LOCUS Exported 7276 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Delivery of dual sgRNA cassettes and Nme2Cas9 in a single AAV vector. ACCESSION . VERSION . KEYWORDS pEJS1099: Dual-sgRNA.Design 4 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7276) AUTHORS Ibraheim R, Tai PWL, Mir A, Javeed N, Wang J, Rodriguez T, Nelson S, Khokhar E, Mintzer E, Maitland S, Cao Y, Tsagkaraki E, Wolfe SA, Wang D, Pai AA, Xue W, Gao G, Sontheimer EJ TITLE Precision Cas9 Genome Editing in vivo with All-in-one, Self-targeting AAV Vectors JOURNAL bioRxiv October 9, 2020 REFERENCE 2 (bases 1 to 7276) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7276 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter complement(285..525) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" regulatory 861..870 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 873..893 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4152..4199 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" promoter 4263..4503 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" repeat_region 4652..4792 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4652..4781 /label=AAV2 ITR rep_origin 4867..5322 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4954..4973) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5164..5185 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5339..5358) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 5491..5595 /gene="bla" /label=AmpR promoter CDS 5596..6456 /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 complement(5814..5833) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6627..7215 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7116..7135 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggcctcta gagtttaaac aaaaaaataa acgatgcccc ttaaagcaga 181 agctttaagg ggcagagcgt tgcggcacat cttttcagac ggccttattg tagcaacgtt 241 tcgggagcta caactgaaga gctggaatgc tcttccggtg tttcgtcctt tccacaagat 301 atataaagcc aagaaatcga aatactttca agttacggta agcatatgat agtccatttt 361 aaaacataat tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt 421 ttgtactaat atctttgtgt ttacagtcaa attaattcta attatctctc taacagcctt 481 gtatcgtata tgcaaatatg aaggaatcat gggaaatagg ccctcttcct gcccgacctt 541 gacgtcgact ctagaatgga ggcggtacta tgtagatgag aattcaggag caaactggga 601 aaagcaactg cttccaaata tttgtgattt ttacagtgta gttttggaaa aactcttagc 661 ctaccaattc ttctaagtgt tttaaaatgt gggagccagt acacatgaag ttatagagtg 721 ttttaatgag gcttaaatat ttaccgtaac tatgaaatgc tacgcatatc atgctgttca 781 ggctccgtgg ccacgcaact catacttaag cagacagtgg ttcaaagttt ttttcttcca 841 tttcaggtgt cgtgaacacc gccaccatgg tgcctaagaa gaagagaaag gtggaagata 901 tggccgcctt caagcctaac ccaatcaatt acatcctggg actggacatc ggaatcgcat 961 ccgtgggatg ggctatggtg gagatcgacg aggaggagaa tcctatccgg ctgatcgatc 1021 tgggcgtgag agtgtttgag agggccgagg tgccaaagac cggcgattct ctggctatgg 1081 cccggagact ggcacggagc gtgaggcgcc tgacacggag aagggcacac aggctgctga 1141 gggcacgccg gctgctgaag agagagggcg tgctgcaggc agcagacttc gatgagaatg 1201 gcctgatcaa gagcctgcca aacaccccct ggcagctgag agcagccgcc ctggacagga 1261 agctgacacc actggagtgg tctgccgtgc tgctgcacct gatcaagcac cgcggctacc 1321 tgagccagcg gaagaacgag ggagagacag cagacaagga gctgggcgcc ctgctgaagg 1381 gagtggccaa caatgcccac gccctgcaga ccggcgattt caggacacct gccgagctgg 1441 ccctgaataa gtttgagaag gagtccggcc acatcagaaa ccagaggggc gactatagcc 1501 acaccttctc ccgcaaggat ctgcaggccg agctgatcct gctgttcgag aagcagaagg 1561 agtttggcaa tccacacgtg agcggaggcc tgaaggaggg aatcgagacc ctgctgatga 1621 cacagaggcc tgccctgtcc ggcgacgcag tgcagaagat gctgggacac tgcaccttcg 1681 agcctgcaga gccaaaggcc gccaagaaca cctacacagc cgagcggttt atctggctga 1741 caaagctgaa caatctgaga atcctggagc agggatccga gaggccactg accgacacag 1801 agagggccac cctgatggat gagccttacc ggaagtctaa gctgacatat gcccaggcca 1861 gaaagctgct gggcctggag gacaccgcct tctttaaggg cctgagatac ggcaaggata 1921 atgccgaggc ctccacactg atggagatga aggcctatca cgccatctct cgcgccctgg 1981 agaaggaggg cctgaaggac aagaagtccc ccctgaacct gagctccgag ctgcaggatg 2041 agatcggcac cgccttctct ctgtttaaga ccgacgagga tatcacaggc cgcctgaagg 2101 acagggtgca gcctgagatc ctggaggccc tgctgaagca catctctttc gataagtttg 2161 tgcagatcag cctgaaggcc ctgagaagga tcgtgccact gatggagcag ggcaagcggt 2221 acgacgaggc ctgcgccgag atctacggcg atcactatgg caagaagaac acagaggaga 2281 agatctatct gccccctatc cctgccgacg agatcagaaa tcctgtggtg ctgagggccc 2341 tgtcccaggc aagaaaagtg atcaacggag tggtgcgccg gtacggatct ccagcccgga 2401 tccacatcga gaccgccaga gaagtgggca agagcttcaa ggaccggaag gagatcgaga 2461 agagacagga ggagaatcgc aaggatcggg agaaggccgc cgccaagttt agggagtact 2521 tccctaactt tgtgggcgag ccaaagtcta aggacatcct gaagctgcgc ctgtacgagc 2581 agcagcacgg caagtgtctg tatagcggca aggagatcaa tctggtgcgg ctgaacgaga 2641 agggctatgt ggagatcgat cacgccctgc ctttctccag aacctgggac gattctttta 2701 acaataaggt gctggtgctg ggcagcgaga accagaataa gggcaatcag