LOCUS       sequence-331412-        6966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for liver-specific expression of AAV SaCas9 with a BbsI 
            cloning site for a gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6966)
  AUTHORS   Li A, Lee CM, Hurley AE, Jarrett KE, De Giorgi M, Lu W, Balderrama 
            KS, Doerfler AM, Deshmukh H, Ray A, Bao G, Lagor WR
  TITLE     A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing.
  JOURNAL   Mol Ther Methods Clin Dev. 2018 Dec 6;12:111-122. doi: 
            10.1016/j.omtm.2018.11.009. eCollection 2019 Mar 15.
  PUBMED    30619914
REFERENCE   2  (bases 1 to 6966)
  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 Ther 
            Methods Clin Dev."; date: "2018-12-6"; volume: "12:111-122. doi"; 
            pages: " 10.1016/j.omtm.2018.11.009. eCollection 2019 Mar 15"
COMMENT     Sequence Label: 
            1313_pAAV-U6-SA-BbsI-MluI-gRNA-HLP-SACas9-HA-OLLAS-spA
FEATURES             Location/Qualifiers
     source          1..6966
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        252..500
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     252..272
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     423..442
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        523..598
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     regulatory      875..884
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             887..907
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4088..4135
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4142..4168
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4169..4210
                     /codon_start=1
                     /product="Escherichia coli OmpF Linker and mouse Langerin 
                     fusion sequence tag (Park et al., 2008)"
                     /label=OLLAS tag
                     /translation="SGFANELGPRLMGK"
     polyA_signal    4228..4276
                     /note="synthetic polyadenylation signal"
     repeat_region   4324..4453
                     /label=AAV2 ITR
     rep_origin      complement(4690..5278)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4770..4789)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5449..6240,6241..6309))
                     /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     6072..6091
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6310..6414)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6440..6895)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6577..6598)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6789..6808
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 ggtgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc ggtaccaagg
      241 tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      301 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
      361 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      421 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      481 tgtggaaagg acgaaacacc gggtcttcac gcgtgaagac cagttttagt actctggaaa
      541 cagaatctac taaaacaagg caaaatgccg tgtttatctc gtcaacttgt tggcgagatt
      601 tttttctaga aagctttgtt tgctgcttgc aatgtttgcc cattttaggg tggacacagg
      661 acgctgtggt ttctgagcca gggggcgact cagatcccag ccagtggact tagcccctgt
      721 ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct cccccgttgc
      781 ccctctggat ccactgctta aatacggacg aggacagggc cctgtctcct cagcttcagg
      841 caccaccact gacctgggac agtgaatcac cggtgccacc atggccccaa agaagaagcg
      901 gaaggtcggt atccacggag tcccagcagc caagcggaac tacatcctgg gcctggacat
      961 cggcatcacc agcgtgggct acggcatcat cgactacgag acacgggacg tgatcgatgc
     1021 cggcgtgcgg ctgttcaaag aggccaacgt ggaaaacaac gagggcaggc ggagcaagag
     1081 aggcgccaga aggctgaagc ggcggaggcg gcatagaatc cagagagtga agaagctgct
     1141 gttcgactac aacctgctga ccgaccacag cgagctgagc ggcatcaacc cctacgaggc
     1201 cagagtgaag ggcctgagcc agaagctgag cgaggaagag ttctctgccg ccctgctgca
     1261 cctggccaag agaagaggcg tgcacaacgt gaacgaggtg gaagaggaca ccggcaacga
     1321 gctgtccacc agagagcaga tcagccggaa cagcaaggcc ctggaagaga aatacgtggc
     1381 cgaactgcag ctggaacggc tgaagaaaga cggcgaagtg cggggcagca tcaacagatt
     1441 caagaccagc gactacgtga aagaagccaa acagctgctg aaggtgcaga aggcctacca
     1501 ccagctggac cagagcttca tcgacaccta catcgacctg ctggaaaccc ggcggaccta
     1561 ctatgaggga cctggcgagg gcagcccctt cggctggaag gacatcaaag aatggtacga
     1621 gatgctgatg ggccactgca cctacttccc cgaggaactg cggagcgtga agtacgccta
     1681 caacgccgac ctgtacaacg ccctgaacga cctgaacaat ctcgtgatca ccagggacga
     1741 gaacgagaag ctggaatatt acgagaagtt ccagatcatc gagaacgtgt tcaagcagaa
     1801 gaagaagccc accctgaagc agatcgccaa agaaatcctc gtgaacgaag aggatattaa
     1861 gggctacaga gtgaccagca ccggcaagcc cgagttcacc aacctgaagg tgtaccacga
     1921 catcaaggac attaccgccc ggaaagagat tattgagaac gccgagctgc tggatcagat
     1981 tgccaagatc ctgaccatct accagagcag cgaggacatc caggaagaac tgaccaatct
     2041 gaactccgag ctgacccagg aagagatcga gcagatctct aatctgaagg gctataccgg
     2101 cacccacaac ctgagcctga aggccatcaa cctgatcctg gacgagctgt ggcacaccaa
     2161 cgacaaccag atcgctatct tcaaccggct gaagctggtg cccaagaagg tggacctgtc
     2221 ccagcagaaa gagatcccca ccaccctggt ggacgacttc atcctgagcc ccgtcgtgaa
