LOCUS       sequence-211122-        7278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Delivery of human-codon-optimized Cas9 from Neisseria meningitidis 
            (NmeCas9) and its single-guide RNA in a single AAV vector for in 
            vivo genome editing..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7278)
  AUTHORS   Ibraheim R, Song CQ, Mir A, Amrani N, Xue W, Sontheimer EJ
  TITLE     All-in-one adeno-associated virus delivery and genome editing by 
            Neisseria meningitidis Cas9 in vivo.
  JOURNAL   Genome Biol. 2018 Sep 19;19(1):137. doi: 10.1186/s13059-018-1515-0.
  PUBMED    30231914
REFERENCE   2  (bases 1 to 7278)
  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: "Genome 
            Biol."; date: "2018-09-19"; volume: "19(1):137. doi"; pages: " 
            10.1186/s13059-018-1515-0"
COMMENT     Sequence Label: pEJS654 All-in-One AAV-sgRNA-hNmeCas9
FEATURES             Location/Qualifiers
     source          1..7278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     misc_RNA        complement(169..261)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(312..560)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(370..389)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(540..560)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     regulatory      896..905
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             908..928
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             935..982
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             983..4225
                     /codon_start=1
                     /product="Cas9 endonuclease from the Neisseria meningitidis
                     Type II CRISPR/Cas system (Esvelt et al., 2013)"
                     /label=NmCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="AAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVF
                     ERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKS
                     LPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVAG
                     NAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFG
                     NPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTK
                     LNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDN
                     AEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK
                     DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTE
                     EKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKE
                     IEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGR
                     LNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFK
                     ARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRV
                     FASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFD
                     GKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAEK
                     LSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDL
                     EKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKT
                     GVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQL
                     IDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEG
                     IGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     CDS             4232..4279
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4280..4369
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             4376..4402
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     primer_bind     complement(4408..4427)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4473..4528
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   4541..4681
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4541..4670
                     /label=AAV2 ITR
     rep_origin      4756..5211
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4843..4862)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5053..5074
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5228..5247)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5407..5425)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5493..5597
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5598..5666,5667..6458)
                     /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(5816..5835)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6629..7217
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7118..7137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggcctct agagtttaaa caaaaaaata aacgatgccc
      181 cttaaagcag aagctttaag gggcagagcg ttgcggcaca tcttttcaga cggccttatt
      241 gtagcaacgg ttctcatttc gtttccgaaa tgagaaaggg agctacaact gaagagctgg
      301 aatgctcttc cggtgtttcg tcctttccac aagatatata aagccaagaa atcgaaatac
      361 tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa aactgcaaac
      421 tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt tgtgtttaca
      481 gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa atatgaagga
      541 atcatgggaa ataggccctc ttcctgcccg accttgacgt cgactctaga atggaggcgg
      601 tactatgtag atgagaattc aggagcaaac tgggaaaagc aactgcttcc aaatatttgt
      661 gatttttaca gtgtagtttt ggaaaaactc ttagcctacc aattcttcta agtgttttaa
      721 aatgtgggag ccagtacaca tgaagttata gagtgtttta atgaggctta aatatttacc
      781 gtaactatga aatgctacgc atatcatgct gttcaggctc cgtggccacg caactcatac
      841 ttaagcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga acaccgccac
      901 catggtgcct aagaagaaga gaaaggtgga agataaacgc ccagcagcta caaagaaggc
      961 aggtcaagcc aagaaaaaga aagcagcatt caagccaaac tcaatcaatt acatcctggg
     1021 actggacatc ggcatcgcat ccgtcgggtg ggctatggtc gaaatcgacg aggaggagaa
     1081 ccccatccgc ctgatcgatc tgggcgtgcg cgtgtttgag agggcagagg tgcctaagac
     1141 cggcgacagc ctggccatgg cacggagact ggcacgctcc gtgaggcgcc tgacccggag
     1201 aagggcccac agactgctga ggacacgccg gctgctgaag agggagggcg tgctgcaggc
     1261 cgccaacttc gatgagaatg gcctgatcaa gtccctgccc aatacccctt ggcagctgag
     1321 ggcagccgcc ctggaccgca agctgacacc tctggagtgg tccgccgtgc tgctgcacct
     1381 gatcaagcac cggggctacc tgtctcagag aaagaacgag ggcgagacag ccgataagga
     1441 gctgggcgcc ctgctgaagg gagtggcagg aaatgcacac gccctgcaga ccggcgactt
     1501 tcgcacacca gccgagctgg ccctgaacaa gttcgagaag gagagcggcc acatccgcaa
     1561 tcagcggtct gactatagcc acaccttctc ccggaaggat ctgcaggccg agctgatcct
