LOCUS Exported 8267 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV genome: expresses the C-terminal of v5 AAV-ABE from the Cbh promoter, and U6-sgRNA. ACCESSION . VERSION . KEYWORDS Cbh_v5 AAV-ABE C-terminal SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8267) AUTHORS Levy JM, Yeh WH, Pendse N, Davis JR, Hennessey E, Butcher R, Koblan LW, Comander J, Liu Q, Liu DR TITLE Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. JOURNAL Nat Biomed Eng. 2020 Jan;4(1):97-110. doi: 10.1038/s41551-019-0501-5. Epub 2020 Jan 14. PUBMED 31937940 REFERENCE 2 (bases 1 to 8267) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8267 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(125..142) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(125..141) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(134..156) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 283..738 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(370..389) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 580..601 /label=F1ori-F /note="F1 origin, forward primer" promoter 764..868 /gene="bla" /label=AmpR promoter CDS 869..1729 /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(1087..1106) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1900..2488 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2389..2408 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2642..2659 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2776..2797 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2812..2842 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2850..2866 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 2855..2877 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2874..2890 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2874..2890 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" repeat_region 2917..3046 /label=AAV2 ITR enhancer 3069..3354 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 3356..3633 /label=chicken beta-actin promoter intron 3634..3861 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 3915..3935 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4041..6425 /codon_start=1 /product="C-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(C) /translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD" CDS 6468..6488 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(6553..6573) /label=WPRE-R /note="WPRE, reverse primer" polyA_signal 6749..6973 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" misc_RNA complement(6987..7062) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 7076..7103 /label=T7Te terminator /note="phage T7 early transcription terminator" terminator 7119..7190 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" CDS complement(7208..7885) /codon_start=1 /product="monomeric derivative of DsRed (Campbell et al., 2002)" /label=mRFP1 /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS TGA" RBS 7892..7903 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" protein_bind 7921..7939 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 7946..7964 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" promoter complement(7980..8220) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(8030..8049) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(8200..8220) /label=hU6-F /note="Human U6 promoter, forward primer" repeat_region 8244..106 /label=AAV2 ITR ORIGIN 1 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 61 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaccta 121 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 181 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 241 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg 301 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 361 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 421 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 481 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 541 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 601 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 661 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 721 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 781 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 841 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 901 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 961 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 1021 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 1081 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 1141 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 1201 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 1261 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 1321 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 1381 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 1441 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 1501 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 1561 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 1621 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1681 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1741 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1801 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1861 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1921 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1981 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 2041 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 2101 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 2161 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 2221 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 2281 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 2341 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 2401 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 2461 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 2521 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 2581 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 2641 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc 2701 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 2761 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt 2821 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 2881 cagctatgac catgattacg ccagatttaa ttaaggctgc gcgctcgctc gctcactgag 2941 gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag 3001 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagatc 3061 agggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc 3121 cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca atgggtggag 3181 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc 3241 cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta catgacctta 3301 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag 3361 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg 3421 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc 3481 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg 3541 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg 3601 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc 3661 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 3721 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg 3781 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg 3841 cctgaaatca ctttttttca ggttggaccg gtgccaccat gaaacggaca gccgacggaa 3901 gcgagttcga gtcaccaaag aagaagcgga aagtcatcaa gattgctaca cggaaatacc 3961 tgggaaagca gaacgtgtac gacatcggcg tggagcggga tcacaacttc gccctgaaga 4021 atggctttat cgccagcaat tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt 4081 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc 4141 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg 4201 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag 4261 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga 4321 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg 4381 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg 4441 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc 4501 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc 4561 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga 4621 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg 4681 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc 4741 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg 4801 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc 4861 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga 4921 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc 4981 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg 5041 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga 5101 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg 5161 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc 5221 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg 5281 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg 5341 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc 5401 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca 5461 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg 5521 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc 5581 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca 5641 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg 5701 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg 5761 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca 5821 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct 5881 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg 5941 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg 6001 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg 6061 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg 6121 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc 6181 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg 6241 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact 6301 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc 6361 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag 6421 gtgactctgg cggctcaaaa agaaccgccg acggcagcga attcgagccc aagaagaaga 6481 ggaaagtcta agatctgata atcaacctct ggattacaaa atttgtgaaa gattgactgg 6541 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta 6601 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttagt 6661 tcttgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 6721 gttgggcact gacaattccg tggtgcgact gtgccttcta gttgccagcc atctgttgtt 6781 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 6841 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 6901 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 6961 gtgggctcta tggctcgaga aaaaaagcac cgactcggtg ccactttttc aagttgataa 7021 cggactagcc ttattttaac ttgctatttc tagctctaaa accgagacgt ataaacgcag 7081 aaaggcccac ccgaaggtga gccagtgtga ctctagtaga gagcgttcac cgacaaacaa 7141 cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctggag 7201 atccttatta agcacccgtg gagtgacgac cttcagcacg ttcgtactgt tcaacgatgg 7261 tgtagtcttc gttgtgggag gtgatgtcca gtttgatgtc ggttttgtaa gcacccggca 7321 gctgaacggg ttttttagcc atgtaggtgg ttttaacttc agcgtcgtag tgaccaccgt 7381 ctttcagttt cagacgcatt ttgatttcac ctttcagagc accgtcttcc gggtacatac 7441 gttcggtgga agcttcccaa cccatggttt ttttctgcat aaccggaccg tcggacggga 7501 agttggtacc acgcagttta actttgtaga tgaactcacc gtcttgcagg gaggagtcct 7561 gggtaacggt aacaacacca ccgtcttcga agttcataac acgttcccat ttgaaacctt 7621 ccgggaagga cagtttcagg tagtccggga tgtcagccgg gtgtttaacg taagctttgg 7681 aaccgtactg gaactgcggg gacaggatgt cccaagcgaa cggcagcgga ccacctttgg 7741 taactttcag tttagcggtc tgggtacctt cgtacggacg accttcacct tcaccttcga 7801 tttcgaactc gtgaccgtta acggaacctt ccatacgaac tttgaaacgc atgaactctt 7861 tgataacgtc ttcgctactc gccatagatc ctttctcctc tttcagatcc gtgctcagta 7921 tctctatcac tgatagggat gtcaatctct atcactgata gggacgtctc gggtgtttcg 7981 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat 8041 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt 8101 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttctaattat 8161 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc 8221 ttcctgcccg accttgcggc cgcaggaacc cctagtgatg gagttgg //