LOCUS       sequence-495341-        7338 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a sgRNA targeting the GRIN2B locus, a SauCas9 and an 
            AcrIIA5-cpGR2 hybrid (insertion before D41) linked through a P2A 
            sequence, under the same pol-III H1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7338)
  AUTHORS   Brenker L, Aschenbrenner S, Bubeck F, Staykov K, Gebhardt C, Wolf B,
            Jendrusch M, Kroll AS, Becker J, Ambiel I, Fackler OT, Grimm D, 
            Mathony J, Niopek D
  TITLE     A versatile anti-CRISPR platform for opto- and chemogenetic control 
            of CRISPR-Cas9 and Cas12 across a wide range of orthologs.
  JOURNAL   Nucleic Acids Res. 2025 Jul 19;53(14):gkaf752. doi: 
            10.1093/nar/gkaf752.
  PUBMED    40794863
REFERENCE   2  (bases 1 to 7338)
  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: "Nucleic 
            Acids Res."; date: "2025-07-19"; volume: "53(14):gkaf752. doi"; 
            pages: " 10.1093/nar/gkaf752"
COMMENT     Sequence Label: H1_sgRNA(GRIN2B)_SauCas9_AcrIIA5-AsLOV2(D41)
FEATURES             Location/Qualifiers
     source          1..7338
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     158..177
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        269..344
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     regulatory      359..368
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             371..391
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             416..3571
                     /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             3572..3619
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3626..3682
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     repeat_region   4601..4741
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4601..4730
                     /label=AAV2 ITR
     rep_origin      4816..5271
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4903..4922)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5113..5134
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5288..5307)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5407..5429
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5467..5485)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5553..5657
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5658..5726,5727..6518)
                     /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(5876..5895)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6689..7277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7178..7197
                     /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 gactccatat ttgcatgtcg ctatgtgttc tgggaaatca
      181 ccataaacgt gaaatgtctt tggatttggg aatcttataa gttctgtatg aggaccacgg
      241 tacaccgaga gtaggctggt agatggaggt tttagtactc tggaaacaga atctactaaa
      301 acaaggcaaa atgccgtgtt tatctcgtca acttgttggc gagatttttt ccaccggtgc
      361 caccatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccaagcg
      421 gaactacatc ctgggcctgg acatcggcat caccagcgtg ggctacggca tcatcgacta
      481 cgagacacgg gacgtgatcg atgccggcgt gcggctgttc aaagaggcca acgtggaaaa
      541 caacgagggc aggcggagca agagaggcgc cagaaggctg aagcggcgga ggcggcatag
      601 aatccagaga gtgaagaagc tgctgttcga ctacaacctg ctgaccgacc acagcgagct
      661 gagcggcatc aacccctacg aggccagagt gaagggcctg agccagaagc tgagcgagga
      721 agagttctct gccgccctgc tgcacctggc caagagaaga ggcgtgcaca acgtgaacga
      781 ggtggaagag gacaccggca acgagctgtc caccaaagag cagatcagcc ggaacagcaa
      841 ggccctggaa gagaaatacg tggccgaact gcagctggaa cggctgaaga aagacggcga
      901 agtgcggggc agcatcaaca gattcaagac cagcgactac gtgaaagaag ccaaacagct
      961 gctgaaggtg cagaaggcct accaccagct ggaccagagc ttcatcgaca cctacatcga
     1021 cctgctggaa acccggcgga cctactatga gggacctggc gagggcagcc ccttcggctg
     1081 gaaggacatc aaagaatggt acgagatgct gatgggccac tgcacctact tccccgagga
     1141 actgcggagc gtgaagtacg cctacaacgc cgacctgtac aacgccctga acgacctgaa
     1201 caatctcgtg atcaccaggg acgagaacga gaagctggaa tattacgaga agttccagat
     1261 catcgagaac gtgttcaagc agaagaagaa gcccaccctg aagcagatcg ccaaagaaat
     1321 cctcgtgaac gaagaggata ttaagggcta cagagtgacc agcaccggca agcccgagtt
     1381 caccaacctg aaggtgtacc acgacatcaa ggacattacc gcccggaaag agattattga
     1441 gaacgccgag ctgctggatc agattgccaa gatcctgacc atctaccaga gcagcgagga
     1501 catccaggaa gaactgacca atctgaactc cgagctgacc caggaagaga tcgagcagat
     1561 ctctaatctg aagggctata ccggcaccca caacctgagc ctgaaggcca tcaacctgat
     1621 cctggacgag ctgtggcaca ccaacgacaa ccagatcgct atcttcaacc ggctgaagct
     1681 ggtgcccaag aaggtggacc tgtcccagca gaaagagatc cccaccaccc tggtggacga
     1741 cttcatcctg agccccgtcg tgaagagaag cttcatccag agcatcaaag tgatcaacgc
     1801 catcatcaag aagtacggcc tgcccaacga catcattatc gagctggccc gcgagaagaa
     1861 ctccaaggac gcccagaaaa tgatcaacga gatgcagaag cggaaccggc agaccaacga
     1921 gcggatcgag gaaatcatcc ggaccaccgg caaagagaac gccaagtacc tgatcgagaa
     1981 gatcaagctg cacgacatgc aggaaggcaa gtgcctgtac agcctggaag ccatccctct
     2041 ggaagatctg ctgaacaacc ccttcaacta tgaggtggac cacatcatcc ccagaagcgt
     2101 gtccttcgac aacagcttca acaacaaggt gctcgtgaag caggaagaaa acagcaagaa
     2161 gggcaaccgg accccattcc agtacctgag cagcagcgac agcaagatca gctacgaaac
     2221 cttcaagaag cacatcctga atctggccaa gggcaagggc agaatcagca agaccaagaa
     2281 agagtatctg ctggaagaac gggacatcaa caggttctcc gtgcagaaag acttcatcaa
     2341 ccggaacctg gtggatacca gatacgccac cagaggcctg atgaacctgc tgcggagcta
