LOCUS Exported 6324 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses the N-terminal split-intein fragment of BE3.9max from the CMV promoter. ACCESSION . VERSION . KEYWORDS CMV_Npu-BE3.9max N-terminal SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6324) 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 6324) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6324 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 132..335 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 286..306 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 377..396 /label=T7 /note="T7 promoter, forward primer" promoter 377..395 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 445..465 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 466..1149 /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 1246..2961 /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 3310..3330 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3339..3356 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3379..3396) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3385..3609 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3680..3696) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3680..3696) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3693..3715) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3704..3720 /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)." promoter complement(3728..3758) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3773..3794 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3911..3928) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4082..4670) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4162..4181) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4841..5701) /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 5464..5483 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5702..5806) /gene="bla" /label=AmpR promoter primer_bind complement(5885..5904) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 6076..131 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca 421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctcctc agagactggg 481 cctgtcgccg tcgatccaac cctgcgccgc cggattgaac ctcacgagtt tgaagtgttc 541 tttgaccccc gggagctgag aaaggagaca tgcctgctgt acgagatcaa ctggggaggc 601 aggcactcca tctggaggca cacctctcag aacacaaata agcacgtgga ggtgaacttc 661 atcgagaagt ttaccacaga gcggtacttc tgccccaata ccagatgtag catcacatgg 721 tttctgagct ggtccccttg cggagagtgt agcagggcca tcaccgagtt cctgtccaga 781 tatccacacg tgacactgtt tatctacatc gccaggctgt atcaccacgc agacccaagg 841 aataggcagg gcctgcgcga tctgatcagc tccggcgtga ccatccagat catgacagag 901 caggagtccg gctactgctg gcggaacttc gtgaattatt ctcctagcaa cgaggcccac 961 tggcctaggt acccacacct gtgggtgcgc ctgtacgtgc tggagctgta ttgcatcatc 1021 ctgggcctgc ccccttgtct gaatatcctg cggagaaagc agccccagct gaccttcttt 1081 acaatcgccc tgcagtcttg tcactatcag aggctgccac cccacatcct gtgggccaca 1141 ggcctgaagt ctggaggatc tagcggagga tcctctggca gcgagacacc aggaacaagc 1201 gagtcagcaa caccagagag cagtggcggc agcagcggcg gcagcgacaa gaagtacagc 1261 atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag 1321 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac 1381 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga 1441 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc 1501 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg 1561 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg 1621 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc 1681 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc 1741 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc 1801 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg 1861 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc 1921 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg 1981 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg 2041 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac 2101 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg 2161 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac 2221 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag 2281 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga 2341 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc 2401 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac 2461 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag 2521 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc 2581 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc 2641 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct 2701 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag 2761 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa 2821 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag 2881 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag 2941 gactacttca agaaaatcga gtgcctgtcc tacgagacag agatcctgac agtggagtat 3001 ggcctgctgc caatcggcaa gatcgtggag aagaggatcg agtgtaccgt gtactctgtg 3061 gataacaatg gcaacatcta tacacagccc gtggcacagt ggcacgatag gggagagcag 3121 gaggtgttcg agtattgcct ggaggacggc agcctgatca gggcaaccaa ggaccacaag 3181 ttcatgacag tggatggcca gatgctgccc atcgacgaga ttttcgagcg ggagctggac 3241 ctgatgagag tggataacct gcctaatagc ggaggcagta aaagaacagc agacgggagt 3301 gagtttgagc ccaagaaaaa gagaaaggtg taaccggtca tcatcaccat caccattgag 3361 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 3421 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 3481 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 3541 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 3601 gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg acctctagct 3661 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 3721 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc taatgagtga 3781 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 3841 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 3901 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 3961 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 4021 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 4081 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 4141 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 4201 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 4261 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 4321 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 4381 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 4441 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 4501 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 4561 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 4621 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 4681 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 4741 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 4801 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 4861 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 4921 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 4981 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 5041 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 5101 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 5161 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 5221 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 5281 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 5341 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 5401 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 5461 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 5521 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 5581 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 5641 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 5701 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 5761 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 5821 aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc 5881 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt 5941 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg 6001 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg 6061 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt 6121 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 6181 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 6241 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 6301 acttggcagt acatcaagtg tatc //