LOCUS Exported 6012 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses the C-terminal split-intein fragment of ABEmax from the CMV promoter. ACCESSION . VERSION . KEYWORDS CMV_Npu-ABEmax C-terminal SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6012) 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 6012) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6012 /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 571..2955 /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 2998..3018 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3027..3044 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3067..3084) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3073..3297 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3368..3384) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3368..3384) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3381..3403) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3392..3408 /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(3416..3446) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3461..3482 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3599..3616) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3770..4358) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3850..3869) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4529..5389) /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 5152..5171 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5390..5494) /gene="bla" /label=AmpR promoter primer_bind complement(5573..5592) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 5764..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 aagtcatcaa gattgctaca 481 cggaaatacc tgggaaagca gaacgtgtac gacatcggcg tggagcggga tcacaacttc 541 gccctgaaga atggctttat cgccagcaat tgcttcgact ccgtggaaat ctccggcgtg 601 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac 661 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg 721 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc 781 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc 841 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg 901 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc 961 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac 1021 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg 1081 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg 1141 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg 1201 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac 1261 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac 1321 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct 1381 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac 1441 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg 1501 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc 1561 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa 1621 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac 1681 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg 1741 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac 1801 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag 1861 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc 1921 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg 1981 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc 2041 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 2101 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 2161 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag 2221 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg 2281 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 2341 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 2401 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 2461 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga 2521 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 2581 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 2641 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 2701 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 2761 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc 2821 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg 2881 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 2941 cagctgggag gtgactctgg cggctcaaaa agaaccgccg acggcagcga attcgagccc 3001 aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 3061 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 3121 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 3181 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 3241 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 3301 ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 3361 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 3421 tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 3481 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 3541 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 3601 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 3661 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 3721 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 3781 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 3841 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3901 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3961 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 4021 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 4081 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 4141 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 4201 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 4261 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 4321 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 4381 cggggtctga cactcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 4441 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 4501 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 4561 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 4621 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 4681 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 4741 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 4801 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 4861 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4921 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4981 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 5041 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 5101 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 5161 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 5221 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 5281 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 5341 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 5401 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 5461 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 5521 acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 5581 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 5641 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 5701 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 5761 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 5821 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 5881 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 5941 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 6001 atcaagtgta tc //