LOCUS       40924_11576        8727 bp DNA     circular SYN 13-MAY-2021
DEFINITION  expresses ABE7.10 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8727)
  AUTHORS   Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu
            DR
  TITLE     Programmable base editing of A*T to G*C in genomic DNA without DNA 
            cleavage.
  JOURNAL   Nature. 2017 Nov 23;551(7681):464-471. doi: 10.1038/nature24644. 
            Epub 2017 Oct 25.
  PUBMED    29160308
REFERENCE   2  (bases 1 to 8727)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8727)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nature24644"; journalName: "Nature"; date: "2017-11-23-
            23"; volume: "551"; issue: "7681"; pages: "464-471"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1098..5198
                     /codon_start=1
                     /label=Cas9(D10A)
                     /note="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /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             5240..5257
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     polyA_signal    5286..5510
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5581..5597)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5605..5621)
                     /label=lac operator
                     /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(5629..5659)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5674..5695)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5983..6571)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6745..7602)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7603..7707)
                     /label=AmpR promoter
     enhancer        7977..8356
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8357..8560
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        8602..8620
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(8634..8727,1..1097)
                     /codon_start=1
                     /label=ABE(7.10)
                     /note="adenine base editor comprising wild-type E. coli
                     TadA fused to the evolved TadA*(7.10) mutant (Gaudelli et 
                     al., 2017)"
                     /translation="MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGW
                     NRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVF
                     GARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQ
                     SSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARD
                     EREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT
                     FEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECA
                     ALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGS"
ORIGIN
        1 cagtactcgt gcataacaat cgcgtaatcg gcgaaggttg gaataggccg atcggacgcc
       61 acgaccccac tgcacatgcg gaaatcatgg cccttcgaca gggagggctt gtgatgcaga
      121 attatcgact tatcgatgcg acgctgtacg tcacgcttga accttgcgta atgtgcgcgg
      181 gagctatgat tcactcccgc attggacgag ttgtattcgg tgcccgcgac gccaagacgg
      241 gtgccgcagg ttcactgatg gacgtgctgc atcacccagg catgaaccac cgggtagaaa
      301 tcacagaagg catattggcg gacgaatgtg cggcgctgtt gtccgacttt tttcgcatgc
      361 ggaggcagga gatcaaggcc cagaaaaaag cacaatcctc tactgactct ggtggttctt
      421 ctggtggttc tagcggcagc gagactcccg ggacctcaga gtccgccaca cccgaaagtt
      481 ctggtggttc ttctggtggt tcttccgaag tcgagttttc ccatgagtac tggatgagac
      541 acgcattgac tctcgcaaag agggctcgag atgaacgcga ggtgcccgtg ggggcagtac
      601 tcgtgctcaa caatcgcgta atcggcgaag gttggaatag ggcaatcgga ctccacgacc
      661 ccactgcaca tgcggaaatc atggcccttc gacagggagg gcttgtgatg cagaattatc
      721 gacttatcga tgcgacgctg tacgtcacgt ttgaaccttg cgtaatgtgc gcgggagcta
      781 tgattcactc ccgcattgga cgagttgtat tcggtgttcg caacgccaag acgggtgccg
      841 caggttcact gatggacgtg ctgcattacc caggcatgaa ccaccgggta gaaatcacag
      901 aaggcatatt ggcggacgaa tgtgcggcgc tgttgtgtta cttttttcgc atgcccaggc
      961 aggtctttaa cgcccagaaa aaagcacaat cctctactga ctctggtggt tcttctggtg
     1021 gttctagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa agttctggtg
     1081 gttcttctgg tggttctgat aaaaagtatt ctattggttt agccatcggc actaattccg
     1141 ttggatgggc tgtcataacc gatgaataca aagtaccttc aaagaaattt aaggtgttgg
     1201 ggaacacaga ccgtcattcg attaaaaaga atcttatcgg tgccctccta ttcgatagtg
