LOCUS       sequence-227329-        7281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one AAV plasmid expressing Nme2Cas9 with sgRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7281)
  AUTHORS   Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, 
            Gallant J, Xue W, Rivera-Perez JA, Sontheimer EJ
  TITLE     A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo 
            Genome Editing.
  JOURNAL   Mol Cell. 2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 
            10.1016/j.molcel.2018.12.003.
  PUBMED    30581144
REFERENCE   2  (bases 1 to 7281)
  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: "Mol Cell. 
            2018 Dec 18. pii: S1097-2765(18)31033-5. doi: 
            10.1016/j.molcel.2018.12.003."
COMMENT     Sequence Label: Nme2Cas9_AAV
FEATURES             Location/Qualifiers
     source          1..7281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..54
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             55..144
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             151..177
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     primer_bind     complement(183..202)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    248..303
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   316..456
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   316..445
                     /label=AAV2 ITR
     rep_origin      531..986
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(618..637)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     828..849
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1003..1022)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1122..1144
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1182..1200)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1268..1372
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1373..1441,1442..2233)
                     /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(1591..1610)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2404..2992
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2893..2912
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   3054..3194
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3065..3194
                     /label=AAV2 ITR
     misc_RNA        complement(3222..3314)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(3365..3613)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3423..3442)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3593..3613)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     regulatory      3949..3958
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3961..3981
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3988..4035
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
ORIGIN
        1 gaggataagc ggcccgcagc aaccaagaag gcaggacagg ccaagaagaa gaagtacccc
       61 tatgacgtgc ctgactacgc cgggtatccc tacgacgtgc ctgattacgc cgggtcctat
      121 ccctacgacg tgccagatta cgctgcagct ccagcagcga agaaaaagaa gctggattaa
      181 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
      241 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
      301 tcactcggcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
      361 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
      421 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
      481 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
      541 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
      601 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
      661 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
      721 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
      781 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
      841 ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt
      901 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
      961 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     1021 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     1081 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     1141 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     1201 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     1261 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1321 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     1381 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     1441 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1501 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     1561 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     1621 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1681 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1741 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1801 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1861 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     1921 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1981 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     2041 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat
     2101 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     2161 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     2221 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     2281 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     2341 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2401 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2461 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2521 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     2581 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2641 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2701 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2761 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2821 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2881 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2941 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3001 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag
