LOCUS       sequence-206102-        7170 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Plasmid for AAV SaCas9 Mammalian Expression with a BbsI cloning site
            for a gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7170)
  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 Jul 19. pii: ATVBAHA.118.311221.
            doi: 10.1161/ATVBAHA.118.311221.
  PUBMED    30026278
REFERENCE   2  (bases 1 to 7170)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Arterioscler Thromb Vasc Biol. 2018 Jul 19. pii: 
            ATVBAHA.118.311221. doi: 10.1161/ATVBAHA.118.311221."
COMMENT     Sequence Label: 
            1255_pAAV-U6-SA-BbsI-MluI-gRNA-CB-SACas9-HA-OLLAS-spA
FEATURES             Location/Qualifiers
     source          1..7170
                     /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"
     CDS             1093..1113
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1138..4293
                     /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             4294..4341
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4348..4374
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4375..4416
                     /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    4434..4482
                     /note="synthetic polyadenylation signal"
     repeat_region   4530..4659
                     /label=AAV2 ITR
     rep_origin      complement(4896..5484)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4976..4995)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5655..6446,6447..6515))
                     /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     6278..6297
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6516..6620)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6647..7102)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6784..6805)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6996..7015
                     /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 accgggtctt cacgcgtgaa gaccagtttt agtactctgg
      541 aaacagaatc tactaaaaca aggcaaaatg ccgtgtttat ctcgtcaact tgttggcgag
      601 atttttttct agacccagct ttcaagcttg attaacccgc catgctactt atctacgtag
      661 ccatgctcta ggaagatcgg aattcgccct taagctagta tgccaagtac gccccctatt
      721 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      781 tttcctactt ggcagtacat ctactcgagg ccacgttctg cttcactctc cccatctccc
      841 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
      901 gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc
      961 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
     1021 cgaagcgcgc ggcgggcggg agcgggatca gaatgatctg atatcatcga tgaattcgag
     1081 ctcaccatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccaag
     1141 cggaactaca tcctgggcct ggacatcggc atcaccagcg tgggctacgg catcatcgac
     1201 tacgagacac gggacgtgat cgatgccggc gtgcggctgt tcaaagaggc caacgtggaa
     1261 aacaacgagg gcaggcggag caagagaggc gccagaaggc tgaagcggcg gaggcggcat
     1321 agaatccaga gagtgaagaa gctgctgttc gactacaacc tgctgaccga ccacagcgag
     1381 ctgagcggca tcaaccccta cgaggccaga gtgaagggcc tgagccagaa gctgagcgag
     1441 gaagagttct ctgccgccct gctgcacctg gccaagagaa gaggcgtgca caacgtgaac
     1501 gaggtggaag aggacaccgg caacgagctg tccaccaaag agcagatcag ccggaacagc
     1561 aaggccctgg aagagaaata cgtggccgaa ctgcagctgg aacggctgaa gaaagacggc
     1621 gaagtgcggg gcagcatcaa cagattcaag accagcgact acgtgaaaga agccaaacag
     1681 ctgctgaagg tgcagaaggc ctaccaccag ctggaccaga gcttcatcga cacctacatc
     1741 gacctgctgg aaacccggcg gacctactat gagggacctg gcgagggcag ccccttcggc
     1801 tggaaggaca tcaaagaatg gtacgagatg ctgatgggcc actgcaccta cttccccgag
     1861 gaactgcgga gcgtgaagta cgcctacaac gccgacctgt acaacgccct gaacgacctg
     1921 aacaatctcg tgatcaccag ggacgagaac gagaagctgg aatattacga gaagttccag
     1981 atcatcgaga acgtgttcaa gcagaagaag aagcccaccc tgaagcagat cgccaaagaa
     2041 atcctcgtga acgaagagga tattaagggc tacagagtga ccagcaccgg caagcccgag
     2101 ttcaccaacc tgaaggtgta ccacgacatc aaggacatta ccgcccggaa agagattatt
     2161 gagaacgccg agctgctgga tcagattgcc aagatcctga ccatctacca gagcagcgag
     2221 gacatccagg aagaactgac caatctgaac tccgagctga cccaggaaga gatcgagcag
     2281 atctctaatc tgaagggcta taccggcacc cacaacctga gcctgaaggc catcaacctg
     2341 atcctggacg agctgtggca caccaacgac aaccagatcg ctatcttcaa ccggctgaag
     2401 ctggtgccca agaaggtgga cctgtcccag cagaaagaga tccccaccac cctggtggac
     2461 gacttcatcc tgagccccgt cgtgaagaga agcttcatcc agagcatcaa agtgatcaac
     2521 gccatcatca agaagtacgg cctgcccaac gacatcatta tcgagctggc ccgcgagaag
     2581 aactccaagg acgcccagaa aatgatcaac gagatgcaga agcggaaccg gcagaccaac
     2641 gagcggatcg aggaaatcat ccggaccacc ggcaaagaga acgccaagta cctgatcgag
     2701 aagatcaagc tgcacgacat gcaggaaggc aagtgcctgt acagcctgga agccatccct
     2761 ctggaagatc tgctgaacaa ccccttcaac tatgaggtgg accacatcat ccccagaagc
