LOCUS       40924_185        8166 bp DNA     circular SYN 13-MAY-2021
DEFINITION  expresses ABE8.20-m in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8166)
  AUTHORS   Gaudelli NM, Lam DK, Rees HA, Sola-Esteves NM, Barrera LA, Born DA, 
            Edwards A, Gehrke JM, Lee SJ, Liquori AJ, Murray R, Packer MS, 
            Rinaldi C, Slaymaker IM, Yen J, Young LE, Ciaramella G
  TITLE     Directed evolution of adenine base editors with increased activity 
            and therapeutic application.
  JOURNAL   Nat Biotechnol. 2020 Apr 13. pii: 10.1038/s41587-020-0491-6. doi: 
            10.1038/s41587-020-0491-6.
  PUBMED    32284586
REFERENCE   2  (bases 1 to 8166)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8166)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2020 Apr 13. pii: 10.1038/s41587-020-0491-6. doi: 
            10.1038/s41587-020-0491-6."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8166
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        103..482
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        483..686
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        728..746
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             763..1260
                     /codon_start=1
                     /product="DNA adenosine deaminase evolved from the E. coli
                     tRNA adenosine deaminase TadA (Gaudelli et al., 2017)"
                     /label=TadA*(7.10)
                     /translation="SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWN
                     RAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATLYSTFEPCVMCAGAMIHSRIGRVVFG
                     VRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQS
                     STD"
     CDS             1357..5457
                     /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             5503..5523
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             5532..5549
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     polyA_signal    5578..5802
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5873..5889)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5897..5913)
                     /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(5921..5951)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5966..5987)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(6104..6121)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6275..6863)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7037..7894)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7895..7999)
                     /label=AmpR promoter
     primer_bind     complement(8078..8097)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
ORIGIN
        1 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
       61 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
      121 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      181 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      241 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      301 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      361 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      421 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      481 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      541 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      601 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      661 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagagatccg
      721 cggccgctaa tacgactcac tatagggaga gccgccacca tgtccgaagt cgagttttcc
      781 catgagtact ggatgagaca cgcattgact ctcgcaaaga gggctcgaga tgaacgcgag
      841 gtgcccgtgg gggcagtact cgtgctcaac aatcgcgtaa tcggcgaagg ttggaatagg
      901 gcaatcggac tccacgaccc cactgcacat gcggaaatca tggcccttcg acagggaggg
      961 cttgtgatgc agaattatcg actttatgat gcgacgctgt actcgacgtt tgaaccttgc
     1021 gtaatgtgcg cgggagctat gattcactcc cgcattggac gagttgtatt cggtgttcgc
     1081 aacgccaaga cgggtgccgc aggttcactg atggacgtgc tgcatcatcc aggcatgaac
     1141 caccgggtag aaatcacaga aggcatattg gcggacgaat gtgcggcgct gttgtgtcgt
     1201 ttttttcgca tgcccaggcg ggtctttaac gcccagaaaa aagcacaatc ctctactgac
     1261 tctggtggtt cttctggtgg ttctagcggc agcgagactc ccgggacctc agagtccgcc
     1321 acacccgaaa gttctggtgg ttcttctggt ggttctgaca agaagtacag catcggcctg
     1381 gccatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc
     1441 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga
     1501 gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga
     1561 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag
     1621 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
     1681 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac
     1741 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc
     1801 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg
     1861 atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg
     1921 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag
     1981 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg
     2041 cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc
     2101 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac
     2161 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg
     2221 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
     2281 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac
     2341 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
     2401 attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag
     2461 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg
     2521 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
     2581 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt
     2641 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
     2701 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc
     2761 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag
     2821 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
     2881 aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac
     2941 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg
     3001 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc
     3061 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
     3121 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
     3181 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
     3241 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
     3301 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
     3361 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
     3421 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
     3481 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
     3541 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
     3601 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
     3661 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
     3721 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
     3781 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
     3841 atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac
     3901 gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac
     3961 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc
     4021 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
     4081 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
     4141 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg
     4201 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
     4261 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg
     4321 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
     4381 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc
     4441 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
     4501 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc
     4561 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg
     4621 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
     4681 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag
     4741 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
     4801 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
     4861 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
     4921 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
     4981 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc
     5041 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc
     5101 gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc
     5161 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa
     5221 gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc
     5281 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc
     5341 accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac
     5401 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggtgacgag
     5461 ggagctgata agcgcaccgc cgatggttcc gagttcgaaa gccccaagaa gaagaggaaa
     5521 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg
     5581 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     5641 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     5701 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     5761 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     5821 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5881 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5941 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     6001 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6061 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     6121 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6181 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6241 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6301 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6361 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6421 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6481 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6541 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6601 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6661 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6721 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6781 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6841 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6901 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6961 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     7021 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     7081 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     7141 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     7201 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7261 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7321 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7381 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7441 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7501 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7561 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7621 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7681 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7741 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7801 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7861 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7921 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7981 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     8041 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat
     8101 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     8161 aaattt
//