     3061 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac
     3121 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     3181 cactaggggt tcctgcggcc tctagagttt aaacaaaaaa ataaacgatg ccccttaaag
     3241 cagaagcttt aaggggcaga gcgttgcggc acatcttttc agacggcctt attgtagcaa
     3301 cggttctcat ttcgtttccg aaatgagaaa gggagctaca actgaagagc tggaatgctc
     3361 ttccggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag
     3421 ttacggtaag catatgatag tccattttaa aacataattt taaaactgca aactacccaa
     3481 gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat
     3541 taattctaat tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg
     3601 gaaataggcc ctcttcctgc ccgaccttga cgtcgactct agaatggagg cggtactatg
     3661 tagatgagaa ttcaggagca aactgggaaa agcaactgct tccaaatatt tgtgattttt
     3721 acagtgtagt tttggaaaaa ctcttagcct accaattctt ctaagtgttt taaaatgtgg
     3781 gagccagtac acatgaagtt atagagtgtt ttaatgaggc ttaaatattt accgtaacta
     3841 tgaaatgcta cgcatatcat gctgttcagg ctccgtggcc acgcaactca tacttaagca
     3901 gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaacaccgc caccatggtg
     3961 cctaagaaga agagaaaggt ggaagataaa cgcccagcag ctacaaagaa ggcaggtcaa
     4021 gccaagaaaa agaaaatggc cgccttcaag cctaacccaa tcaattacat cctgggactg
     4081 gacatcggaa tcgcatccgt gggatgggct atggtggaga tcgacgagga ggagaatcct
     4141 atccggctga tcgatctggg cgtgagagtg tttgagaggg ccgaggtgcc aaagaccggc
     4201 gattctctgg ctatggcccg gagactggca cggagcgtga ggcgcctgac acggagaagg
     4261 gcacacaggc tgctgagggc acgccggctg ctgaagagag agggcgtgct gcaggcagca
     4321 gacttcgatg agaatggcct gatcaagagc ctgccaaaca ccccctggca gctgagagca
     4381 gccgccctgg acaggaagct gacaccactg gagtggtctg ccgtgctgct gcacctgatc
     4441 aagcaccgcg gctacctgag ccagcggaag aacgagggag agacagcaga caaggagctg
     4501 ggcgccctgc tgaagggagt ggccaacaat gcccacgccc tgcagaccgg cgatttcagg
     4561 acacctgccg agctggccct gaataagttt gagaaggagt ccggccacat cagaaaccag
     4621 aggggcgact atagccacac cttctcccgc aaggatctgc aggccgagct gatcctgctg
     4681 ttcgagaagc agaaggagtt tggcaatcca cacgtgagcg gaggcctgaa ggagggaatc
     4741 gagaccctgc tgatgacaca gaggcctgcc ctgtccggcg acgcagtgca gaagatgctg
     4801 ggacactgca ccttcgagcc tgcagagcca aaggccgcca agaacaccta cacagccgag
     4861 cggtttatct ggctgacaaa gctgaacaat ctgagaatcc tggagcaggg atccgagagg
     4921 ccactgaccg acacagagag ggccaccctg atggatgagc cttaccggaa gtctaagctg
     4981 acatatgccc aggccagaaa gctgctgggc ctggaggaca ccgccttctt taagggcctg
     5041 agatacggca aggataatgc cgaggcctcc acactgatgg agatgaaggc ctatcacgcc
     5101 atctctcgcg ccctggagaa ggagggcctg aaggacaaga agtcccccct gaacctgagc
     5161 tccgagctgc aggatgagat cggcaccgcc ttctctctgt ttaagaccga cgaggatatc
     5221 acaggccgcc tgaaggacag ggtgcagcct gagatcctgg aggccctgct gaagcacatc
     5281 tctttcgata agtttgtgca gatcagcctg aaggccctga gaaggatcgt gccactgatg
     5341 gagcagggca agcggtacga cgaggcctgc gccgagatct acggcgatca ctatggcaag
     5401 aagaacacag aggagaagat ctatctgccc cctatccctg ccgacgagat cagaaatcct
     5461 gtggtgctga gggccctgtc ccaggcaaga aaagtgatca acggagtggt gcgccggtac
     5521 ggatctccag cccggatcca catcgagacc gccagagaag tgggcaagag cttcaaggac
     5581 cggaaggaga tcgagaagag acaggaggag aatcgcaagg atcgggagaa ggccgccgcc
     5641 aagtttaggg agtacttccc taactttgtg ggcgagccaa agtctaagga catcctgaag
     5701 ctgcgcctgt acgagcagca gcacggcaag tgtctgtata gcggcaagga gatcaatctg
     5761 gtgcggctga acgagaaggg ctatgtggag atcgatcacg ccctgccttt ctccagaacc
     5821 tgggacgatt cttttaacaa taaggtgctg gtgctgggca gcgagaacca gaataagggc
     5881 aatcagacac catacgagta tttcaatggc aaggacaact ccagggagtg gcaggagttc
     5941 aaggcccgcg tggagacctc tagatttccc aggagcaaga agcagcggat cctgctgcag
     6001 aagttcgacg aggatggctt taaggagtgc aacctgaatg acaccagata cgtgaaccgg
     6061 ttcctgtgcc agtttgtggc cgatcacatc ctgctgaccg gcaagggcaa gagaagggtg
     6121 ttcgcctcta atggccagat cacaaacctg ctgaggggat tttggggact gaggaaggtg
     6181 cgggcagaga atgacagaca ccacgcactg gatgcagtgg tggtggcatg cagcaccgtg
     6241 gcaatgcagc agaagatcac aagattcgtg aggtataagg agatgaacgc ctttgacggc
     6301 aagaccatcg ataaggagac aggcaaggtg ctgcaccaga agacccactt cccccagcct
     6361 tgggagttct ttgcccagga agtgatgatc cgggtgttcg gcaagccaga cggcaagcct
     6421 gagtttgagg aggccgatac cccagagaag ctgaggacac tgctggcaga gaagctgtct
     6481 agcaggccag aggcagtgca cgagtacgtg accccactgt tcgtgtccag ggcacccaat
     6541 cggaagatgt ctggcgccca caaggacaca ctgagaagcg ccaagaggtt tgtgaagcac
     6601 aacgagaaga tctccgtgaa gagagtgtgg ctgaccgaga tcaagctggc cgatctggag
     6661 aacatggtga attacaagaa cggcagggag atcgagctgt atgaggccct gaaggcaagg
     6721 ctggaggcct acggaggaaa tgccaagcag gccttcgacc caaaggataa ccccttttat
     6781 aagaagggag gacagctggt gaaggccgtg cgggtggaga agacccagga gagcggcgtg
     6841 ctgctgaata agaagaacgc ctacacaatc gccgacaatg gcgatatggt gagagtggac
     6901 gtgttctgta aggtggataa gaagggcaag aatcagtact ttatcgtgcc tatctatgcc
     6961 tggcaggtgg ccgagaacat cctgccagac atcgattgca agggctacag aatcgacgat
     7021 agctatacat tctgtttttc cctgcacaag tatgacctga tcgccttcca gaaggatgag
     7081 aagtccaagg tggagtttgc ctactatatc aattgcgact cctctaacgg caggttctac
     7141 ctggcctggc acgataaggg cagcaaggag cagcagtttc gcatctccac ccagaatctg
     7201 gtgctgatcc agaagtatca ggtgaacgag ctgggcaagg agatcaggcc atgtcggctg
     7261 aagaagcgcc cacccgtgcg g
//