     1261 gcgaaacggc agaggcgact cgcctgaaac gaaccgctcg gagaaggtat acacgtcgca
     1321 agaaccgaat atgttactta caagaaattt ttagcaatga gatggccaaa gttgacgatt
     1381 ctttctttca ccgtttggaa gagtccttcc ttgtcgaaga ggacaagaaa catgaacggc
     1441 accccatctt tggaaacata gtagatgagg tggcatatca tgaaaagtac ccaacgattt
     1501 atcacctcag aaaaaagcta gttgactcaa ctgataaagc ggacctgagg ttaatctact
     1561 tggctcttgc ccatatgata aagttccgtg ggcactttct cattgagggt gatctaaatc
     1621 cggacaactc ggatgtcgac aaactgttca tccagttagt acaaacctat aatcagttgt
     1681 ttgaagagaa ccctataaat gcaagtggcg tggatgcgaa ggctattctt agcgcccgcc
     1741 tctctaaatc ccgacggcta gaaaacctga tcgcacaatt acccggagag aagaaaaatg
     1801 ggttgttcgg taaccttata gcgctctcac taggcctgac accaaatttt aagtcgaact
     1861 tcgacttagc tgaagatgcc aaattgcagc ttagtaagga cacgtacgat gacgatctcg
     1921 acaatctact ggcacaaatt ggagatcagt atgcggactt atttttggct gccaaaaacc
     1981 ttagcgatgc aatcctccta tctgacatac tgagagttaa tactgagatt accaaggcgc
     2041 cgttatccgc ttcaatgatc aaaaggtacg atgaacatca ccaagacttg acacttctca
     2101 aggccctagt ccgtcagcaa ctgcctgaga aatataagga aatattcttt gatcagtcga
     2161 aaaacgggta cgcaggttat attgacggcg gagcgagtca agaggaattc tacaagttta
     2221 tcaaacccat attagagaag atggatggga cggaagagtt gcttgtaaaa ctcaatcgcg
     2281 aagatctact gcgaaagcag cggactttcg acaacggtag cattccacat caaatccact
     2341 taggcgaatt gcatgctata cttagaaggc aggaggattt ttatccgttc ctcaaagaca
     2401 atcgtgaaaa gattgagaaa atcctaacct ttcgcatacc ttactatgtg ggacccctgg
     2461 cccgagggaa ctctcggttc gcatggatga caagaaagtc cgaagaaacg attactccat
     2521 ggaattttga ggaagttgtc gataaaggtg cgtcagctca atcgttcatc gagaggatga
     2581 ccaactttga caagaattta ccgaacgaaa aagtattgcc taagcacagt ttactttacg
     2641 agtatttcac agtgtacaat gaactcacga aagttaagta tgtcactgag ggcatgcgta
     2701 aacccgcctt tctaagcgga gaacagaaga aagcaatagt agatctgtta ttcaagacca
     2761 accgcaaagt gacagttaag caattgaaag aggactactt taagaaaatt gaatgcttcg
     2821 attctgtcga gatctccggg gtagaagatc gatttaatgc gtcacttggt acgtatcatg
     2881 acctcctaaa gataattaaa gataaggact tcctggataa cgaagagaat gaagatatct
     2941 tagaagatat agtgttgact cttaccctct ttgaagatcg ggaaatgatt gaggaaagac
     3001 taaaaacata cgctcacctg ttcgacgata aggttatgaa acagttaaag aggcgtcgct
     3061 atacgggctg gggacgattg tcgcggaaac ttatcaacgg gataagagac aagcaaagtg
     3121 gtaaaactat tctcgatttt ctaaagagcg acggcttcgc caataggaac tttatgcagc
     3181 tgatccatga tgactcttta accttcaaag aggatataca aaaggcacag gtttccggac
     3241 aaggggactc attgcacgaa catattgcga atcttgctgg ttcgccagcc atcaaaaagg
     3301 gcatactcca gacagtcaaa gtagtggatg agctagttaa ggtcatggga cgtcacaaac
     3361 cggaaaacat tgtaatcgag atggcacgcg aaaatcaaac gactcagaag gggcaaaaaa
     3421 acagtcgaga gcggatgaag agaatagaag agggtattaa agaactgggc agccagatct
     3481 taaaggagca tcctgtggaa aatacccaat tgcagaacga gaaactttac ctctattacc
     3541 tacaaaatgg aagggacatg tatgttgatc aggaactgga cataaaccgt ttatctgatt
     3601 acgacgtcga tcacattgta ccccaatcct ttttgaagga cgattcaatc gacaataaag
     3661 tgcttacacg ctcggataag aaccgaggga aaagtgacaa tgttccaagc gaggaagtcg
     3721 taaagaaaat gaagaactat tggcggcagc tcctaaatgc gaaactgata acgcaaagaa
     3781 agttcgataa cttaactaaa gctgagaggg gtggcttgtc tgaacttgac aaggccggat
     3841 ttattaaacg tcagctcgtg gaaacccgcc aaatcacaaa gcatgttgca cagatactag
     3901 attcccgaat gaatacgaaa tacgacgaga acgataagct gattcgggaa gtcaaagtaa
     3961 tcactttaaa gtcaaaattg gtgtcggact tcagaaagga ttttcaattc tataaagtta
     4021 gggagataaa taactaccac catgcgcacg acgcttatct taatgccgtc gtagggaccg
     4081 cactcattaa gaaatacccg aagctagaaa gtgagtttgt gtatggtgat tacaaagttt
     4141 atgacgtccg taagatgatc gcgaaaagcg aacaggagat aggcaaggct acagccaaat
     4201 acttctttta ttctaacatt atgaatttct ttaagacgga aatcactctg gcaaacggag
     4261 agatacgcaa acgaccttta attgaaacca atggggagac aggtgaaatc gtatgggata
     4321 agggccggga cttcgcgacg gtgagaaaag ttttgtccat gccccaagtc aacatagtaa
     4381 agaaaactga ggtgcagacc ggagggtttt caaaggaatc gattcttcca aaaaggaata
     4441 gtgataagct catcgctcgt aaaaaggact gggacccgaa aaagtacggt ggcttcgata
     4501 gccctacagt tgcctattct gtcctagtag tggcaaaagt tgagaaggga aaatccaaga
     4561 aactgaagtc agtcaaagaa ttattgggga taacgattat ggagcgctcg tcttttgaaa
     4621 agaaccccat cgacttcctt gaggcgaaag gttacaagga agtaaaaaag gatctcataa
