LOCUS       Exported                8217 bp DNA     circular SYN 25-SEP-2024
DEFINITION  A-to-G base editor.
ACCESSION   .
VERSION     .
KEYWORDS    ABE8e
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8217)
  AUTHORS   Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, 
            Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR
  TITLE     Phage-assisted evolution of an adenine base editor with improved Cas
            domain compatibility and activity.
  JOURNAL   Nat Biotechnol. 2020 Jul;38(7):883-891. doi: 
            10.1038/s41587-020-0453-z. Epub 2020 Mar 16.
  PUBMED    32433547
REFERENCE   2  (bases 1 to 8217)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 25, 2024 from SnapGene 6.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41587-020-0453-z"; journalName: "Nat Biotechnol"; date: 
            "2020-07"; volume: "38"; issue: "7"; pages: "883-891"
FEATURES             Location/Qualifiers
     source          1..8217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        168..371
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     322..342
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     413..432
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        413..431
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             481..501
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1096..5196
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5239..5259
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5268..5285
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5308..5325)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5314..5538
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5609..5625)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5609..5625)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5622..5644)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5633..5649
                     /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(5657..5687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5702..5723
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5840..5857)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6011..6599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6091..6110)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6770..7630)
                     /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     7393..7412
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7631..7735)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7814..7833)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8005..8217,1..167)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
       61 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      121 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      181 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      241 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      301 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
      361 taagcagagc tggtttagtg aaccgtcaga tccgctagag atccgcggcc gctaatacga
      421 ctcactatag ggagagccgc caccatgaaa cggacagccg acggaagcga gttcgagtca
      481 ccaaagaaga agcggaaagt ctctgaggtg gagttttccc acgagtactg gatgagacat
      541 gccctgaccc tggccaagag ggcacgggat gagagggagg tgcctgtggg agccgtgctg
      601 gtgctgaaca atagagtgat cggcgagggc tggaacagag ccatcggcct gcacgaccca
      661 acagcccatg ccgaaattat ggccctgaga cagggcggcc tggtcatgca gaactacaga
      721 ctgattgacg ccaccctgta cgtgacattc gagccttgcg tgatgtgcgc cggcgccatg
      781 atccactcta ggatcggccg cgtggtgttt ggcgtgagga actcaaaaag aggcgccgca
      841 ggctccctga tgaacgtgct gaactacccc ggcatgaatc accgcgtcga aattaccgag
      901 ggaatcctgg cagatgaatg tgccgccctg ctgtgcgatt tctatcggat gcctagacag
      961 gtgttcaatg ctcagaagaa ggcccagagc tccatcaact ccggaggatc tagcggaggc
     1021 tcctctggct ctgagacacc tggcacaagc gagagcgcaa cacctgaaag cagcgggggc
     1081 agcagcgggg ggtcagacaa gaagtacagc atcggcctgg ccatcggcac caactctgtg
     1141 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc
     1201 aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc
     1261 gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag
     1321 aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc
     1381 ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac
     1441 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac
     1501 cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg
     1561 gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc
     1621 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc
     1681 gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg
     1741 agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc
     1801 ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc
     1861 gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac
     1921 aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg
     1981 tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc
     2041 ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa
     2101 gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag
     2161 aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc
     2221 aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag
     2281 gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg
     2341 ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac
     2401 cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc
     2461 aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg
     2521 aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc
     2581 aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag
     2641 tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag
     2701 cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac
     2761 cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac
     2821 tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat
     2881 ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg
     2941 gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg
     3001 aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac
     3061 accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc
     3121 aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg
     3181 atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag
     3241 ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc
     3301 atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc
     3361 gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac
     3421 agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg
     3481 aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg
     3541 cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac
     3601 gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg
     3661 ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg
     3721 aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag
     3781 ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc
     3841 atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac
     3901 tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc
     3961 accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc
     4021 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc
     4081 ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac
     4141 gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac
     4201 ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag
     4261 atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag
     4321 ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa
     4381 aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc
     4441 gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc
     4501 cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa
     4561 ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag
     4621 aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc
     4681 aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct
     4741 gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg
     4801 tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag
     4861 ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc
     4921 tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag
     4981 caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc
     5041 aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac
     5101 accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac
     5161 gagacacgga tcgacctgtc tcagctggga ggtgactctg gcggctcaaa aagaaccgcc
     5221 gacggcagcg aattcgagcc caagaagaag aggaaagtct aaccggtcat catcaccatc
     5281 accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     5341 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     5401 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     5461 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     5521 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg ataccgtcga
     5581 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5641 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tagggtgcct
     5701 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5761 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5821 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5881 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5941 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6001 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6061 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6121 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6181 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6241 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6301 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6361 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6421 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6481 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6541 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6601 aagatccttt gatcttttct acggggtctg acactcagtg gaacgaaaac tcacgttaag
     6661 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6721 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6781 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6841 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6901 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6961 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7021 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7081 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7141 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7201 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7261 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7321 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7381 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7441 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7501 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7561 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7621 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7681 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7741 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcgatctccc gatcccctag
     7801 ggtcgactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     7861 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
     7921 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
     7981 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
     8041 ttacggggtc attagttcat agcccatata ttgagttccg cgttacataa cttacggtaa
     8101 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     8161 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttac
//
