LOCUS       sequence-438443-        6966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV plasmid with U6 promoter driving a gRNA against LDLR and 
            liver specific HLP promoter driving SaCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6966)
  AUTHORS   De Giorgi M, Jarrett KE, de Aguiar Vallim TQ, Lagor WR
  TITLE     In Vivo Gene Editing in Lipid and Atherosclerosis Research.
  JOURNAL   Methods Mol Biol. 2022;2419:673-713. doi: 
            10.1007/978-1-0716-1924-7_42.
  PUBMED    35237996
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: "Methods Mol
            Biol."; date: "2022"; volume: "2419:673-713. doi"; pages: " 
            10.1007/978-1-0716-1924-7_42"
COMMENT     Sequence Label: 
            pAAV-U6-SA-WTmLdlrEx14-gRNA2-N22-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        526..601
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     regulatory      878..887
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             890..910
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4091..4138
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4145..4171
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4172..4213
                     /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    4231..4279
                     /note="synthetic polyadenylation signal"
     repeat_region   4327..4456
                     /label=AAV2 ITR
     rep_origin      complement(4693..5281)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4773..4792)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5452..6243,6244..6312))
                     /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     6075..6094
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6313..6417)
                     /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 accgggcagg cgcaggtgaa tttgggtttt agtactctgg
      541 aaacagaatc tactaaaaca aggcaaaatg ccgtgtttat ctcgtcaact tgttggcgag
      601 atttttttct agaaagcttt gtttgctgct tgcaatgttt gcccatttta gggtggacac
      661 aggacgctgt ggtttctgag ccagggggcg actcagatcc cagccagtgg acttagcccc
      721 tgtttgctcc tccgataact ggggtgacct tggttaatat tcaccagcag cctcccccgt
      781 tgcccctctg gatccactgc ttaaatacgg acgaggacag ggccctgtct cctcagcttc
      841 aggcaccacc actgacctgg gacagtgaat caccggtgcc accatggccc caaagaagaa
      901 gcggaaggtc ggtatccacg gagtcccagc agccaagcgg aactacatcc tgggcctgga
      961 catcggcatc accagcgtgg gctacggcat catcgactac gagacacggg acgtgatcga
     1021 tgccggcgtg cggctgttca aagaggccaa cgtggaaaac aacgagggca ggcggagcaa
     1081 gagaggcgcc agaaggctga agcggcggag gcggcataga atccagagag tgaagaagct
     1141 gctgttcgac tacaacctgc tgaccgacca cagcgagctg agcggcatca acccctacga
     1201 ggccagagtg aagggcctga gccagaagct gagcgaggaa gagttctctg ccgccctgct
     1261 gcacctggcc aagagaagag gcgtgcacaa cgtgaacgag gtggaagagg acaccggcaa
     1321 cgagctgtcc accagagagc agatcagccg gaacagcaag gccctggaag agaaatacgt
     1381 ggccgaactg cagctggaac ggctgaagaa agacggcgaa gtgcggggca gcatcaacag
     1441 attcaagacc agcgactacg tgaaagaagc caaacagctg ctgaaggtgc agaaggccta
     1501 ccaccagctg gaccagagct tcatcgacac ctacatcgac ctgctggaaa cccggcggac
     1561 ctactatgag ggacctggcg agggcagccc cttcggctgg aaggacatca aagaatggta
     1621 cgagatgctg atgggccact gcacctactt ccccgaggaa ctgcggagcg tgaagtacgc
     1681 ctacaacgcc gacctgtaca acgccctgaa cgacctgaac aatctcgtga tcaccaggga
     1741 cgagaacgag aagctggaat attacgagaa gttccagatc atcgagaacg tgttcaagca
     1801 gaagaagaag cccaccctga agcagatcgc caaagaaatc ctcgtgaacg aagaggatat
     1861 taagggctac agagtgacca gcaccggcaa gcccgagttc accaacctga aggtgtacca
     1921 cgacatcaag gacattaccg cccggaaaga gattattgag aacgccgagc tgctggatca
     1981 gattgccaag atcctgacca tctaccagag cagcgaggac atccaggaag aactgaccaa
     2041 tctgaactcc gagctgaccc aggaagagat cgagcagatc tctaatctga agggctatac
     2101 cggcacccac aacctgagcc tgaaggccat caacctgatc ctggacgagc tgtggcacac
     2161 caacgacaac cagatcgcta tcttcaaccg gctgaagctg gtgcccaaga aggtggacct
     2221 gtcccagcag aaagagatcc ccaccaccct ggtggacgac ttcatcctga gccccgtcgt
     2281 gaagagaagc ttcatccaga gcatcaaagt gatcaacgcc atcatcaaga agtacggcct
     2341 gcccaacgac atcattatcg agctggcccg cgagaagaac tccaaggacg cccagaaaat
     2401 gatcaacgag atgcagaagc ggaaccggca gaccaacgag cggatcgagg aaatcatccg
     2461 gaccaccggc aaagagaacg ccaagtacct gatcgagaag atcaagctgc acgacatgca
     2521 ggaaggcaag tgcctgtaca gcctggaagc catccctctg gaagatctgc tgaacaaccc
     2581 cttcaactat gaggtggacc acatcatccc cagaagcgtg tccttcgaca acagcttcaa
