LOCUS Exported 7691 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV genome: expresses the N-terminal of v5 AAV-CBE from the Cbh promoter, and U6-sgRNA. ACCESSION . VERSION . KEYWORDS Cbh_v5 AAV-CBE N-terminal SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7691) 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 7691) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7691 /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 3936..4619 /codon_start=1 /product="cytidine deaminase (C to U editing enzyme) from rat" /label=APOBEC-1 /note="can use ssDNA as a substrate (Komor et al., 2016)" /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL WATGLK" CDS 4716..6431 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" CDS 6780..6800 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(6865..6885) /label=WPRE-R /note="WPRE, reverse primer" polyA_signal 7061..7285 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" misc_RNA complement(7299..7374) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(7404..7644) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(7454..7473) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(7624..7644) /label=hU6-F /note="Human U6 promoter, forward primer" repeat_region 7668..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 aagtctcctc agagactggg cctgtcgccg 3961 tcgatccaac cctgcgccgc cggattgaac ctcacgagtt tgaagtgttc tttgaccccc 4021 gggagctgag aaaggagaca tgcctgctgt acgagatcaa ctggggaggc aggcactcca 4081 tctggaggca cacctctcag aacacaaata agcacgtgga ggtgaacttc atcgagaagt 4141 ttaccacaga gcggtacttc tgccccaata ccagatgtag catcacatgg tttctgagct 4201 ggtccccttg cggagagtgt agcagggcca tcaccgagtt cctgtccaga tatccacacg 4261 tgacactgtt tatctacatc gccaggctgt atcaccacgc agacccaagg aataggcagg 4321 gcctgcgcga tctgatcagc tccggcgtga ccatccagat catgacagag caggagtccg 4381 gctactgctg gcggaacttc gtgaattatt ctcctagcaa cgaggcccac tggcctaggt 4441 acccacacct gtgggtgcgc ctgtacgtgc tggagctgta ttgcatcatc ctgggcctgc 4501 ccccttgtct gaatatcctg cggagaaagc agccccagct gaccttcttt acaatcgccc 4561 tgcagtcttg tcactatcag aggctgccac cccacatcct gtgggccaca ggcctgaagt 4621 ctggaggatc tagcggagga tcctctggca gcgagacacc aggaacaagc gagtcagcaa 4681 caccagagag cagtggcggc agcagcggcg gcagcgacaa gaagtacagc atcggcctgg 4741 ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca 4801 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag 4861 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa 4921 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga 4981 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg 5041 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg 5101 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg 5161 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga 5221 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc 5281 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg 5341 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc 5401 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc 5461 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca 5521 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt 5581 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca 5641 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc 5701 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga 5761 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg 5821 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc 5881 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca 5941 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt 6001 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct 6061 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg 6121 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga 6181 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca 6241 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg 6301 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg 6361 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca 6421 agaaaatcga gtgcctgtcc tacgagacag agatcctgac agtggagtat ggcctgctgc 6481 caatcggcaa gatcgtggag aagaggatcg agtgtaccgt gtactctgtg gataacaatg 6541 gcaacatcta tacacagccc gtggcacagt ggcacgatag gggagagcag gaggtgttcg 6601 agtattgcct ggaggacggc agcctgatca gggcaaccaa ggaccacaag ttcatgacag 6661 tggatggcca gatgctgccc atcgacgaga ttttcgagcg ggagctggac ctgatgagag 6721 tggataacct gcctaatagc ggaggcagta aaagaacagc agacgggagt gagtttgagc 6781 ccaagaaaaa gagaaaggtg taagatctga taatcaacct ctggattaca aaatttgtga 6841 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt 6901 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa 6961 atcctggtta gttcttgcca cggcggaact catcgccgcc tgccttgccc gctgctggac 7021 aggggctcgg ctgttgggca ctgacaattc cgtggtgcga ctgtgccttc tagttgccag 7081 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 7141 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 7201 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat 7261 gctggggatg cggtgggctc tatggctcga gaaaaaaagc accgactcgg tgccactttt 7321 tcaagttgat aacggactag ccttatttta acttgctatt tctagctcta aaaccgagac 7381 gcattaatcg tctcgggtgt ttcgtccttt ccacaagata tataaagcca agaaatcgaa 7441 atactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt ttaaaactgc 7501 aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata tctttgtgtt 7561 tacagtcaaa ttaattctaa ttatctctct aacagccttg tatcgtatat gcaaatatga 7621 aggaatcatg ggaaataggc cctcttcctg cccgaccttg cggccgcagg aacccctagt 7681 gatggagttg g //