LOCUS       sequence-311973-        7276 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Delivery of dual sgRNA cassettes and Nme2Cas9 in a single AAV 
            vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7276)
  AUTHORS   Ibraheim R, Tai PWL, Mir A, Javeed N, Wang J, Rodriguez TC, Namkung 
            S, Nelson S, Khokhar ES, Mintzer E, Maitland S, Chen Z, Cao Y, 
            Tsagkaraki E, Wolfe SA, Wang D, Pai AA, Xue W, Gao G, Sontheimer EJ
  TITLE     Self-inactivating, all-in-one AAV vectors for precision Cas9 genome 
            editing via homology-directed repair in vivo.
  JOURNAL   Nat Commun. 2021 Nov 1;12(1):6267. doi: 10.1038/s41467-021-26518-y.
  PUBMED    34725353
REFERENCE   2  (bases 1 to 7276)
  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: "Nat 
            Commun."; date: "2021-11-1"; volume: "12(1):6267. doi"; pages: " 
            10.1038/s41467-021-26518-y"
COMMENT     Sequence Label: pEJS1096 Dual-sgRNA.Design 1
FEATURES             Location/Qualifiers
     source          1..7276
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        complement(277..525)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(683..931)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     regulatory      1250..1259
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1262..1282
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4541..4588
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     repeat_region   4652..4792
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4652..4781
                     /label=AAV2 ITR
     rep_origin      4867..5322
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4954..4973)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5164..5185
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5339..5358)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5491..5595
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5596..5664,5665..6456)
                     /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(5814..5833)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6627..7215
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7116..7135
                     /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 gagtttaaac aaaaaaataa acgatgcccc ttaaagcaga
      181 agctttaagg ggcagagcgt tgcggcacat cttttcagac ggccttattg tagcaacgtt
      241 tcgggagcta caactgaaga gctggaatgc tcttccggtg tttcgtcctt tccacaagat
      301 atataaagcc aagaaatcga aatactttca agttacggta agcatatgat agtccatttt
      361 aaaacataat tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt
      421 ttgtactaat atctttgtgt ttacagtcaa attaattcta attatctctc taacagcctt
      481 gtatcgtata tgcaaatatg aaggaatcat gggaaatagg ccctcttcct gcccgacctt
      541 gacgggtagt gtttaaacaa aaaaataaac gatgcccctt aaagcagaag ctttaagggg
      601 cagagcgttg cggcacatct tttcagacgg ccttattgta gcaacgtttc gggagctaca
      661 actgagacgt ggaatcgtct ccggtgtttc gtcctttcca caagatatat aaagccaaga
      721 aatcgaaata ctttcaagtt acggtaagca tatgatagtc cattttaaaa cataatttta
      781 aaactgcaaa ctacccaaga aattattact ttctacgtca cgtattttgt actaatatct
      841 ttgtgtttac agtcaaatta attctaatta tctctctaac agccttgtat cgtatatgca
      901 aatatgaagg aatcatggga aataggccct cattatcctc tagaatggag gcggtactat
      961 gtagatgaga attcaggagc aaactgggaa aagcaactgc ttccaaatat ttgtgatttt
     1021 tacagtgtag ttttggaaaa actcttagcc taccaattct tctaagtgtt ttaaaatgtg
     1081 ggagccagta cacatgaagt tatagagtgt tttaatgagg cttaaatatt taccgtaact
     1141 atgaaatgct acgcatatca tgctgttcag gctccgtggc cacgcaactc atacttaagc
     1201 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgaacaccg ccaccatggt
     1261 gcctaagaag aagagaaagg tggaagatat ggccgccttc aagcctaacc caatcaatta
     1321 catcctggga ctggacatcg gaatcgcatc cgtgggatgg gctatggtgg agatcgacga
     1381 ggaggagaat cctatccggc tgatcgatct gggcgtgaga gtgtttgaga gggccgaggt
     1441 gccaaagacc ggcgattctc tggctatggc ccggagactg gcacggagcg tgaggcgcct
     1501 gacacggaga agggcacaca ggctgctgag ggcacgccgg ctgctgaaga gagagggcgt
     1561 gctgcaggca gcagacttcg atgagaatgg cctgatcaag agcctgccaa acaccccctg
     1621 gcagctgaga gcagccgccc tggacaggaa gctgacacca ctggagtggt ctgccgtgct
     1681 gctgcacctg atcaagcacc gcggctacct gagccagcgg aagaacgagg gagagacagc
     1741 agacaaggag ctgggcgccc tgctgaaggg agtggccaac aatgcccacg ccctgcagac
     1801 cggcgatttc aggacacctg ccgagctggc cctgaataag tttgagaagg agtccggcca
     1861 catcagaaac cagaggggcg actatagcca caccttctcc cgcaaggatc tgcaggccga
     1921 gctgatcctg ctgttcgaga agcagaagga gtttggcaat ccacacgtga gcggaggcct
     1981 gaaggaggga atcgagaccc tgctgatgac acagaggcct gccctgtccg gcgacgcagt
     2041 gcagaagatg ctgggacact gcaccttcga gcctgcagag ccaaaggccg ccaagaacac
     2101 ctacacagcc gagcggttta tctggctgac aaagctgaac aatctgagaa tcctggagca
     2161 gggatccgag aggccactga ccgacacaga gagggccacc ctgatggatg agccttaccg
     2221 gaagtctaag ctgacatatg cccaggccag aaagctgctg ggcctggagg acaccgcctt
     2281 ctttaagggc ctgagatacg gcaaggataa tgccgaggcc tccacactga tggagatgaa
     2341 ggcctatcac gccatctctc gcgccctgga gaaggagggc ctgaaggaca agaagtcccc
     2401 cctgaacctg agctccgagc tgcaggatga gatcggcacc gccttctctc tgtttaagac
     2461 cgacgaggat atcacaggcc gcctgaagga cagggtgcag cctgagatcc tggaggccct