     2821 gtgtccttcg acaacagctt caacaacaag gtgctcgtga agcaggaaga aaacagcaag
     2881 aagggcaacc ggaccccatt ccagtacctg agcagcagcg acagcaagat cagctacgaa
     2941 accttcaaga agcacatcct gaatctggcc aagggcaagg gcagaatcag caagaccaag
     3001 aaagagtatc tgctggaaga acgggacatc aacaggttct ccgtgcagaa agacttcatc
     3061 aaccggaacc tggtggatac cagatacgcc accagaggcc tgatgaacct gctgcggagc
     3121 tacttcagag tgaacaacct ggacgtgaaa gtgaagtcca tcaatggcgg cttcaccagc
     3181 tttctgcggc ggaagtggaa gtttaagaaa gagcggaaca aggggtacaa gcaccacgcc
     3241 gaggacgccc tgatcattgc caacgccgat ttcatcttca aagagtggaa gaaactggac
     3301 aaggccaaaa aagtgatgga aaaccagatg ttcgaggaaa agcaggccga gagcatgccc
     3361 gagatcgaaa ccgagcagga gtacaaagag atcttcatca ccccccacca gatcaagcac
     3421 attaaggact tcaaggacta caagtacagc caccgggtgg acaagaagcc taatagagag
     3481 ctgattaacg acaccctgta ctccacccgg aaggacgaca agggcaacac cctgatcgtg
     3541 aacaatctga acggcctgta cgacaaggac aatgacaagc tgaaaaagct gatcaacaag
     3601 agccccgaaa agctgctgat gtaccaccac gacccccaga cctaccagaa actgaagctg
     3661 attatggaac agtacggcga cgagaagaat cccctgtaca agtactacga ggaaaccggg
     3721 aactacctga ccaagtactc caaaaaggac aacggccccg tgatcaagaa gattaagtat
     3781 tacggcaaca aactgaacgc ccatctggac atcaccgacg actaccccaa cagcagaaac
     3841 aaggtcgtga agctgtccct gaagccctac agattcgacg tgtacctgga caatggcgtg
     3901 tacaagttcg tgaccgtgaa gaatctggat gtgatcaaaa aagaaaacta ctacgaagtg
     3961 aatagcaagt gctatgagga agctaagaag ctgaagaaga tcagcaacca ggccgagttt
     4021 atcgcctcct tctacaacaa cgatctgatc aagatcaacg gcgagctgta tagagtgatc
     4081 ggcgtgaaca acgacctgct gaaccggatc gaagtgaaca tgatcgacat cacctaccgc
     4141 gagtacctgg aaaacatgaa cgacaagagg ccccccagga tcattaagac aatcgcctcc
     4201 aagacccaga gcattaagaa gtacagcaca gacattctgg gcaacctgta tgaagtgaaa
     4261 tctaagaagc accctcagat catcaaaaag ggcaaaaggc cggcggccac gaaaaaggcc
     4321 ggccaggcaa aaaagaaaaa gggatcctac ccatacgatg ttccagatta cgctagcggc
     4381 ttcgccaacg agcttggacc caggttgatg ggaaagtaag aattcctaga gcgaataaaa
     4441 gatctttatt ttcattagat ctgtgtgttg gttttttgtg tgatgcagct acgtagataa
     4501 gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc
     4561 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4621 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcattaatg aatcggccaa
     4681 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4741 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4801 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaacc gcagcaaaag
     4861 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4921 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4981 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5041 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5101 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5161 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5221 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5281 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     5341 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5401 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5461 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5521 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5581 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5641 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5701 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5761 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5821 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5881 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5941 aatagtttgc gcaacgttgt taccattact acaggcatcg tggtgtcacg ctcgtcgttt
     6001 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6061 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6121 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6181 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6241 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6301 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6361 ccactattga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6421 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6481 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6541 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6601 aaacaaatag gggttccgcg cacatttccc cgaaagatgc cacctgaaat tataaacgtt
     6661 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     6721 gccgaaatcg gaaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     6781 gttccagttt ggaacaagag tccactattg aggaacgtga actccagcgt caaagggcga
     6841 aaaaccgtct atcggggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     6901 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     6961 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     7021 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     7081 aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg
     7141 cgatcggtgc gggcctcttc gctattacgc
//