     2281 gagaagcttc atccagagca tcaaagtgat caacgccatc atcaagaagt acggcctgcc
     2341 caacgacatc attatcgagc tggcccgcga gaagaactcc aaggacgccc agaaaatgat
     2401 caacgagatg cagaagcgga accggcagac caacgagcgg atcgaggaaa tcatccggac
     2461 caccggcaaa gagaacgcca agtacctgat cgagaagatc aagctgcacg acatgcagga
     2521 aggcaagtgc ctgtacagcc tggaagccat ccctctggaa gatctgctga acaacccctt
     2581 caactatgag gtggaccaca tcatccccag aagcgtgtcc ttcgacaaca gcttcaacaa
     2641 caaggtgctc gtgaagcagg aagaaaacag caagaagggc aaccggaccc cattccagta
     2701 cctgagcagc agcgacagca agatcagcta cgaaaccttc aagaagcaca tcctgaatct
     2761 ggccaagggc aagggcagaa tcagcaagac caagaaagag tatctgctgg aagaacggga
     2821 catcaacagg ttctccgtgc agaaagactt catcaaccgg aacctggtgg ataccagata
     2881 cgccaccaga ggcctgatga acctgctgcg gagctacttc agagtgaaca acctggacgt
     2941 gaaagtgaag tccatcaatg gcggcttcac cagctttctg cggcggaagt ggaagtttaa
     3001 gaaagagcgg aacaaggggt acaagcacca cgccgaggac gccctgatca ttgccaacgc
     3061 cgatttcatc ttcaaagagt ggaagaaact ggacaaggcc aaaaaagtga tggaaaacca
     3121 gatgttcgag gaaaggcagg ccgagagcat gcccgagatc gaaaccgagc aggagtacaa
     3181 agagatcttc atcacccccc accagatcaa gcacattaag gacttcaagg actacaagta
     3241 cagccaccgg gtggacaaga agcctaatag agagctgatt aacgacaccc tgtactccac
     3301 ccggaaggac gacaagggca acaccctgat cgtgaacaat ctgaacggcc tgtacgacaa
     3361 ggacaatgac aagctgaaaa agctgatcaa caagagcccc gaaaagctgc tgatgtacca
     3421 ccacgacccc cagacctacc agaaactgaa gctgattatg gaacagtacg gcgacgagaa
     3481 gaatcccctg tacaagtact acgaggaaac cgggaactac ctgaccaagt actccaaaaa
     3541 ggacaacggc cccgtgatca agaagattaa gtattacggc aacaaactga acgcccatct
     3601 ggacatcacc gacgactacc ccaacagcag aaacaaggtc gtgaagctgt ccctgaagcc
     3661 ctacagattc gacgtgtacc tggacaatgg cgtgtacaag ttcgtgaccg tgaagaatct
     3721 ggatgtgatc aaaaaagaaa actactacga agtgaatagc aagtgctatg aggaagctaa
     3781 gaagctgaag aagatcagca accaggccga gtttatcgcc tccttctaca acaacgatct
     3841 gatcaagatc aacggcgagc tgtatagagt gatcggcgtg aacaacgacc tgctgaaccg
     3901 gatcgaagtg aacatgatcg acatcaccta ccgcgagtac ctggaaaaca tgaacgacaa
     3961 gaggcccccc aggatcatta agacaatcgc ctccaagacc cagagcatta agaagtacag
     4021 cacagacatt ctgggcaacc tgtatgaagt gaaatctaag aagcaccctc agatcatcaa
     4081 aaagggcaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc
     4141 ctacccatac gatgttccag attacgctag cggcttcgcc aacgagcttg gacccaggtt
     4201 gatgggaaag taagaattcc tagagcgaat aaaagatctt tattttcatt agatctgtgt
     4261 gttggttttt tgtgtgatgc agctacgtag ataagtagca tggcgggtta atcattaact
     4321 acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     4381 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     4441 agcgagcgcg cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4501 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4561 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4621 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4681 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4741 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4801 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4861 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4921 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4981 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5041 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5101 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5161 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5221 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5281 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5341 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5401 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5461 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5521 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5581 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5641 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5701 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5761 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5821 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5881 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5941 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6001 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6061 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6121 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6181 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6241 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6301 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6361 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6421 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     6481 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     6541 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     6601 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     6661 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     6721 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     6781 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     6841 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     6901 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     6961 cgccag
//