     4681 ttaaactacc aaagtatagt ctgtttgagt tagaaaatgg ccgaaaacgg atgttggcta
     4741 gcgccggaga gcttcaaaag gggaacgaac tcgcactacc gtctaaatac gtgaatttcc
     4801 tgtatttagc gtcccattac gagaagttga aaggttcacc tgaagataac gaacagaagc
     4861 aactttttgt tgagcagcac aaacattatc tcgacgaaat catagagcaa atttcggaat
     4921 tcagtaagag agtcatccta gctgatgcca atctggacaa agtattaagc gcatacaaca
     4981 agcacaggga taaacccata cgtgagcagg cggaaaatat tatccatttg tttactctta
     5041 ccaacctcgg cgctccagcc gcattcaagt attttgacac aacgatagat cgcaaacgat
     5101 acacttctac caaggaggtg ctagacgcga cactgattca ccaatccatc acgggattat
     5161 atgaaactcg gatagatttg tcacagcttg ggggtgactc tggtggttct cccaagaaga
     5221 agaggaaagt ctaaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc
     5281 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     5341 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     5401 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     5461 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     5521 ggaaagaacc agctggggct cgataccgtc gacctctagc tagagcttgg cgtaatcatg
     5581 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5641 cggaagcata aagtgtaaag cctagggtgc ctaatgagtg agctaactca cattaattgc
     5701 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5761 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5821 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5881 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5941 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6001 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6061 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6121 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6181 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6241 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6301 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6361 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6421 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6481 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6541 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6601 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6661 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6721 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6781 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6841 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6901 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6961 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7021 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7081 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7141 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7201 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7261 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7321 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7381 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7441 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7501 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7561 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7621 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7681 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     7741 cggatcggga gatcgatctc ccgatcccct agggtcgact ctcagtacaa tctgctctga
     7801 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     7861 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     7921 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
     7981 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     8041 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     8101 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     8161 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     8221 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     8281 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     8341 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     8401 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     8461 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     8521 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
     8581 ccgctagaga tccgcggccg ctaatacgac tcactatagg gagagccgcc accatgtccg
     8641 aagtcgagtt ttcccatgag tactggatga gacacgcatt gactctcgca aagagggctt
     8701 gggatgaacg cgaggtgccc gtggggg
//
