LOCUS Exported 7165 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid for AAV SaCas9 Mammalian Expression with a gRNA against Ldlr. ACCESSION . VERSION . KEYWORDS 1375_pAAV-U6-SA-WTmLdlrEx14-gRNA2-N22-CB-SACas9-HA-OLLAS-spA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7165) 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 7165) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7165 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 4..133 /label=AAV2 ITR promoter 255..495 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" 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 primer_bind complement(4720..4737) /label=L4440 /note="L4440 vector, forward primer" 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(5650..6510) /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 6273..6292 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6511..6615) /gene="bla" /label=AmpR promoter rep_origin complement(6642..7097) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6779..6800) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 6991..7010 /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 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 caaagagcag 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 aaaccgcagc 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 ggacagtatt 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 gttgttacca ttactacagg 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 tcttaccact 6361 attgagatcc 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 gatgccacct gaaattataa acgttaatat 6661 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga 6721 aatcggaaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc 6781 agtttggaac aagagtccac tattgaggaa cgtgaactcc agcgtcaaag ggcgaaaaac 6841 cgtctatcgg ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc 6901 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 6961 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag 7021 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc 7081 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc 7141 ggtgcgggcc tcttcgctat tacgc //