LOCUS       sequence-206099-        6966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for liver-specific expression of AAV SaCas9 with a gRNA 
            against eGFP.
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: 1518_pAAV-U6-SA-eGFP-gRNA-HLP-SACas9-HA-OLLAS-spA
FEATURES             Location/Qualifiers
     source          1..6966
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   4..133
                     /label=AAV2 ITR
     promoter        255..503
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     255..275
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     426..445
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        525..600
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     regulatory      877..886
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             889..909
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             934..4089
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             4090..4137
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4144..4170
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4171..4212
                     /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    4230..4278
                     /note="synthetic polyadenylation signal"
     repeat_region   4326..4455
                     /label=AAV2 ITR
     rep_origin      complement(4692..5280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4772..4791)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5451..6242,6243..6311))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6074..6093
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6312..6416)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6443..6898)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6580..6601)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6792..6811
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
       61 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      121 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
      181 tctggtgtac aaaaaagcag gctttaaagg aaccaattca gtcgactgga tccggtacca
      241 aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca
      301 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
      361 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      421 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
      481 tcttgtggaa aggacgaaac accggggtct ttgctcaggg cggagtttta gtactctgga
      541 aacagaatct actaaaacaa ggcaaaatgc cgtgtttatc tcgtcaactt gttggcgaga
      601 tttttttcta gaaagctttg tttgctgctt gcaatgtttg cccattttag ggtggacaca
      661 ggacgctgtg gtttctgagc cagggggcga ctcagatccc agccagtgga cttagcccct
      721 gtttgctcct ccgataactg gggtgacctt ggttaatatt caccagcagc ctcccccgtt
      781 gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc ctcagcttca
      841 ggcaccacca ctgacctggg acagtgaatc accggtgcca ccatggcccc aaagaagaag
      901 cggaaggtcg gtatccacgg agtcccagca gccaagcgga actacatcct gggcctggac
      961 atcggcatca ccagcgtggg ctacggcatc atcgactacg agacacggga cgtgatcgat
     1021 gccggcgtgc ggctgttcaa agaggccaac gtggaaaaca acgagggcag gcggagcaag
     1081 agaggcgcca gaaggctgaa gcggcggagg cggcatagaa tccagagagt gaagaagctg
     1141 ctgttcgact acaacctgct gaccgaccac agcgagctga gcggcatcaa cccctacgag
     1201 gccagagtga agggcctgag ccagaagctg agcgaggaag agttctctgc cgccctgctg
     1261 cacctggcca agagaagagg cgtgcacaac gtgaacgagg tggaagagga caccggcaac
     1321 gagctgtcca ccaaagagca gatcagccgg aacagcaagg ccctggaaga gaaatacgtg
     1381 gccgaactgc agctggaacg gctgaagaaa gacggcgaag tgcggggcag catcaacaga
     1441 ttcaagacca gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca gaaggcctac
     1501 caccagctgg accagagctt catcgacacc tacatcgacc tgctggaaac ccggcggacc
     1561 tactatgagg gacctggcga gggcagcccc ttcggctgga aggacatcaa agaatggtac
     1621 gagatgctga tgggccactg cacctacttc cccgaggaac tgcggagcgt gaagtacgcc
     1681 tacaacgccg acctgtacaa cgccctgaac gacctgaaca atctcgtgat caccagggac
     1741 gagaacgaga agctggaata ttacgagaag ttccagatca tcgagaacgt gttcaagcag
     1801 aagaagaagc ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga agaggatatt
     1861 aagggctaca gagtgaccag caccggcaag cccgagttca ccaacctgaa ggtgtaccac
     1921 gacatcaagg acattaccgc ccggaaagag attattgaga acgccgagct gctggatcag
     1981 attgccaaga tcctgaccat ctaccagagc agcgaggaca tccaggaaga actgaccaat
     2041 ctgaactccg agctgaccca ggaagagatc gagcagatct ctaatctgaa gggctatacc
     2101 ggcacccaca acctgagcct gaaggccatc aacctgatcc tggacgagct gtggcacacc
     2161 aacgacaacc agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa ggtggacctg
     2221 tcccagcaga aagagatccc caccaccctg gtggacgact tcatcctgag ccccgtcgtg
     2281 aagagaagct tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg
     2341 cccaacgaca tcattatcga gctggcccgc gagaagaact ccaaggacgc ccagaaaatg
     2401 atcaacgaga tgcagaagcg gaaccggcag accaacgagc ggatcgagga aatcatccgg
     2461 accaccggca aagagaacgc caagtacctg atcgagaaga tcaagctgca cgacatgcag