     2641 caacaaggtg ctcgtgaagc aggaagaaaa cagcaagaag ggcaaccgga ccccattcca
     2701 gtacctgagc agcagcgaca gcaagatcag ctacgaaacc ttcaagaagc acatcctgaa
     2761 tctggccaag ggcaagggca gaatcagcaa gaccaagaaa gagtatctgc tggaagaacg
     2821 ggacatcaac aggttctccg tgcagaaaga cttcatcaac cggaacctgg tggataccag
     2881 atacgccacc agaggcctga tgaacctgct gcggagctac ttcagagtga acaacctgga
     2941 cgtgaaagtg aagtccatca atggcggctt caccagcttt ctgcggcgga agtggaagtt
     3001 taagaaagag cggaacaagg ggtacaagca ccacgccgag gacgccctga tcattgccaa
     3061 cgccgatttc atcttcaaag agtggaagaa actggacaag gccaaaaaag tgatggaaaa
     3121 ccagatgttc gaggaaaggc aggccgagag catgcccgag atcgaaaccg agcaggagta
     3181 caaagagatc ttcatcaccc cccaccagat caagcacatt aaggacttca aggactacaa
     3241 gtacagccac cgggtggaca agaagcctaa tagagagctg attaacgaca ccctgtactc
     3301 cacccggaag gacgacaagg gcaacaccct gatcgtgaac aatctgaacg gcctgtacga
     3361 caaggacaat gacaagctga aaaagctgat caacaagagc cccgaaaagc tgctgatgta
     3421 ccaccacgac ccccagacct accagaaact gaagctgatt atggaacagt acggcgacga
     3481 gaagaatccc ctgtacaagt actacgagga aaccgggaac tacctgacca agtactccaa
     3541 aaaggacaac ggccccgtga tcaagaagat taagtattac ggcaacaaac tgaacgccca
     3601 tctggacatc accgacgact accccaacag cagaaacaag gtcgtgaagc tgtccctgaa
     3661 gccctacaga ttcgacgtgt acctggacaa tggcgtgtac aagttcgtga ccgtgaagaa
     3721 tctggatgtg atcaaaaaag aaaactacta cgaagtgaat agcaagtgct atgaggaagc
     3781 taagaagctg aagaagatca gcaaccaggc cgagtttatc gcctccttct acaacaacga
     3841 tctgatcaag atcaacggcg agctgtatag agtgatcggc gtgaacaacg acctgctgaa
     3901 ccggatcgaa gtgaacatga tcgacatcac ctaccgcgag tacctggaaa acatgaacga
     3961 caagaggccc cccaggatca ttaagacaat cgcctccaag acccagagca ttaagaagta
     4021 cagcacagac attctgggca acctgtatga agtgaaatct aagaagcacc ctcagatcat
     4081 caaaaagggc aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaaggg
     4141 atcctaccca tacgatgttc cagattacgc tagcggcttc gccaacgagc ttggacccag
     4201 gttgatggga aagtaagaat tcctagagcg aataaaagat ctttattttc attagatctg
     4261 tgtgttggtt ttttgtgtga tgcagctacg tagataagta gcatggcggg ttaatcatta
     4321 actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4381 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4441 gcgagcgagc gcgcagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4501 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4561 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4621 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4681 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4741 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4801 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4861 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4921 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4981 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5041 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5101 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5161 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5221 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5281 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5341 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5401 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5461 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5521 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5581 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5641 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5701 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5761 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5821 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5881 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5941 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6001 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6061 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6121 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6181 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6241 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6301 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6361 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6421 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     6481 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     6541 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     6601 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca 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
//