     2401 cttcagagtg aacaacctgg acgtgaaagt gaagtccatc aatggcggct tcaccagctt
     2461 tctgcggcgg aagtggaagt ttaagaaaga gcggaacaag gggtacaagc accacgccga
     2521 ggacgccctg atcattgcca acgccgattt catcttcaaa gagtggaaga aactggacaa
     2581 ggccaaaaaa gtgatggaaa accagatgtt cgaggaaaag caggccgaga gcatgcccga
     2641 gatcgaaacc gagcaggagt acaaagagat cttcatcacc ccccaccaga tcaagcacat
     2701 taaggacttc aaggactaca agtacagcca ccgggtggac aagaagccta atagagagct
     2761 gattaacgac accctgtact ccacccggaa ggacgacaag ggcaacaccc tgatcgtgaa
     2821 caatctgaac ggcctgtacg acaaggacaa tgacaagctg aaaaagctga tcaacaagag
     2881 ccccgaaaag ctgctgatgt accaccacga cccccagacc taccagaaac tgaagctgat
     2941 tatggaacag tacggcgacg agaagaatcc cctgtacaag tactacgagg aaaccgggaa
     3001 ctacctgacc aagtactcca aaaaggacaa cggccccgtg atcaagaaga ttaagtatta
     3061 cggcaacaaa ctgaacgccc atctggacat caccgacgac taccccaaca gcagaaacaa
     3121 ggtcgtgaag ctgtccctga agccctacag attcgacgtg tacctggaca atggcgtgta
     3181 caagttcgtg accgtgaaga atctggatgt gatcaaaaaa gaaaactact acgaagtgaa
     3241 tagcaagtgc tatgaggaag ctaagaagct gaagaagatc agcaaccagg ccgagtttat
     3301 cgcctccttc tacaacaacg atctgatcaa gatcaacggc gagctgtata gagtgatcgg
     3361 cgtgaacaac gacctgctga accggatcga agtgaacatg atcgacatca cctaccgcga
     3421 gtacctggaa aacatgaacg acaagaggcc ccccaggatc attaagacaa tcgcctccaa
     3481 gacccagagc attaagaagt acagcacaga cattctgggc aacctgtatg aagtgaaatc
     3541 taagaagcac cctcagatca tcaaaaaggg caaaaggccg gcggccacga aaaaggccgg
     3601 ccaggcaaaa aagaaaaagg gatccgctac aaactttagc cttctcaaac aagcaggaga
     3661 tgtcgaagag aatcctggcc cggcatacgg taaaagccga tataactctt accggaaacg
     3721 aaattttagt atcagtgaca accagagaag agaatacgcc aaaaaaatga aggaattgga
     3781 gcaagcgttt gagaatttgc ttgctacaac tttggagcgg attgagaaga atttcgtcat
     3841 aactgatccc aggttgcctg ataatcctat aatattcgcc tccgatagtt ttcttcaact
     3901 tacggagtac tccagagagg agatactggg aaggaattgc agattcctcc aaggtccaga
     3961 aactgataga gctactgtga ggaagatacg ggatgccatt gacaatcaaa ctgaagtgac
     4021 agtgcaactt atcaattaca ccaagtcagg gaaaaaattc tggaacctgt tccatttgca
     4081 acccatgagg gaccaaaagg gcgatgtgca atatttcata ggcgttcaac ttgatggtac
     4141 tgaacacgtc agagatgcgg cggagcgaga aggagtgatg ctgattaaga aaacagcaga
     4201 gaatatcgac gaagcggcga aagagctgga cgggtggtac ctctcatcca tgaaggatag
     4261 cgcctacaaa gacttcggaa agtacgagat aagactgtca aaccacagtg cagacaatag
     4321 gtatcacgac cttgaaaatg gtcgccttat cgttaatgta aaggcttcaa aacttaattt
     4381 cgttgatatt atagagaaca agctgggcaa aattattgag aagatcgata cgcttgattt
     4441 ggataagtac agattcatta acgctactaa actggaaagg gatataaaat gttactataa
     4501 ggggtataaa acaaaaaagg atgttatctg agaattctag caataaagga tcgtttattt
     4561 tcattggaag cgtgtgttgg ttttttgatc aggcgcgggc aggaacccct agtgatggag
     4621 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     4681 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     4741 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     4801 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4861 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     4921 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4981 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     5041 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5101 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     5161 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     5221 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     5281 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     5341 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5401 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     5461 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     5521 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5581 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5641 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5701 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5761 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5821 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5881 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5941 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     6001 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     6061 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6121 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6181 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6241 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6301 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6361 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6421 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6481 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6541 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6601 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6661 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6721 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6781 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6841 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6901 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6961 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     7021 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     7081 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7141 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7201 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7261 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7321 ggccttttgc tcacatgt
//
