LOCUS       pAAV-U6-SA-WTmLd        6967 bp DNA     circular SYN 27-JUL-2023
DEFINITION  This is the gRNA from William Lagor Lab LDLR-AAV CRISPR #1375 
            https://www.addgene.org/109311/, cloned into William Lagor Lab #1313
            https://www.addgene.org/109314/ for liver specific expression.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-U6-SA-WTmLdlrEx14-gRNA2-N22-HLP-SACas9-HA-OLLAS-spA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6967)
  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 2019;12:111-122.
  PUBMED    30619914
REFERENCE   2  (bases 1 to 6967)
  AUTHORS   Jarrett KE, Lee C, De Giorgi M, Hurley A, Gillard BK, Doerfler AM, 
            Li A, Pownall HJ, Bao G, Lagor WR.
  TITLE     Somatic Editing of Ldlr With Adeno-Associated Viral-CRISPR Is an 
            Efficient Tool for Atherosclerosis Research.
  JOURNAL   Arterioscler Thromb Vasc Biol 2018;38:1997-2006.
  PUBMED    30026278
REFERENCE   3  (bases 1 to 6967)
  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.
  PUBMED    35237996
REFERENCE   4  (bases 1 to 6967)
  AUTHORS   K Jarrett
  TITLE     Direct Submission
  JOURNAL   Exported Jul 27, 2023 from SnapGene 7.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Ther 
            Methods Clin Dev"; date: "2019"; volume: "12"; pages: "111-122"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: 
            "Arterioscler Thromb Vasc Biol"; date: "2018"; volume: "38"; pages: 
            "1997-2006"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Methods Mol
            Biol"; date: "2022"; volume: "2419"; pages: "673-713"
COMMENT     Component Fragments
            #1: FRAGMENT of pZAC2.1rhEPO.36.Q-L
             parent position: from 1309 to 239
             original length: 3894
             molecule position: from 1 to 3894
            Left Terminus
              XhoI site #1
            Right Terminus
              BglII site #1
            #2: FRAGMENT of pAAVNEWTBG-BI-aGAL
             parent position: from 5114 to 316
             original length: 896
             molecule position: from 3895 to 4790
            Left Terminus
              BamHI site #1
            Right Terminus
              XhoI site #1
            http://www.informaxinc.com/
FEATURES             Location/Qualifiers
     source          1..6967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..185
                     /label=5'ITR
                     /label=ITR
     promoter        255..495
                     /label=U6 Promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     276..310
                     /label=U6ForSeq
     primer_bind     complement(428..455)
                     /label=U6RevSeq
     misc_feature    504..525
                     /label=SA-mLdlrEx14-gRNA2-N22
     misc_feature    526..606
                     /label=sgRNA scaffold
     misc_feature    526..539
                     /label=crRNA Direct Repeat Portion +1 to +14
     misc_feature    540..543
                     /label=synthetic tetraloop
     misc_feature    544..606
                     /label=TracrRNA
     misc_feature    607
                     /label=Additional ""T"" introduced in gblock
     misc_feature    608..613
                     /label=Cloning Site for Additional Guide RNA cassettes
     misc_feature    620..871
                     /label=HLP Promoter (liver specific)
     primer_bind     718..739
                     /label=HLPForSeq
     primer_bind     complement(837..859)
                     /label=HLPReverseSeq
     regulatory      878..887
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    882..4228
                     /label=cloned from px602 by NcoI and Eco53k1/NruI fusion
     CDS             890..910
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    935..4090
                     /label=hSaCas9
     misc_feature    complement(953..973)
                     /label=anti-SaCas9 gRNA1 near D10
     misc_feature    1334..1336
                     /label=Ub high (AAA = WT, AGA = deUb)
     misc_feature    2908..2931
                     /label=WRL_0065
     misc_feature    3138
                     /label=New Seq shows G
     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"
     misc_feature    4172..4213
                     /label=OLLAS tag
     polyA_signal    4229..4286
                     /label=Syn PolyA
     primer_bind     complement(4250..4279)
                     /label=SynPolyARevSeq
     misc_feature    4287..4459
                     /label=3'ITR
                     /label=ITR
     misc_feature    complement(4484..6899)
                     /label=pEMBL8 Plasmid Backbone
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 accaaagagc 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 gaggaaaagc 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 caaaaccgca 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 aaggacagta 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 acgttgttac
     5761 cattactaca 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 gatcttacca ctattgagat 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 aagatgccac ctgaaattat aaacgttaat attttgttaa aattcgcgtt
     6481 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggaa aaatccctta
     6541 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     6601 actattgagg aacgtgaact ccagcgtcaa agggcgaaaa accgtctatc ggggcgatgg
     6661 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     6721 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     6781 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     6841 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     6901 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     6961 attacgc
//
