LOCUS       Exported                7794 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  A-to-G base editor.
ACCESSION   .
VERSION     .
KEYWORDS    LbABE8e
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7794)
  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 7794)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7794
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        19..398
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        399..602
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     553..573
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     644..663
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        644..662
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             712..732
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1327..5004
                     /codon_start=1
                     /product="CRISPR-associated protein from Lachnospiraceae 
                     bacterium ND2006 (Zetsche et al., 2015)"
                     /label=LbCpf1
                     /translation="SKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAED
                     YKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEI
                     AKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSE
                     EAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGE
                     FFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLS
                     DRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNG
                     PAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQL
                     QEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDL
                     LDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSK
                     DKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGN
                     YEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLI
                     DFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVE
                     EGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKE
                     ELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKIN
                     TEVRVLLKHDDNPYVIGIARGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDY
                     HSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGF
                     KNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQN
                     GFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDY
                     KNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQ
                     GDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNY
                     EAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSV
                     K"
     CDS             5047..5067
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5076..5093
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5116..5133)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5122..5346
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5417..5433)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5417..5433)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5430..5452)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5441..5457
                     /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(5465..5495)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5510..5531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5648..5665)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5819..6407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5899..5918)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6578..7438)
                     /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     7201..7220
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7439..7543)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7622..7641)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
       61 ggtcattagt tcatagccca tatattgagt tccgcgttac ataacttacg gtaaatggcc
      121 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
      181 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      241 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
      301 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
      361 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
      421 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
      481 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
      541 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
      601 ctggtttagt gaaccgtcag atccgctaga gatccgcggc cgctaatacg actcactata
      661 gggagagccg ccaccatgaa acggacagcc gacggaagcg agttcgagtc accaaagaag
      721 aagcggaaag tctctgaggt ggagttttcc cacgagtact ggatgagaca tgccctgacc
      781 ctggccaaga gggcacggga tgagagggag gtgcctgtgg gagccgtgct ggtgctgaac
      841 aatagagtga tcggcgaggg ctggaacaga gccatcggcc tgcacgaccc aacagcccat
      901 gccgaaatta tggccctgag acagggcggc ctggtcatgc agaactacag actgattgac
      961 gccaccctgt acgtgacatt cgagccttgc gtgatgtgcg ccggcgccat gatccactct
     1021 aggatcggcc gcgtggtgtt tggcgtgagg aactcaaaaa gaggcgccgc aggctccctg
     1081 atgaacgtgc tgaactaccc cggcatgaat caccgcgtcg aaattaccga gggaatcctg
     1141 gcagatgaat gtgccgccct gctgtgcgat ttctatcgga tgcctagaca ggtgttcaat
     1201 gctcagaaga aggcccagag ctccatcaac tccggaggat ctagcggagg atcctctggc
     1261 agcgagacac caggaacaag cgagtcagca acaccagaga gcagtggcgg cagcagcggc
     1321 ggcagcagca agctggagaa gtttacaaac tgctactccc tgtctaagac cctgaggttc
     1381 aaggccatcc ctgtgggcaa gacccaggag aacatcgaca ataagcggct gctggtggag
     1441 gacgagaaga gagccgagga ttataagggc gtgaagaagc tgctggatcg ctactatctg
     1501 tcttttatca acgacgtgct gcacagcatc aagctgaaga atctgaacaa ttacatcagc
     1561 ctgttccgga agaaaaccag aaccgagaag gagaataagg agctggagaa cctggagatc
     1621 aatctgcgga aggagatcgc caaggccttc aagggcaacg agggctacaa gtccctgttt
     1681 aagaaggata tcatcgagac aatcctgcca gagttcctgg acgataagga cgagatcgcc
     1741 ctggtgaaca gcttcaatgg ctttaccaca gccttcaccg gcttctttga taacagagag
     1801 aatatgtttt ccgaggaggc caagagcaca tccatcgcct tcaggtgtat caacgagaat
     1861 ctgacccgct acatctctaa tatggacatc ttcgagaagg tggacgccat ctttgataag
     1921 cacgaggtgc aggagatcaa ggagaagatc ctgaacagcg actatgatgt ggaggatttc
