LOCUS       sequence-213156-        7164 bp    DNA     circular SYN 15-DEC-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 7164)
  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 7164)
  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: 
            "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..7164
                     /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             1088..1108
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4289..4336
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4343..4369
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4370..4411
                     /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    4429..4477
                     /note="synthetic polyadenylation signal"
     repeat_region   4525..4654
                     /label=AAV2 ITR
     rep_origin      complement(4891..5479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4971..4990)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5650..6441,6442..6510))
                     /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     6273..6292
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6511..6615)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6641..7096)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6778..6799)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6990..7009
                     /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 gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc
      961 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
     1021 cgcgcggcgg gcgggagcgg gatcagaatg atctgatatc atcgatgaat tcgagctcac
     1081 catggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa
     1141 ctacatcctg ggcctggaca tcggcatcac cagcgtgggc tacggcatca tcgactacga
     1201 gacacgggac gtgatcgatg ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa
     1261 cgagggcagg cggagcaaga gaggcgccag aaggctgaag cggcggaggc ggcatagaat
     1321 ccagagagtg aagaagctgc tgttcgacta caacctgctg accgaccaca gcgagctgag
     1381 cggcatcaac ccctacgagg ccagagtgaa gggcctgagc cagaagctga gcgaggaaga
     1441 gttctctgcc gccctgctgc acctggccaa gagaagaggc gtgcacaacg tgaacgaggt
     1501 ggaagaggac accggcaacg agctgtccac cagagagcag atcagccgga acagcaaggc
     1561 cctggaagag aaatacgtgg ccgaactgca gctggaacgg ctgaagaaag acggcgaagt
     1621 gcggggcagc atcaacagat tcaagaccag cgactacgtg aaagaagcca aacagctgct
     1681 gaaggtgcag aaggcctacc accagctgga ccagagcttc atcgacacct acatcgacct
     1741 gctggaaacc cggcggacct actatgaggg acctggcgag ggcagcccct tcggctggaa
     1801 ggacatcaaa gaatggtacg agatgctgat gggccactgc acctacttcc ccgaggaact
     1861 gcggagcgtg aagtacgcct acaacgccga cctgtacaac gccctgaacg acctgaacaa
     1921 tctcgtgatc accagggacg agaacgagaa gctggaatat tacgagaagt tccagatcat
     1981 cgagaacgtg ttcaagcaga agaagaagcc caccctgaag cagatcgcca aagaaatcct
     2041 cgtgaacgaa gaggatatta agggctacag agtgaccagc accggcaagc ccgagttcac
     2101 caacctgaag gtgtaccacg acatcaagga cattaccgcc cggaaagaga ttattgagaa
     2161 cgccgagctg ctggatcaga ttgccaagat cctgaccatc taccagagca gcgaggacat
     2221 ccaggaagaa ctgaccaatc tgaactccga gctgacccag gaagagatcg agcagatctc
     2281 taatctgaag ggctataccg gcacccacaa cctgagcctg aaggccatca acctgatcct
     2341 ggacgagctg tggcacacca acgacaacca gatcgctatc ttcaaccggc tgaagctggt
     2401 gcccaagaag gtggacctgt cccagcagaa agagatcccc accaccctgg tggacgactt
     2461 catcctgagc cccgtcgtga agagaagctt catccagagc atcaaagtga tcaacgccat
     2521 catcaagaag tacggcctgc ccaacgacat cattatcgag ctggcccgcg agaagaactc
     2581 caaggacgcc cagaaaatga tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg
     2641 gatcgaggaa atcatccgga ccaccggcaa agagaacgcc aagtacctga tcgagaagat
     2701 caagctgcac gacatgcagg aaggcaagtg cctgtacagc ctggaagcca tccctctgga
     2761 agatctgctg aacaacccct tcaactatga ggtggaccac atcatcccca gaagcgtgtc
     2821 cttcgacaac agcttcaaca acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg
     2881 caaccggacc ccattccagt acctgagcag cagcgacagc aagatcagct acgaaacctt
     2941 caagaagcac atcctgaatc tggccaaggg caagggcaga atcagcaaga ccaagaaaga
     3001 gtatctgctg gaagaacggg acatcaacag gttctccgtg cagaaagact tcatcaaccg
     3061 gaacctggtg gataccagat acgccaccag aggcctgatg aacctgctgc ggagctactt
     3121 cagagtgaac aacctggacg tgaaagtgaa gtccatcaat ggcggcttca ccagctttct
     3181 gcggcggaag tggaagttta agaaagagcg gaacaagggg tacaagcacc acgccgagga
     3241 cgccctgatc attgccaacg ccgatttcat cttcaaagag tggaagaaac tggacaaggc
     3301 caaaaaagtg atggaaaacc agatgttcga ggaaaggcag gccgagagca tgcccgagat
     3361 cgaaaccgag caggagtaca aagagatctt catcaccccc caccagatca agcacattaa
     3421 ggacttcaag gactacaagt acagccaccg ggtggacaag aagcctaata gagagctgat
     3481 taacgacacc ctgtactcca cccggaagga cgacaagggc aacaccctga tcgtgaacaa
     3541 tctgaacggc ctgtacgaca aggacaatga caagctgaaa aagctgatca acaagagccc
     3601 cgaaaagctg ctgatgtacc accacgaccc ccagacctac cagaaactga agctgattat
     3661 ggaacagtac ggcgacgaga agaatcccct gtacaagtac tacgaggaaa ccgggaacta
     3721 cctgaccaag tactccaaaa aggacaacgg ccccgtgatc aagaagatta agtattacgg
     3781 caacaaactg aacgcccatc tggacatcac cgacgactac cccaacagca gaaacaaggt
     3841 cgtgaagctg tccctgaagc cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa
     3901 gttcgtgacc gtgaagaatc tggatgtgat caaaaaagaa aactactacg aagtgaatag
     3961 caagtgctat gaggaagcta agaagctgaa gaagatcagc aaccaggccg agtttatcgc
     4021 ctccttctac aacaacgatc tgatcaagat caacggcgag ctgtatagag tgatcggcgt
     4081 gaacaacgac ctgctgaacc ggatcgaagt gaacatgatc gacatcacct accgcgagta
     4141 cctggaaaac atgaacgaca agaggccccc caggatcatt aagacaatcg cctccaagac
     4201 ccagagcatt aagaagtaca gcacagacat tctgggcaac ctgtatgaag tgaaatctaa
     4261 gaagcaccct cagatcatca aaaagggcaa aaggccggcg gccacgaaaa aggccggcca
     4321 ggcaaaaaag aaaaagggat cctacccata cgatgttcca gattacgcta gcggcttcgc
     4381 caacgagctt ggacccaggt tgatgggaaa gtaagaattc ctagagcgaa taaaagatct
     4441 ttattttcat tagatctgtg tgttggtttt ttgtgtgatg cagctacgta gataagtagc
     4501 atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc
     4561 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     4621 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcat taatgaatcg gccaacgcgc
     4681 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4741 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4801 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4861 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4921 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4981 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5041 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5101 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5161 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5221 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5281 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5341 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5401 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5461 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5521 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5581 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5641 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5701 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5761 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5821 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5881 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5941 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6001 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6061 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6121 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6181 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6241 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6301 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6361 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6421 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6481 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6541 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6601 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     6661 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     6721 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     6781 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     6841 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     6901 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     6961 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     7021 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     7081 ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg
     7141 gtgcgggcct cttcgctatt acgc
//
