LOCUS Exported 6840 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV genome: expresses the C-terminal of S. aureus v5 AAV-CBE, and U6-sgRNA (KKH variant).. ACCESSION . VERSION . KEYWORDS Cbh_v5 AAV-saCBE_KKH C-terminal SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6840) 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 6840) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6840 /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..3632 /label=chicken beta-actin promoter intron 3633..3860 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 3914..3934 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5630..5878 /codon_start=1 /product="uracil-DNA glycosylase inhibitor from a Bacillus subtilis bacteriophage (Mol et al., 1995)" /label=UGI /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML" CDS 5921..5941 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(6006..6026) /label=WPRE-R /note="WPRE, reverse primer" polyA_signal 6202..6426 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter complement(6553..6793) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(6603..6622) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(6773..6793) /label=hU6-F /note="Human U6 promoter, forward primer" repeat_region 6817..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 ggggggcgcg 3481 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg 3541 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc 3601 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg 3661 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc 3721 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt 3781 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc 3841 ctgaaatcac tttttttcag gttggaccgg tgccaccatg aaacggacag ccgacggaag 3901 cgagttcgag tcaccaaaga agaagcggaa agtcatcaag attgctacac ggaaatacct 3961 gggaaagcag aacgtgtacg acatcggcgt ggagcgggat cacaacttcg ccctgaagaa 4021 tggctttatc gccagcaatt gtctgtattc tctggaggcc atccctctgg aggacctgct 4081 gaacaatcca ttcaactacg aggtggatca catcatcccc cggagcgtga gcttcgacaa 4141 ttcttttaac aataaggtgc tggtgaagca ggaggagaac agcaagaagg gcaataggac 4201 ccctttccag tacctgtcta gctccgattc taagatcagc tacgagacat tcaagaagca 4261 catcctgaat ctggccaagg gcaagggccg catcagcaag accaagaagg agtacctgct 4321 ggaggagcgg gacatcaaca gattctccgt gcagaaggac ttcatcaacc ggaatctggt 4381 ggacaccaga tacgccacac gcggcctgat gaatctgctg cggtcttatt tcagagtgaa 4441 caatctggat gtgaaggtga agagcatcaa cggcggcttc acctcctttc tgcggagaaa 4501 gtggaagttt aagaaggagc gcaacaaggg ctataagcac cacgccgagg atgccctgat 4561 catcgccaat gccgacttca tctttaagga gtggaagaag ctggacaagg ccaagaaagt 4621 gatggagaac cagatgttcg aggagaagca ggccgagagc atgcccgaga tcgagacaga 4681 gcaggagtac aaggagattt tcatcacacc tcaccagatc aagcacatca aggacttcaa 4741 ggactacaag tattctcaca gggtggataa gaagcccaac cgcaagctga tcaatgacac 4801 cctgtatagc acacggaagg acgataaggg caataccctg atcgtgaaca atctgaacgg 4861 cctgtacgac aaggataatg acaagctgaa gaagctgatc aacaagtctc ccgagaagct 4921 gctgatgtac caccacgatc ctcagacata tcagaagctg aagctgatca tggagcagta 4981 cggcgacgag aagaacccac tgtataagta ctatgaggag acaggcaact acctgacaaa 5041 gtatagcaag aaggataatg gccccgtgat caagaagatc aagtactatg gcaacaagct 5101 gaatgcccac ctggacatca ccgacgatta ccctaactct cgcaataagg tggtgaagct 5161 gagcctgaag ccataccggt tcgacgtgta cctggacaac ggcgtgtata agtttgtgac 5221 agtgaagaat ctggatgtga tcaagaagga gaactactat gaggtgaaca gcaagtgcta 5281 cgaggaggcc aagaagctga agaagatcag caaccaggcc gagttcatcg cctcttttta 5341 caagaatgac ctgatcaaga tcaatggcga gctgtataga gtgatcggcg tgaacaatga 5401 tctgctgaac agaatcgaag tgaatatgat cgacatcacc tacagggagt atctggagaa 5461 catgaatgat aagaggcccc ctcatatcat caagaccatc gcctctaaga cacagagcat 5521 caagaagtac agcacagaca tcctggggaa cctgtatgaa gtcaagagca agaaacatcc 5581 tcagattatc aagaaaggca gcggcgggag cggcgggagc ggggggagca ctaatctgag 5641 cgacatcatt gagaaggaga ctgggaaaca gctggtcatt caggagtcca tcctgatgct 5701 gcctgaggag gtggaggaag tgatcggcaa caagccagag tctgacatcc tggtgcacac 5761 cgcctacgac gagtccacag atgagaatgt gatgctgctg acctctgacg cccccgagta 5821 taagccttgg gccctggtca tccaggattc taacggcgag aataagatca agatgctgag 5881 cggaggatcc aaaagaaccg ccgacggcag cgaattcgag cccaagaaga agaggaaagt 5941 ctaagatctg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 6001 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 6061 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt agttcttgcc 6121 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc 6181 actgacaatt ccgtggtgcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 6241 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 6301 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 6361 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 6421 ctatggctcg agaaaaaaat ctcgccaaca agttgacgag ataaacacgg cattttgcct 6481 tgttttagta gattctgtaa ttttcattac agagtactaa aaccgagacg cattaatcgt 6541 ctcgggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag 6601 ttacggtaag catatgatag tccattttaa aacataattt taaaactgca aactacccaa 6661 gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat 6721 taattctaat tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg 6781 gaaataggcc ctcttcctgc ccgaccttgc ggccgcagga acccctagtg atggagttgg //