LOCUS       sequence-206098-        6977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for liver-specific expression of AAV SaCas9 with a gRNA 
            against Mttp.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6977)
  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 6977)
  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: 1476_pAAV-U6-SA-mMttp-gRNA-N21-HLP-SACas9-spA
FEATURES             Location/Qualifiers
     source          1..6977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   4..133
                     /label=AAV2 ITR
     promoter        266..514
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     266..286
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     437..456
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        536..611
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     regulatory      888..897
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             900..920
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             945..4100
                     /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             4101..4148
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4155..4181
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4182..4223
                     /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    4241..4289
                     /note="synthetic polyadenylation signal"
     repeat_region   4337..4466
                     /label=AAV2 ITR
     rep_origin      complement(4703..5291)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4783..4802)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5462..6253,6254..6322))
                     /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     6085..6104
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6323..6427)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6454..6909)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6591..6612)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6803..6822
                     /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 gtcgacatcc accggtgcgg
      241 ccgcggtacc aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca
      301 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
      361 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
      421 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
      481 ggctttatat atcttgtgga aaggacgaaa caccgtggac gttgtgttac tgtgggttta
      541 agtactctgg aaacagaatc tacttaaaca aggcaaaatg ccgtgtttat ctcgtcaact
      601 tgttggcgag atttttttct agaaagcttt gtttgctgct tgcaatgttt gcccatttta
      661 gggtggacac aggacgctgt ggtttctgag ccagggggcg actcagatcc cagccagtgg
      721 acttagcccc tgtttgctcc tccgataact ggggtgacct tggttaatat tcaccagcag
      781 cctcccccgt tgcccctctg gatccactgc ttaaatacgg acgaggacag ggccctgtct
      841 cctcagcttc aggcaccacc actgacctgg gacagtgaat caccggtgcc accatggccc
      901 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccaagcgg aactacatcc
      961 tgggcctgga catcggcatc accagcgtgg gctacggcat catcgactac gagacacggg
     1021 acgtgatcga tgccggcgtg cggctgttca aagaggccaa cgtggaaaac aacgagggca
     1081 ggcggagcaa gagaggcgcc agaaggctga agcggcggag gcggcataga atccagagag
     1141 tgaagaagct gctgttcgac tacaacctgc tgaccgacca cagcgagctg agcggcatca
     1201 acccctacga ggccagagtg aagggcctga gccagaagct gagcgaggaa gagttctctg
     1261 ccgccctgct gcacctggcc aagagaagag gcgtgcacaa cgtgaacgag gtggaagagg
     1321 acaccggcaa cgagctgtcc accaaagagc agatcagccg gaacagcaag gccctggaag
     1381 agaaatacgt ggccgaactg cagctggaac ggctgaagaa agacggcgaa gtgcggggca
     1441 gcatcaacag attcaagacc agcgactacg tgaaagaagc caaacagctg ctgaaggtgc
     1501 agaaggccta ccaccagctg gaccagagct tcatcgacac ctacatcgac ctgctggaaa
     1561 cccggcggac ctactatgag ggacctggcg agggcagccc cttcggctgg aaggacatca
     1621 aagaatggta cgagatgctg atgggccact gcacctactt ccccgaggaa ctgcggagcg
     1681 tgaagtacgc ctacaacgcc gacctgtaca acgccctgaa cgacctgaac aatctcgtga
     1741 tcaccaggga cgagaacgag aagctggaat attacgagaa gttccagatc atcgagaacg
     1801 tgttcaagca gaagaagaag cccaccctga agcagatcgc caaagaaatc ctcgtgaacg
     1861 aagaggatat taagggctac agagtgacca gcaccggcaa gcccgagttc accaacctga
     1921 aggtgtacca cgacatcaag gacattaccg cccggaaaga gattattgag aacgccgagc
     1981 tgctggatca gattgccaag atcctgacca tctaccagag cagcgaggac atccaggaag
     2041 aactgaccaa tctgaactcc gagctgaccc aggaagagat cgagcagatc tctaatctga
     2101 agggctatac cggcacccac aacctgagcc tgaaggccat caacctgatc ctggacgagc
     2161 tgtggcacac caacgacaac cagatcgcta tcttcaaccg gctgaagctg gtgcccaaga
     2221 aggtggacct gtcccagcag aaagagatcc ccaccaccct ggtggacgac ttcatcctga
     2281 gccccgtcgt gaagagaagc ttcatccaga gcatcaaagt gatcaacgcc atcatcaaga
     2341 agtacggcct gcccaacgac atcattatcg agctggcccg cgagaagaac tccaaggacg
     2401 cccagaaaat gatcaacgag atgcagaagc ggaaccggca gaccaacgag cggatcgagg
     2461 aaatcatccg gaccaccggc aaagagaacg ccaagtacct gatcgagaag atcaagctgc
     2521 acgacatgca ggaaggcaag tgcctgtaca gcctggaagc catccctctg gaagatctgc
