LOCUS       sequence-495340-        6508 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a sgRNA targeting the GRIN2B locus and SauCas9 under 
            the same pol-III H1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6508)
  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 6508)
  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
FEATURES             Location/Qualifiers
     source          1..6508
                     /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   3771..3911
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3771..3900
                     /label=AAV2 ITR
     rep_origin      3986..4441
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4073..4092)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4283..4304
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4458..4477)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4577..4599
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4637..4655)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4723..4827
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4828..4896,4897..5688)
                     /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(5046..5065)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5859..6447
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6348..6367
                     /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 cggtcttctt ttgaagactg agaattctag caataaagga
     3721 tcgtttattt tcattggaag cgtgtgttgg ttttttgatc aggcgcgggc aggaacccct
     3781 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     3841 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     3901 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3961 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4021 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4081 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4141 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4201 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4261 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     4321 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     4381 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     4441 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     4501 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4561 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4621 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4681 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4741 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4801 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4861 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4921 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4981 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5041 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5101 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5161 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5221 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5281 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5341 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5401 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5461 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5521 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
     5581 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5641 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5701 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5761 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5821 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5881 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5941 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6001 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6061 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6121 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6181 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6241 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6301 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6361 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6421 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6481 gccttttgct ggccttttgc tcacatgt
//
