LOCUS       sequence-482051-        7525 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GfaABC1D promoter driven HA-tagged SaCas9 together with U6 driven 
            expression of sgRNA targeting exon-1 of mouse Ezrin; for 
            knocking-out ezrin in murine astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7525)
  AUTHORS   Badia-Soteras A, Heistek TS, Kater MSJ, Mak A, Negrean A, van den 
            Oever MC, Mansvelder HD, Khakh BS, Min R, Smit AB, Verheijen MHG
  TITLE     Retraction of Astrocyte Leaflets From the Synapse Enhances Fear 
            Memory.
  JOURNAL   Biol Psychiatry. 2023 Aug 1;94(3):226-238. doi: 
            10.1016/j.biopsych.2022.10.013. Epub 2022 Oct 29.
  PUBMED    36702661
REFERENCE   2  (bases 1 to 7525)
  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.biopsych.2022.10.013"; journalName: "Biol Psychiatry"; 
            date: "2023-08-1- 1"; volume: "94"; issue: "3"; pages: "226-238"
COMMENT     Sequence Label: pAAV- GfaABC1D::Cas9-HA-sgRNA-Ezr-seq2
FEATURES             Location/Qualifiers
     source          1..7525
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      834..843
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             846..866
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             891..4046
                     /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             4047..4094
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4101..4181
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4209..4226)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4215..4422
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4429..4677
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4429..4449
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4600..4619
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4699..4774
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4788..4928
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4788..4917
                     /label=AAV2 ITR
     rep_origin      5003..5458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5090..5109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5300..5321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5475..5494)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5594..5616
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5654..5672)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5740..5844
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5845..5913,5914..6705)
                     /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"
     rep_origin      6876..7464
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7365..7384
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgagga acatatcctg gtgtggagta ggggacgctg
      181 ctctgacaga ggctcggggg cctgagctgg ctctgtgagc tggggaggag gcagacagcc
      241 aggccttgtc tgcaagcaga cctggcagca ttgggctggc cgccccccag ggcctcctct
      301 tcatgcccag tgaatgactc accttggcac agacacaatg ttcggggtgg gcacagtgcc
      361 tgcttcccgc cgcaccccag cccccctcaa atgccttccg agaagcccat tgagcagggg
      421 gcttgcattg caccccagcc tgacagcctg gcatcttggg ataaaagcag cacagccccc
      481 taggggctgc ccttgctgtg tggcgccacc ggcggtggag aacaaggctc tattcagcct
      541 gtgcccagga aaggggatca ggggatgccc aggcatggac agtgggtggc agggggggag
      601 aggagggctg tctgcttccc agaagtccaa ggacacaaat gggtgagggg agagctctcc
      661 ccatagctgg gctgcggccc aaccccaccc cctcaggcta tgccaggggg tgttgccagg
      721 ggcacccggg catcgccagt ctagcccact ccttcataaa gccctcgcat cccaggagcg
      781 agcagagcca gagcaggttg gagaggagac gcatcacctc cgctgctcgc ggtgccacca
      841 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc aagcggaact
      901 acatcctggg cctggacatc ggcatcacca gcgtgggcta cggcatcatc gactacgaga
      961 cacgggacgt gatcgatgcc ggcgtgcggc tgttcaaaga ggccaacgtg gaaaacaacg
     1021 agggcaggcg gagcaagaga ggcgccagaa ggctgaagcg gcggaggcgg catagaatcc
     1081 agagagtgaa gaagctgctg ttcgactaca acctgctgac cgaccacagc gagctgagcg
     1141 gcatcaaccc ctacgaggcc agagtgaagg gcctgagcca gaagctgagc gaggaagagt
     1201 tctctgccgc cctgctgcac ctggccaaga gaagaggcgt gcacaacgtg aacgaggtgg
     1261 aagaggacac cggcaacgag ctgtccacca aagagcagat cagccggaac agcaaggccc
     1321 tggaagagaa atacgtggcc gaactgcagc tggaacggct gaagaaagac ggcgaagtgc
     1381 ggggcagcat caacagattc aagaccagcg actacgtgaa agaagccaaa cagctgctga
     1441 aggtgcagaa ggcctaccac cagctggacc agagcttcat cgacacctac atcgacctgc
     1501 tggaaacccg gcggacctac tatgagggac ctggcgaggg cagccccttc ggctggaagg
     1561 acatcaaaga atggtacgag atgctgatgg gccactgcac ctacttcccc gaggaactgc
     1621 ggagcgtgaa gtacgcctac aacgccgacc tgtacaacgc cctgaacgac ctgaacaatc
     1681 tcgtgatcac cagggacgag aacgagaagc tggaatatta cgagaagttc cagatcatcg
     1741 agaacgtgtt caagcagaag aagaagccca ccctgaagca gatcgccaaa gaaatcctcg
     1801 tgaacgaaga ggatattaag ggctacagag tgaccagcac cggcaagccc gagttcacca
     1861 acctgaaggt gtaccacgac atcaaggaca ttaccgcccg gaaagagatt attgagaacg
     1921 ccgagctgct ggatcagatt gccaagatcc tgaccatcta ccagagcagc gaggacatcc
     1981 aggaagaact gaccaatctg aactccgagc tgacccagga agagatcgag cagatctcta
     2041 atctgaaggg ctataccggc acccacaacc tgagcctgaa ggccatcaac ctgatcctgg
     2101 acgagctgtg gcacaccaac gacaaccaga tcgctatctt caaccggctg aagctggtgc
     2161 ccaagaaggt ggacctgtcc cagcagaaag agatccccac caccctggtg gacgacttca
     2221 tcctgagccc cgtcgtgaag agaagcttca tccagagcat caaagtgatc aacgccatca
     2281 tcaagaagta cggcctgccc aacgacatca ttatcgagct ggcccgcgag aagaactcca
     2341 aggacgccca gaaaatgatc aacgagatgc agaagcggaa ccggcagacc aacgagcgga
     2401 tcgaggaaat catccggacc accggcaaag agaacgccaa gtacctgatc gagaagatca