     2581 tgaacaaccc cttcaactat gaggtggacc acatcatccc cagaagcgtg tccttcgaca
     2641 acagcttcaa caacaaggtg ctcgtgaagc aggaagaaaa cagcaagaag ggcaaccgga
     2701 ccccattcca gtacctgagc agcagcgaca gcaagatcag ctacgaaacc ttcaagaagc
     2761 acatcctgaa tctggccaag ggcaagggca gaatcagcaa gaccaagaaa gagtatctgc
     2821 tggaagaacg ggacatcaac aggttctccg tgcagaaaga cttcatcaac cggaacctgg
     2881 tggataccag atacgccacc agaggcctga tgaacctgct gcggagctac ttcagagtga
     2941 acaacctgga cgtgaaagtg aagtccatca atggcggctt caccagcttt ctgcggcgga
     3001 agtggaagtt taagaaagag cggaacaagg ggtacaagca ccacgccgag gacgccctga
     3061 tcattgccaa cgccgatttc atcttcaaag agtggaagaa actggacaag gccaaaaaag
     3121 tgatggaaaa ccagatgttc gaggaaaagc aggccgagag catgcccgag atcgaaaccg
     3181 agcaggagta caaagagatc ttcatcaccc cccaccagat caagcacatt aaggacttca
     3241 aggactacaa gtacagccac cgggtggaca agaagcctaa tagagagctg attaacgaca
     3301 ccctgtactc cacccggaag gacgacaagg gcaacaccct gatcgtgaac aatctgaacg
     3361 gcctgtacga caaggacaat gacaagctga aaaagctgat caacaagagc cccgaaaagc
     3421 tgctgatgta ccaccacgac ccccagacct accagaaact gaagctgatt atggaacagt
     3481 acggcgacga gaagaatccc ctgtacaagt actacgagga aaccgggaac tacctgacca
     3541 agtactccaa aaaggacaac ggccccgtga tcaagaagat taagtattac ggcaacaaac
     3601 tgaacgccca tctggacatc accgacgact accccaacag cagaaacaag gtcgtgaagc
     3661 tgtccctgaa gccctacaga ttcgacgtgt acctggacaa tggcgtgtac aagttcgtga
     3721 ccgtgaagaa tctggatgtg atcaaaaaag aaaactacta cgaagtgaat agcaagtgct
     3781 atgaggaagc taagaagctg aagaagatca gcaaccaggc cgagtttatc gcctccttct
     3841 acaacaacga tctgatcaag atcaacggcg agctgtatag agtgatcggc gtgaacaacg
     3901 acctgctgaa ccggatcgaa gtgaacatga tcgacatcac ctaccgcgag tacctggaaa
     3961 acatgaacga caagaggccc cccaggatca ttaagacaat cgcctccaag acccagagca
     4021 ttaagaagta cagcacagac attctgggca acctgtatga agtgaaatct aagaagcacc
     4081 ctcagatcat caaaaagggc aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa
     4141 agaaaaaggg atcctaccca tacgatgttc cagattacgc tagcggcttc gccaacgagc
     4201 ttggacccag gttgatggga aagtaagaat tcctagagcg aataaaagat ctttattttc
     4261 attagatctg tgtgttggtt ttttgtgtga tgcagctacg tagataagta gcatggcggg
     4321 ttaatcatta actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     4381 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     4441 gcctcagtga gcgagcgagc gcgcagctgc attaatgaat cggccaacgc gcggggagag
     4501 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4561 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4621 caggggataa cgcaggaaag aacatgtgag caaaaccgca gcaaaaggcc aggaaccgta
     4681 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4741 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4801 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4861 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4921 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4981 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5041 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5101 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     5161 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5221 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5281 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5341 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5401 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5461 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5521 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5581 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5641 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5701 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5761 acgttgttac cattactaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5821 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5881 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5941 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6001 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6061 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6121 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttacca ctattgagat
     6181 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6241 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6301 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6361 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6421 ttccgcgcac atttccccga aagatgccac ctgaaattat aaacgttaat attttgttaa
     6481 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggaa
     6541 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     6601 acaagagtcc actattgagg aacgtgaact ccagcgtcaa agggcgaaaa accgtctatc
     6661 ggggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     6721 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     6781 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     6841 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     6901 agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     6961 cctcttcgct attacgc
//
