LOCUS       Exported                8217 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  A-to-G base editor.
ACCESSION   .
VERSION     .
KEYWORDS    ABE8e(TadA-8e K20A R21A)
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)
REFERENCE   2  (bases 1 to 8217)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8217
                     /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             1060..5160
                     /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             5203..5223
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5232..5249
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5272..5289)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5278..5502
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5573..5589)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5573..5589)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5586..5608)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5597..5613
                     /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(5621..5651)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5666..5687
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5804..5821)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5975..6563)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6055..6074)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6734..7594)
                     /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     7357..7376
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7595..7699)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7778..7797)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7969..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 aagtctctga ggtggagttt
      481 tcccacgagt actggatgag acatgccctg accctggcag cagctgctcg ggatgagagg
      541 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac
      601 agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc
      661 ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct
      721 tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg
      781 aggaactcaa aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg
      841 aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc
      901 gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc
      961 aactccggag gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc
     1021 gcaacacctg aaagcagcgg gggcagcagc ggggggtcag acaagaagta cagcatcggc
     1081 ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
     1141 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
     1201 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
     1261 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     1321 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     1381 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     1441 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     1501 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     1561 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     1621 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     1681 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     1741 ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg
     1801 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     1861 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     1921 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     1981 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
     2041 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     2101 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
     2161 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     2221 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     2281 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
     2341 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     2401 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     2461 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
     2521 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
     2581 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
     2641 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc
     2701 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
     2761 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
     2821 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
     2881 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
     2941 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
     3001 aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac
     3061 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
     3121 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
     3181 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
     3241 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
     3301 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
     3361 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
     3421 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
     3481 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
     3541 gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag
     3601 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac
     3661 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac
     3721 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg
     3781 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
     3841 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag
     3901 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
     3961 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
     4021 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc
     4081 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
     4141 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
     4201 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
     4261 accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc
     4321 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag
     4381 tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct
     4441 aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa
     4501 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc
     4561 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa
     4621 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac
     4681 ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg
     4741 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc
     4801 cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag
     4861 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac
     4921 aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat
     4981 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac
     5041 accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc
     5101 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggtgac
     5161 tctggcggct caaaaagaac cgccgacggc agcgaattcg agcccaagaa gaagaggaaa
     5221 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg
     5281 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     5341 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     5401 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     5461 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     5521 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5581 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5641 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5701 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5761 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5821 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5881 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5941 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6001 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6061 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6121 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6181 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6241 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6301 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6361 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6421 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6481 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6541 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacactc
     6601 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6661 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6721 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6781 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6841 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6901 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6961 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7021 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7081 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7141 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7201 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7261 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7321 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7381 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7441 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7501 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7561 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7621 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7681 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     7741 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat
     7801 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     7861 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     7921 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     7981 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatattgagt
     8041 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     8101 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     8161 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc
//