     2461 agctgcacga catgcaggaa ggcaagtgcc tgtacagcct ggaagccatc cctctggaag
     2521 atctgctgaa caaccccttc aactatgagg tggaccacat catccccaga agcgtgtcct
     2581 tcgacaacag cttcaacaac aaggtgctcg tgaagcagga agaaaacagc aagaagggca
     2641 accggacccc attccagtac ctgagcagca gcgacagcaa gatcagctac gaaaccttca
     2701 agaagcacat cctgaatctg gccaagggca agggcagaat cagcaagacc aagaaagagt
     2761 atctgctgga agaacgggac atcaacaggt tctccgtgca gaaagacttc atcaaccgga
     2821 acctggtgga taccagatac gccaccagag gcctgatgaa cctgctgcgg agctacttca
     2881 gagtgaacaa cctggacgtg aaagtgaagt ccatcaatgg cggcttcacc agctttctgc
     2941 ggcggaagtg gaagtttaag aaagagcgga acaaggggta caagcaccac gccgaggacg
     3001 ccctgatcat tgccaacgcc gatttcatct tcaaagagtg gaagaaactg gacaaggcca
     3061 aaaaagtgat ggaaaaccag atgttcgagg aaaagcaggc cgagagcatg cccgagatcg
     3121 aaaccgagca ggagtacaaa gagatcttca tcacccccca ccagatcaag cacattaagg
     3181 acttcaagga ctacaagtac agccaccggg tggacaagaa gcctaataga gagctgatta
     3241 acgacaccct gtactccacc cggaaggacg acaagggcaa caccctgatc gtgaacaatc
     3301 tgaacggcct gtacgacaag gacaatgaca agctgaaaaa gctgatcaac aagagccccg
     3361 aaaagctgct gatgtaccac cacgaccccc agacctacca gaaactgaag ctgattatgg
     3421 aacagtacgg cgacgagaag aatcccctgt acaagtacta cgaggaaacc gggaactacc
     3481 tgaccaagta ctccaaaaag gacaacggcc ccgtgatcaa gaagattaag tattacggca
     3541 acaaactgaa cgcccatctg gacatcaccg acgactaccc caacagcaga aacaaggtcg
     3601 tgaagctgtc cctgaagccc tacagattcg acgtgtacct ggacaatggc gtgtacaagt
     3661 tcgtgaccgt gaagaatctg gatgtgatca aaaaagaaaa ctactacgaa gtgaatagca
     3721 agtgctatga ggaagctaag aagctgaaga agatcagcaa ccaggccgag tttatcgcct
     3781 ccttctacaa caacgatctg atcaagatca acggcgagct gtatagagtg atcggcgtga
     3841 acaacgacct gctgaaccgg atcgaagtga acatgatcga catcacctac cgcgagtacc
     3901 tggaaaacat gaacgacaag aggcccccca ggatcattaa gacaatcgcc tccaagaccc
     3961 agagcattaa gaagtacagc acagacattc tgggcaacct gtatgaagtg aaatctaaga
     4021 agcaccctca gatcatcaaa aagggcaaaa ggccggcggc cacgaaaaag gccggccagg
     4081 caaaaaagaa aaagggatcc tacccatacg atgttccaga ttacgcttac ccatacgatg
     4141 ttccagatta cgcttaccca tacgatgttc cagattacgc ttaagaattc ctagagctcg
     4201 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     4261 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     4321 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     4381 caagggggag gattgggaag agaatagcag gcatgctggg gaggtaccga gggcctattt
     4441 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
     4501 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     4561 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     4621 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgcc
     4681 gtcgcctccg ccgtacaggt tttagtactc tggaaacaga atctactaaa acaaggcaaa
     4741 atgccgtgtt tatctcgtca acttgttggc gagatttttg cggccgcagg aacccctagt
     4801 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     4861 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     4921 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4981 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5041 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     5101 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5161 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     5221 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5281 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     5341 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     5401 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     5461 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     5521 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5581 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5641 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     5701 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5761 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5821 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5881 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5941 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6001 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6061 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6121 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6181 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6241 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6301 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6361 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6421 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6481 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6541 tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag
     6601 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6661 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6721 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6781 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6841 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6901 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6961 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     7021 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     7081 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7141 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7201 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7261 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7321 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7381 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7441 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7501 ttttgctggc cttttgctca catgt
//