     1621 gctgtttgag aagcagaagg agttcggcaa cccacacgtg tctggcggcc tgaaggaggg
     1681 catcgagaca ctgctgatga cacagcggcc cgccctgagc ggcgacgcag tgcagaagat
     1741 gctgggacac tgcacctttg agccagccga gcccaaggcc gccaagaata cctacacagc
     1801 cgagcggttc atctggctga caaagctgaa caatctgagg atcctggagc agggaagcga
     1861 gcgcccactg accgacacag agagggccac cctgatggat gagccctacc gcaagtccaa
     1921 gctgacatat gcacaggcaa ggaagctgct gggcctggag gacaccgcct tctttaaggg
     1981 cctgagatac ggcaaggata acgccgaggc ctctacactg atggagatga aggcctatca
     2041 cgccatcagc agggccctgg agaaggaggg cctgaaggac aagaagtccc cactgaatct
     2101 gtctcccgag ctgcaggatg agatcggcac cgcctttagc ctgttcaaga ccgacgagga
     2161 tatcacaggc agactgaagg acaggatcca gccagagatc ctggaggccc tgctgaagca
     2221 catcagcttt gataagttcg tgcagatcag cctgaaggcc ctgcggagga tcgtgccact
     2281 gatggagcag ggcaagaggt acgacgaggc ctgcgccgaa atctacggcg atcactatgg
     2341 caagaagaac acagaggaga aaatctacct gccccctatc cccgccgatg agatcaggaa
     2401 ccctgtggtg ctgcgcgccc tgtctcaggc aagaaaagtg atcaacggag tggtgcgccg
     2461 gtacggcagc cccgccagaa tccacatcga gacagccagg gaagtgggca agtcctttaa
     2521 ggacagaaag gagatcgaga agaggcagga ggagaacaga aaggataggg agaaggccgc
     2581 cgccaagttc agagagtact ttcctaattt cgtgggcgag ccaaagtcca aggatatcct
     2641 gaagctgagg ctgtacgagc agcagcacgg caagtgtctg tattctggca aggagatcaa
     2701 cctgggccgc ctgaatgaga agggctatgt ggagatcgac cacgccctgc ctttttctcg
     2761 gacctgggac gatagcttca acaataaggt gctggtgctg ggctctgaga accagaataa
     2821 gggcaaccag acaccctacg agtatttcaa cggcaaggac aatagccgcg agtggcagga
     2881 gtttaaggca agggtggaga caagcaggtt ccctcggtcc aagaagcaga gaatcctgct
     2941 gcagaagttt gacgaggatg gcttcaagga gaggaacctg aatgacaccc gctacgtgaa
     3001 tcggtttctg tgccagttcg tggccgatag aatgaggctg accggcaagg gcaagaagag
     3061 agtgtttgcc tccaacggcc agatcacaaa tctgctgagg ggcttctggg gcctgagaaa
     3121 ggtgagggca gagaacgaca ggcaccacgc actggatgca gtggtggtgg catgttctac
     3181 cgtggccatg cagcagaaga tcacacgctt tgtgcggtat aaggagatga atgccttcga
     3241 cggcaagacc atcgataagg agacaggcga ggtgctgcac cagaagacac actttcctca
     3301 gccatgggag ttctttgccc aggaagtgat gatccgggtg tttggcaagc ctgacggcaa
     3361 gccagagttc gaggaggccg ataccctgga gaagctgaga acactgctgg cagagaagct
     3421 gagctccagg cccgaggcag tgcacgagta cgtgacccca ctgttcgtgt ctagagcccc
     3481 caacaggaag atgagcggcc agggccacat ggagacagtg aagtccgcca agagactgga
     3541 cgagggcgtg tctgtgctga gggtgcctct gacacagctg aagctgaagg atctggagaa
     3601 gatggtgaat cgcgagcggg agccaaagct gtatgaggcc ctgaaggcaa ggctggaggc
     3661 acacaaggac gatcctgcca aggcctttgc cgagccattc tacaagtatg ataaggccgg
     3721 caacagaacc cagcaggtga aggccgtgag ggtggagcag gtgcagaaga caggcgtgtg
     3781 ggtgcgcaac cacaatggca tcgccgacaa tgctaccatg gtgcgggtgg acgtgtttga
     3841 gaagggcgat aagtactatc tggtgcccat ctacagctgg caggtggcca agggcatcct
     3901 gcctgataga gccgtggtgc agggcaagga cgaggaggat tggcagctga tcgacgattc
     3961 cttcaacttt aagttctctc tgcaccccaa tgacctggtg gaagtgatca ccaagaaggc
     4021 caggatgttt ggctacttcg cctcctgcca ccgcggcaca ggcaacatca atatccggat
     4081 ccacgacctg gatcacaaga tcggcaagaa cggcatcctg gagggcatcg gcgtgaagac
     4141 agccctgagc ttccagaagt atcagatcga cgagctgggc aaggagatca gaccttgtag
     4201 gctgaagaag cgcccacccg tgcgggagga taagcggccc gcagcaacca agaaggcagg
     4261 acaggccaag aagaagaagt acccctatga cgtgcctgac tacgccgggt atccctacga
     4321 cgtgcctgat tacgccgggt cctatcccta cgacgtgcca gattacgctg cagctccagc
     4381 agcgaagaaa aagaagctgg attaagatct ttttccctct gccaaaaatt atggggacat
     4441 catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat
     4501 agtgtgttgg aattttttgt gtctctcact cggcggccgc aggaacccct agtgatggag
     4561 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     4621 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     4681 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     4741 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4801 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4861 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4921 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     4981 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5041 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct
     5101 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5161 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     5221 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     5281 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5341 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     5401 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     5461 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5521 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5581 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5641 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5701 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5761 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5821 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5881 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5941 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     6001 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6061 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6121 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6181 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6241 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6301 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6361 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6421 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6481 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6541 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6601 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6661 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6721 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     6781 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6841 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6901 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6961 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     7021 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7081 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7141 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7201 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7261 ggccttttgc tcacatgt
//