     2521 gctgaagcac atctctttcg ataagtttgt gcagatcagc ctgaaggccc tgagaaggat
     2581 cgtgccactg atggagcagg gcaagcggta cgacgaggcc tgcgccgaga tctacggcga
     2641 tcactatggc aagaagaaca cagaggagaa gatctatctg ccccctatcc ctgccgacga
     2701 gatcagaaat cctgtggtgc tgagggccct gtcccaggca agaaaagtga tcaacggagt
     2761 ggtgcgccgg tacggatctc cagcccggat ccacatcgag accgccagag aagtgggcaa
     2821 gagcttcaag gaccggaagg agatcgagaa gagacaggag gagaatcgca aggatcggga
     2881 gaaggccgcc gccaagttta gggagtactt ccctaacttt gtgggcgagc caaagtctaa
     2941 ggacatcctg aagctgcgcc tgtacgagca gcagcacggc aagtgtctgt atagcggcaa
     3001 ggagatcaat ctggtgcggc tgaacgagaa gggctatgtg gagatcgatc acgccctgcc
     3061 tttctccaga acctgggacg attcttttaa caataaggtg ctggtgctgg gcagcgagaa
     3121 ccagaataag ggcaatcaga caccatacga gtatttcaat ggcaaggaca actccaggga
     3181 gtggcaggag ttcaaggccc gcgtggagac ctctagattt cccaggagca agaagcagcg
     3241 gatcctgctg cagaagttcg acgaggatgg ctttaaggag tgcaacctga atgacaccag
     3301 atacgtgaac cggttcctgt gccagtttgt ggccgatcac atcctgctga ccggcaaggg
     3361 caagagaagg gtgttcgcct ctaatggcca gatcacaaac ctgctgaggg gattttgggg
     3421 actgaggaag gtgcgggcag agaatgacag acaccacgca ctggatgcag tggtggtggc
     3481 atgcagcacc gtggcaatgc agcagaagat cacaagattc gtgaggtata aggagatgaa
     3541 cgcctttgac ggcaagacca tcgataagga gacaggcaag gtgctgcacc agaagaccca
     3601 cttcccccag ccttgggagt tctttgccca ggaagtgatg atccgggtgt tcggcaagcc
     3661 agacggcaag cctgagtttg aggaggccga taccccagag aagctgagga cactgctggc
     3721 agagaagctg tctagcaggc cagaggcagt gcacgagtac gtgaccccac tgttcgtgtc
     3781 cagggcaccc aatcggaaga tgtctggcgc ccacaaggac acactgagaa gcgccaagag
     3841 gtttgtgaag cacaacgaga agatctccgt gaagagagtg tggctgaccg agatcaagct
     3901 ggccgatctg gagaacatgg tgaattacaa gaacggcagg gagatcgagc tgtatgaggc
     3961 cctgaaggca aggctggagg cctacggagg aaatgccaag caggccttcg acccaaagga
     4021 taaccccttt tataagaagg gaggacagct ggtgaaggcc gtgcgggtgg agaagaccca
     4081 ggagagcggc gtgctgctga ataagaagaa cgcctacaca atcgccgaca atggcgatat
     4141 ggtgagagtg gacgtgttct gtaaggtgga taagaagggc aagaatcagt actttatcgt
     4201 gcctatctat gcctggcagg tggccgagaa catcctgcca gacatcgatt gcaagggcta
     4261 cagaattgac gatagctata cattctgttt ttccctgcac aagtatgacc tgatcgcctt
     4321 ccagaaggat gagaagtcca aggtggagtt tgcctactat atcaattgcg actcctctaa
     4381 cggcaggttc tacctggcct ggcacgataa gggcagcaag gagcagcagt ttcgcatctc
     4441 cacccagaat ctggtgctga tccagaagta tcaggtgaac gagctgggca aggagatcag
     4501 gccatgtcgg ctgaagaagc gcccacccgt gcgggaggat aagcggcccg cagcaaccaa
     4561 gaaggcagga caggccaaga agaagaagta aaataaaaga tctttatttt cattagatct
     4621 gtgtgttggt tttttgtgta agcgcggccg caggaacccc tagtgatgga gttggccact
     4681 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     4741 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     4801 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     4861 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4921 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     4981 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5041 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     5101 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5161 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     5221 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     5281 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     5341 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     5401 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg ctcgagttag
     5461 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5521 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5581 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5641 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5701 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5761 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5821 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5881 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5941 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     6001 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6061 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6121 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6181 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6241 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6301 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6361 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6421 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6481 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6541 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6601 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6661 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6721 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6781 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6841 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6901 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6961 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7021 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7081 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7141 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7201 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7261 ccttttgctc acatgt
//