acaccatacg 2761 agtatttcaa tggcaaggac aactccaggg agtggcagga gttcaaggcc cgcgtggaga 2821 cctctagatt tcccaggagc aagaagcagc ggatcctgct gcagaagttc gacgaggatg 2881 gctttaagga gtgcaacctg aatgacacca gatacgtgaa ccggttcctg tgccagtttg 2941 tggccgatca catcctgctg accggcaagg gcaagagaag ggtgttcgcc tctaatggcc 3001 agatcacaaa cctgctgagg ggattttggg gactgaggaa ggtgcgggca gagaatgaca 3061 gacaccacgc actggatgca gtggtggtgg catgcagcac cgtggcaatg cagcagaaga 3121 tcacaagatt cgtgaggtat aaggagatga acgcctttga cggcaagacc atcgataagg 3181 agacaggcaa ggtgctgcac cagaagaccc acttccccca gccttgggag ttctttgccc 3241 aggaagtgat gatccgggtg ttcggcaagc cagacggcaa gcctgagttt gaggaggccg 3301 ataccccaga gaagctgagg acactgctgg cagagaagct gtctagcagg ccagaggcag 3361 tgcacgagta cgtgacccca ctgttcgtgt ccagggcacc caatcggaag atgtctggcg 3421 cccacaagga cacactgaga agcgccaaga ggtttgtgaa gcacaacgag aagatctccg 3481 tgaagagagt gtggctgacc gagatcaagc tggccgatct ggagaacatg gtgaattaca 3541 agaacggcag ggagatcgag ctgtatgagg ccctgaaggc aaggctggag gcctacggag 3601 gaaatgccaa gcaggccttc gacccaaagg ataacccctt ttataagaag ggaggacagc 3661 tggtgaaggc cgtgcgggtg gagaagaccc aggagagcgg cgtgctgctg aataagaaga 3721 acgcctacac aatcgccgac aatggcgata tggtgagagt ggacgtgttc tgtaaggtgg 3781 ataagaaggg caagaatcag tactttatcg tgcctatcta tgcctggcag gtggccgaga 3841 acatcctgcc agacatcgat tgcaagggct acagaattga cgatagctat acattctgtt 3901 tttccctgca caagtatgac ctgatcgcct tccagaagga tgagaagtcc aaggtggagt 3961 ttgcctacta tatcaattgc gactcctcta acggcaggtt ctacctggcc tggcacgata 4021 agggcagcaa ggagcagcag tttcgcatct ccacccagaa tctggtgctg atccagaagt 4081 atcaggtgaa cgagctgggc aaggagatca ggccatgtcg gctgaagaag cgcccacccg 4141 tgcgggagga taagcggccc gcagcaacca agaaggcagg acaggccaag aagaagaagt 4201 aaaataaaag atctttattt tcattagatc tgtgtgttgg ttttttgtgt aagcgcacga 4261 atgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag 4321 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta 4381 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc 4441 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag 4501 gacgaaacac cggagacgat tccacgtctc agttgtagct cccgaaacgt tgctacaata 4561 aggccgtctg aaaagatgtg ccgcaacgct ctgcccctta aagcttctgc tttaaggggc 4621 atcgtttatt tttttgttta aacactaccg caggaacccc tagtgatgga gttggccact 4681 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg 4741 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg 4801 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac 4861 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 4921 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc 4981 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 5041 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta 5101 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 5161 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg 5221 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa 5281 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta 5341 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg 5401 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg ctcgagttag 5461 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 5521 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 5581 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 5641 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 5701 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 5761 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 5821 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 5881 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 5941 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 6001 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 6061 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 6121 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa 6181 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 6241 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 6301 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 6361 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 6421 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 6481 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 6541 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 6601 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 6661 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 6721 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta 6781 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 6841 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 6901 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 6961 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 7021 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 7081 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 7141 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 7201 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 7261 ccttttgctc acatgt //