     2521 gaaggcaagt gcctgtacag cctggaagcc atccctctgg aagatctgct gaacaacccc
     2581 ttcaactatg aggtggacca catcatcccc agaagcgtgt ccttcgacaa cagcttcaac
     2641 aacaaggtgc tcgtgaagca ggaagaaaac agcaagaagg gcaaccggac cccattccag
     2701 tacctgagca gcagcgacag caagatcagc tacgaaacct tcaagaagca catcctgaat
     2761 ctggccaagg gcaagggcag aatcagcaag accaagaaag agtatctgct ggaagaacgg
     2821 gacatcaaca ggttctccgt gcagaaagac ttcatcaacc ggaacctggt ggataccaga
     2881 tacgccacca gaggcctgat gaacctgctg cggagctact tcagagtgaa caacctggac
     2941 gtgaaagtga agtccatcaa tggcggcttc accagctttc tgcggcggaa gtggaagttt
     3001 aagaaagagc ggaacaaggg gtacaagcac cacgccgagg acgccctgat cattgccaac
     3061 gccgatttca tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt gatggaaaac
     3121 cagatgttcg aggaaaagca ggccgagagc atgcccgaga tcgaaaccga gcaggagtac
     3181 aaagagatct tcatcacccc ccaccagatc aagcacatta aggacttcaa ggactacaag
     3241 tacagccacc gggtggacaa gaagcctaat agagagctga ttaacgacac cctgtactcc
     3301 acccggaagg acgacaaggg caacaccctg atcgtgaaca atctgaacgg cctgtacgac
     3361 aaggacaatg acaagctgaa aaagctgatc aacaagagcc ccgaaaagct gctgatgtac
     3421 caccacgacc cccagaccta ccagaaactg aagctgatta tggaacagta cggcgacgag
     3481 aagaatcccc tgtacaagta ctacgaggaa accgggaact acctgaccaa gtactccaaa
     3541 aaggacaacg gccccgtgat caagaagatt aagtattacg gcaacaaact gaacgcccat
     3601 ctggacatca ccgacgacta ccccaacagc agaaacaagg tcgtgaagct gtccctgaag
     3661 ccctacagat tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac cgtgaagaat
     3721 ctggatgtga tcaaaaaaga aaactactac gaagtgaata gcaagtgcta tgaggaagct
     3781 aagaagctga agaagatcag caaccaggcc gagtttatcg cctccttcta caacaacgat
     3841 ctgatcaaga tcaacggcga gctgtataga gtgatcggcg tgaacaacga cctgctgaac
     3901 cggatcgaag tgaacatgat cgacatcacc taccgcgagt acctggaaaa catgaacgac
     3961 aagaggcccc ccaggatcat taagacaatc gcctccaaga cccagagcat taagaagtac
     4021 agcacagaca ttctgggcaa cctgtatgaa gtgaaatcta agaagcaccc tcagatcatc
     4081 aaaaagggca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggga
     4141 tcctacccat acgatgttcc agattacgct agcggcttcg ccaacgagct tggacccagg
     4201 ttgatgggaa agtaagaatt cctagagcga ataaaagatc tttattttca ttagatctgt
     4261 gtgttggttt tttgtgtgat gcagctacgt agataagtag catggcgggt taatcattaa
     4321 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4381 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4441 cgagcgagcg cgcagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4501 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4561 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4621 gcaggaaaga acatgtgagc aaaaccgcag caaaaggcca ggaaccgtaa aaaggccgcg
     4681 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4741 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4801 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4861 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4921 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4981 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5041 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5101 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     5161 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5221 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5281 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5341 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5401 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5461 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5521 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5581 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5641 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5701 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttacc
     5761 attactacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5821 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     5881 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     5941 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6001 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6061 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6121 aaacgttctt cggggcgaaa actctcaagg atcttaccac tattgagatc cagttcgatg
     6181 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6241 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6301 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6361 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6421 tttccccgaa agatgccacc tgaaattata aacgttaata ttttgttaaa attcgcgtta
     6481 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggaaa aatcccttat
     6541 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     6601 ctattgagga acgtgaactc cagcgtcaaa gggcgaaaaa ccgtctatcg gggcgatggc
     6661 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     6721 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     6781 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     6841 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     6901 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     6961 ttacgc
//
