LOCUS Exported 6841 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV genome: expresses the C-terminal of S. aureus v5 AAV-CBE from the Cbh promoter, and U6-sgRNA. ACCESSION . VERSION . KEYWORDS Cbh_v5 AAV-saCBE C-terminal SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6841) 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 6841) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6841 /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 5631..5879 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5922..5942 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(6007..6027) /label=WPRE-R /note="WPRE, reverse primer" polyA_signal 6203..6427 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter complement(6554..6794) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(6604..6623) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(6774..6794) /label=hU6-F /note="Human U6 promoter, forward primer" repeat_region 6818..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 tgtctgtatt ctctggaggc catccctctg gaggacctgc 4081 tgaacaatcc attcaactac gaggtggatc acatcatccc ccggagcgtg agcttcgaca 4141 attcttttaa caataaggtg ctggtgaagc aggaggagaa cagcaagaag ggcaatagga 4201 cccctttcca gtacctgtct agctccgatt ctaagatcag ctacgagaca ttcaagaagc 4261 acatcctgaa tctggccaag ggcaagggcc gcatcagcaa gaccaagaag gagtacctgc 4321 tggaggagcg ggacatcaac agattctccg tgcagaagga cttcatcaac cggaatctgg 4381 tggacaccag atacgccaca cgcggcctga tgaatctgct gcggtcttat ttcagagtga 4441 acaatctgga tgtgaaggtg aagagcatca acggcggctt cacctccttt ctgcggagaa 4501 agtggaagtt taagaaggag cgcaacaagg gctataagca ccacgccgag gatgccctga 4561 tcatcgccaa tgccgacttc atctttaagg agtggaagaa gctggacaag gccaagaaag 4621 tgatggagaa ccagatgttc gaggagaagc aggccgagag catgcccgag atcgagacag 4681 agcaggagta caaggagatt ttcatcacac ctcaccagat caagcacatc aaggacttca 4741 aggactacaa gtattctcac agggtggata agaagcccaa ccgcgagctg atcaatgaca 4801 ccctgtatag cacacggaag gacgataagg gcaataccct gatcgtgaac aatctgaacg 4861 gcctgtacga caaggataat gacaagctga agaagctgat caacaagtct cccgagaagc 4921 tgctgatgta ccaccacgat cctcagacat atcagaagct gaagctgatc atggagcagt 4981 acggcgacga gaagaaccca ctgtataagt actatgagga gacaggcaac tacctgacaa 5041 agtatagcaa gaaggataat ggccccgtga tcaagaagat caagtactat ggcaacaagc 5101 tgaatgccca cctggacatc accgacgatt accctaactc tcgcaataag gtggtgaagc 5161 tgagcctgaa gccataccgg ttcgacgtgt acctggacaa cggcgtgtat aagtttgtga 5221 cagtgaagaa tctggatgtg atcaagaagg agaactacta tgaggtgaac agcaagtgct 5281 acgaggaggc caagaagctg aagaagatca gcaaccaggc cgagttcatc gcctcttttt 5341 acaacaatga cctgatcaag atcaatggcg agctgtatag agtgatcggc gtgaacaatg 5401 atctgctgaa cagaatcgaa gtgaatatga tcgacatcac ctacagggag tatctggaga 5461 acatgaatga taagaggccc cctcgcatca tcaagaccat cgcctctaag acacagagca 5521 tcaagaagta cagcacagac atcctgggga acctgtatga agtcaagagc aagaaacatc 5581 ctcagattat caagaaaggc agcggcggga gcggcgggag cggggggagc actaatctga 5641 gcgacatcat tgagaaggag actgggaaac agctggtcat tcaggagtcc atcctgatgc 5701 tgcctgagga ggtggaggaa gtgatcggca acaagccaga gtctgacatc ctggtgcaca 5761 ccgcctacga cgagtccaca gatgagaatg tgatgctgct gacctctgac gcccccgagt 5821 ataagccttg ggccctggtc atccaggatt ctaacggcga gaataagatc aagatgctga 5881 gcggaggatc caaaagaacc gccgacggca gcgaattcga gcccaagaag aagaggaaag 5941 tctaagatct gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct 6001 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc 6061 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tagttcttgc 6121 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg 6181 cactgacaat tccgtggtgc gactgtgcct tctagttgcc agccatctgt tgtttgcccc 6241 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat 6301 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg 6361 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc 6421 tctatggctc gagaaaaaaa tctcgccaac aagttgacga gataaacacg gcattttgcc 6481 ttgttttagt agattctgta attttcatta cagagtacta aaaccgagac gcattaatcg 6541 tctcgggtgt ttcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa 6601 gttacggtaa gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca 6661 agaaattatt actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa 6721 ttaattctaa ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg 6781 ggaaataggc cctcttcctg cccgaccttg cggccgcagg aacccctagt gatggagttg 6841 g //