LOCUS       sequence-311994-        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: pEJS1099: Dual-sgRNA.Design 4
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)"
     regulatory      861..870
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             873..893
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4152..4199
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     promoter        4263..4511
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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 gacgtcgact ctagaatgga ggcggtacta tgtagatgag aattcaggag caaactggga
      601 aaagcaactg cttccaaata tttgtgattt ttacagtgta gttttggaaa aactcttagc
      661 ctaccaattc ttctaagtgt tttaaaatgt gggagccagt acacatgaag ttatagagtg
      721 ttttaatgag gcttaaatat ttaccgtaac tatgaaatgc tacgcatatc atgctgttca
      781 ggctccgtgg ccacgcaact catacttaag cagacagtgg ttcaaagttt ttttcttcca
      841 tttcaggtgt cgtgaacacc gccaccatgg tgcctaagaa gaagagaaag gtggaagata
      901 tggccgcctt caagcctaac ccaatcaatt acatcctggg actggacatc ggaatcgcat
      961 ccgtgggatg ggctatggtg gagatcgacg aggaggagaa tcctatccgg ctgatcgatc
     1021 tgggcgtgag agtgtttgag agggccgagg tgccaaagac cggcgattct ctggctatgg
     1081 cccggagact ggcacggagc gtgaggcgcc tgacacggag aagggcacac aggctgctga
     1141 gggcacgccg gctgctgaag agagagggcg tgctgcaggc agcagacttc gatgagaatg
     1201 gcctgatcaa gagcctgcca aacaccccct ggcagctgag agcagccgcc ctggacagga
     1261 agctgacacc actggagtgg tctgccgtgc tgctgcacct gatcaagcac cgcggctacc
     1321 tgagccagcg gaagaacgag ggagagacag cagacaagga gctgggcgcc ctgctgaagg
     1381 gagtggccaa caatgcccac gccctgcaga ccggcgattt caggacacct gccgagctgg
     1441 ccctgaataa gtttgagaag gagtccggcc acatcagaaa ccagaggggc gactatagcc
     1501 acaccttctc ccgcaaggat ctgcaggccg agctgatcct gctgttcgag aagcagaagg
     1561 agtttggcaa tccacacgtg agcggaggcc tgaaggaggg aatcgagacc ctgctgatga
     1621 cacagaggcc tgccctgtcc ggcgacgcag tgcagaagat gctgggacac tgcaccttcg
     1681 agcctgcaga gccaaaggcc gccaagaaca cctacacagc cgagcggttt atctggctga
     1741 caaagctgaa caatctgaga atcctggagc agggatccga gaggccactg accgacacag
     1801 agagggccac cctgatggat gagccttacc ggaagtctaa gctgacatat gcccaggcca
     1861 gaaagctgct gggcctggag gacaccgcct tctttaaggg cctgagatac ggcaaggata
     1921 atgccgaggc ctccacactg atggagatga aggcctatca cgccatctct cgcgccctgg
     1981 agaaggaggg cctgaaggac aagaagtccc ccctgaacct gagctccgag ctgcaggatg
     2041 agatcggcac cgccttctct ctgtttaaga ccgacgagga tatcacaggc cgcctgaagg
     2101 acagggtgca gcctgagatc ctggaggccc tgctgaagca catctctttc gataagtttg
     2161 tgcagatcag cctgaaggcc ctgagaagga tcgtgccact gatggagcag ggcaagcggt
     2221 acgacgaggc ctgcgccgag atctacggcg atcactatgg caagaagaac acagaggaga
     2281 agatctatct gccccctatc cctgccgacg agatcagaaa tcctgtggtg ctgagggccc
     2341 tgtcccaggc aagaaaagtg atcaacggag tggtgcgccg gtacggatct ccagcccgga
     2401 tccacatcga gaccgccaga gaagtgggca agagcttcaa ggaccggaag gagatcgaga
     2461 agagacagga ggagaatcgc aaggatcggg agaaggccgc cgccaagttt agggagtact
     2521 tccctaactt tgtgggcgag ccaaagtcta aggacatcct gaagctgcgc ctgtacgagc
     2581 agcagcacgg caagtgtctg tatagcggca aggagatcaa tctggtgcgg ctgaacgaga
     2641 agggctatgt ggagatcgat cacgccctgc ctttctccag aacctgggac gattctttta
     2701 acaataaggt gctggtgctg ggcagcgaga accagaataa gggcaatcag acaccatacg
     2761 agtatttcaa tggcaaggac aactccaggg agtggcagga gttcaaggcc cgcgtggaga
     2821 cctctagatt tcccaggagc aagaagcagc ggatcctgct gcagaagttc gacgaggatg
     2881 gctttaagga gtgcaacctg aatgacacca gatacgtgaa ccggttcctg tgccagtttg
     2941 tggccgatca catcctgctg accggcaagg gcaagagaag ggtgttcgcc tctaatggcc
     3001 agatcacaaa cctgctgagg ggattttggg gactgaggaa ggtgcgggca gagaatgaca
     3061 gacaccacgc actggatgca gtggtggtgg catgcagcac cgtggcaatg cagcagaaga
     3121 tcacaagatt cgtgaggtat aaggagatga acgcctttga cggcaagacc atcgataagg
     3181 agacaggcaa ggtgctgcac cagaagaccc acttccccca gccttgggag ttctttgccc
     3241 aggaagtgat gatccgggtg ttcggcaagc cagacggcaa gcctgagttt gaggaggccg
     3301 ataccccaga gaagctgagg acactgctgg cagagaagct gtctagcagg ccagaggcag
     3361 tgcacgagta cgtgacccca ctgttcgtgt ccagggcacc caatcggaag atgtctggcg
     3421 cccacaagga cacactgaga agcgccaaga ggtttgtgaa gcacaacgag aagatctccg
     3481 tgaagagagt gtggctgacc gagatcaagc tggccgatct ggagaacatg gtgaattaca
     3541 agaacggcag ggagatcgag ctgtatgagg ccctgaaggc aaggctggag gcctacggag
     3601 gaaatgccaa gcaggccttc gacccaaagg ataacccctt ttataagaag ggaggacagc
     3661 tggtgaaggc cgtgcgggtg gagaagaccc aggagagcgg cgtgctgctg aataagaaga
     3721 acgcctacac aatcgccgac aatggcgata tggtgagagt ggacgtgttc tgtaaggtgg
     3781 ataagaaggg caagaatcag tactttatcg tgcctatcta tgcctggcag gtggccgaga
     3841 acatcctgcc agacatcgat tgcaagggct acagaattga cgatagctat acattctgtt
     3901 tttccctgca caagtatgac ctgatcgcct tccagaagga tgagaagtcc aaggtggagt
     3961 ttgcctacta tatcaattgc gactcctcta acggcaggtt ctacctggcc tggcacgata
     4021 agggcagcaa ggagcagcag tttcgcatct ccacccagaa tctggtgctg atccagaagt
     4081 atcaggtgaa cgagctgggc aaggagatca ggccatgtcg gctgaagaag cgcccacccg
     4141 tgcgggagga taagcggccc gcagcaacca agaaggcagg acaggccaag aagaagaagt
     4201 aaaataaaag atctttattt tcattagatc tgtgtgttgg ttttttgtgt aagcgcacga
     4261 atgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     4321 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     4381 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     4441 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     4501 gacgaaacac cggagacgat tccacgtctc agttgtagct cccgaaacgt tgctacaata
     4561 aggccgtctg aaaagatgtg ccgcaacgct ctgcccctta aagcttctgc tttaaggggc
     4621 atcgtttatt tttttgttta aacactaccg 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
//
