3121 tcactctgta gcccttaata tcctcttcgt tcacgaggat ttctttggcg atctgcttca
     3181 gggtgggctt cttcttctgc ttgaacacgt tctcgatgat ctggaacttc tcgtaatatt
     3241 ccagcttctc gttctcgtcc ctggtgatca cgagattgtt caggtcgttc agggcgttgt
     3301 acaggtcggc gttgtaggcg tacttcacgc tccgcagttc ctcggggaag taggtgcagt
     3361 ggcccatcag catctcgtac cattctttga tgtccttcca gccgaagggg ctgccctcgc
     3421 caggtccctc atagtaggtc cgccgggttt ccagcaggtc gatgtaggtg tcgatgaagc
     3481 tctggtccag ctggtggtag gccttctgca ccttcagcag ctgtttggct tctttcacgt
     3541 agtcgctggt cttgaatctg ttgatgctgc cccgcacttc gccgtctttc ttcagccgtt
     3601 ccagctgcag ttcggccacg tatttctctt ccagggcctt gctgttccgg ctgatctgct
     3661 ctttggtgga cagctcgttg ccggtgtcct cttccacctc gttcacgttg tgcacgcctc
     3721 ttctcttggc caggtgcagc agggcggcag agaactcttc ctcgctcagc ttctggctca
     3781 ggcccttcac tctggcctcg taggggttga tgccgctcag ctcgctgtgg tcggtcagca
     3841 ggttgtagtc gaacagcagc ttcttcactc tctggattct atgccgcctc cgccgcttca
     3901 gccttctggc gcctctcttg ctccgcctgc cctcgttgtt ttccacgttg gcctctttga
     3961 acagccgcac gccggcatcg atcacgtccc gtgtctcgta gtcgatgatg ccgtagccca
     4021 cgctggtgat gccgatgtcc aggcccagga tgtagttccg cttggctgct gggactccgt
     4081 ggataccgac cttccgcttc ttctttgggg ccatgcggcc gctgcagacc ggtgctagca
     4141 taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt
     4201 tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg aagttatgaa
     4261 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     4321 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     4381 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     4441 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggaggtac
     4501 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     4561 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     4621 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     4681 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     4741 acgaaacacc gtatttccaa ggggcgctga tcgttttagt actctggaaa cagaatctac
     4801 taaaacaagg caaaatgccg tgtttatctc gtcaacttgt tggcgagatt tttgcggccg
     4861 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     4921 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     4981 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5041 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     5101 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     5161 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     5221 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5281 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5341 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5401 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     5461 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5521 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     5581 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5641 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5701 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     5761 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5821 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5881 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5941 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6001 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6061 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6121 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6181 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6241 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6301 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6361 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6421 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6481 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6541 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6601 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     6661 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6721 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6781 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6841 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6901 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6961 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7021 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7081 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7141 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7201 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7261 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7321 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7381 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7441 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7501 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7561 tacggttcct ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc
     7621 gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc
     7681 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcct
//
