LOCUS       sequence-311970-        6887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Delivery of Nme2Cas9 and its sgRNA in a single AAV vector with 
            overall packaging size of 4.4 Kb.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6887)
  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 6887)
  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: pEJS1089: mini-AAV.sgRNA.Nme2Cas9
FEATURES             Location/Qualifiers
     source          1..6887
                     /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)"
     primer_bind     complement(335..354)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(505..525)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     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"
     repeat_region   4263..4403
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4263..4392
                     /label=AAV2 ITR
     rep_origin      4478..4933
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4565..4584)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4775..4796
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4950..4969)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5102..5206
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5207..5275,5276..6067)
                     /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(5425..5444)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6238..6826
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6727..6746
                     /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 aagcgcggcc
     4261 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4321 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     4381 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     4441 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4501 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     4561 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4621 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4681 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4741 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4801 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     4861 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4921 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4981 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5041 tccgcttaca gacaagctgt gctcgagtta gacgtcaggt ggcacttttc ggggaaatgt
     5101 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5161 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5221 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5281 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5341 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5401 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5461 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5521 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5581 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5641 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5701 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5761 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5821 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5881 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     5941 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6001 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6061 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6121 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6181 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6241 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6301 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6361 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6421 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6481 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6541 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6601 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6661 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6721 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6781 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6841 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