     1981 tttgagggcg agttctttaa ctttgtgctg acacaggagg gcatcgacgt gtataacgcc
     2041 atcatcggcg gcttcgtgac cgagagcggc gagaagatca agggcctgaa cgagtacatc
     2101 aacctgtata atcagaaaac caagcagaag ctgcctaagt ttaagccact gtataagcag
     2161 gtgctgagcg atcgggagtc tctgagcttc tacggcgagg gctatacatc cgatgaggag
     2221 gtgctggagg tgtttagaaa caccctgaac aagaacagcg agatcttcag ctccatcaag
     2281 aagctggaga agctgttcaa gaattttgac gagtactcta gcgccggcat ctttgtgaag
     2341 aacggccccg ccatcagcac aatctccaag gatatcttcg gcgagtggaa cgtgatccgg
     2401 gacaagtgga atgccgagta tgacgatatc cacctgaaga agaaggccgt ggtgaccgag
     2461 aagtacgagg acgatcggag aaagtccttc aagaagatcg gctccttttc tctggagcag
     2521 ctgcaggagt acgccgacgc cgatctgtct gtggtggaga agctgaagga gatcatcatc
     2581 cagaaggtgg atgagatcta caaggtgtat ggctcctctg agaagctgtt cgacgccgat
     2641 tttgtgctgg agaagagcct gaagaagaac gacgccgtgg tggccatcat gaaggacctg
     2701 ctggattctg tgaagagctt cgagaattac atcaaggcct tctttggcga gggcaaggag
     2761 acaaacaggg acgagtcctt ctatggcgat tttgtgctgg cctacgacat cctgctgaag
     2821 gtggaccaca tctacgatgc catccgcaat tatgtgaccc agaagcccta ctctaaggat
     2881 aagttcaagc tgtattttca gaaccctcag ttcatgggcg gctgggacaa ggataaggag
     2941 acagactatc gggccaccat cctgagatac ggctccaagt actatctggc catcatggat
     3001 aagaagtacg ccaagtgcct gcagaagatc gacaaggacg atgtgaacgg caattacgag
     3061 aagatcaact ataagctgct gcccggccct aataagatgc tgccaaaggt gttcttttct
     3121 aagaagtgga tggcctacta taaccccagc gaggacatcc agaagatcta caagaatggc
     3181 acattcaaga agggcgatat gtttaacctg aatgactgtc acaagctgat cgacttcttt
     3241 aaggatagca tctcccggta tccaaagtgg tccaatgcct acgatttcaa cttttctgag
     3301 acagagaagt ataaggacat cgccggcttt tacagagagg tggaggagca gggctataag
     3361 gtgagcttcg agtctgccag caagaaggag gtggataagc tggtggagga gggcaagctg
     3421 tatatgttcc agatctataa caaggacttt tccgataagt ctcacggcac acccaatctg
     3481 cacaccatgt acttcaagct gctgtttgac gagaacaatc acggacagat caggctgagc
     3541 ggaggagcag agctgttcat gaggcgcgcc tccctgaaga aggaggagct ggtggtgcac
     3601 ccagccaact cccctatcgc caacaagaat ccagataatc ccaagaaaac cacaaccctg
     3661 tcctacgacg tgtataagga taagaggttt tctgaggacc agtacgagct gcacatccca
     3721 atcgccatca ataagtgccc caagaacatc ttcaagatca atacagaggt gcgcgtgctg
     3781 ctgaagcacg acgataaccc ctatgtgatc ggcatcgcca ggggtgagcg caatctgctg
     3841 tatatcgtgg tggtggacgg caagggcaac atcgtggagc agtattccct gaacgagatc
     3901 atcaacaact tcaacggcat caggatcaag acagattacc actctctgct ggacaagaag
     3961 gagaaggaga ggttcgaggc ccgccagaac tggacctcca tcgagaatat caaggagctg
     4021 aaggccggct atatctctca ggtggtgcac aagatctgcg agctggtgga gaagtacgat
     4081 gccgtgatcg ccctggagga cctgaactct ggctttaaga atagccgcgt gaaggtggag
     4141 aagcaggtgt atcagaagtt cgagaagatg ctgatcgata agctgaacta catggtggac
     4201 aagaagtcta atccttgtgc aacaggcggc gccctgaagg gctatcagat caccaataag
     4261 ttcgagagct ttaagtccat gtctacccag aacggcttca tcttttacat ccctgcctgg
     4321 ctgacatcca agatcgatcc atctaccggc tttgtgaacc tgctgaaaac caagtatacc
     4381 agcatcgccg attccaagaa gttcatcagc tcctttgaca ggatcatgta cgtgcccgag
     4441 gaggatctgt tcgagtttgc cctggactat aagaacttct ctcgcacaga cgccgattac
     4501 atcaagaagt ggaagctgta ctcctacggc aaccggatca gaatcttccg gaatcctaag
     4561 aagaacaacg tgttcgactg ggaggaggtg tgcctgacca gcgcctataa ggagctgttc
     4621 aacaagtacg gcatcaatta tcagcagggc gatatcagag ccctgctgtg cgagcagtcc
     4681 gacaaggcct tctactctag ctttatggcc ctgatgagcc tgatgctgca gatgcggaac
     4741 agcatcacag gccgcaccga cgtggatttt ctgatcagcc ctgtgaagaa ctccgacggc
     4801 atcttctacg atagccggaa ctatgaggcc caggagaatg ccatcctgcc aaagaacgcc
     4861 gacgccaatg gcgcctataa catcgccaga aaggtgctgt gggccatcgg ccagttcaag
     4921 aaggccgagg acgagaagct ggataaggtg aagatcgcca tctctaacaa ggagtggctg
     4981 gagtacgccc agaccagcgt gaagtctggc ggctcaaaaa gaaccgccga cggcagcgaa
     5041 ttcgagccca agaagaagag gaaagtctaa ccggtcatca tcaccatcac cattgagttt
     5101 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     5161 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     5221 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     5281 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     5341 ctatggcttc tgaggcggaa agaaccagct ggggctcgat accgtcgacc tctagctaga
     5401 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5461 cacacaacat acgagccgga agcataaagt gtaaagccta gggtgcctaa tgagtgagct
     5521 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5581 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5641 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5701 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5761 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5821 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5881 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5941 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6001 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6061 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6121 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6181 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6241 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6301 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6361 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6421 tcttttctac ggggtctgac actcagtgga acgaaaactc acgttaaggg attttggtca
     6481 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6541 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6601 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6661 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6721 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6781 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6841 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6901 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6961 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7021 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7081 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7141 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7201 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7261 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7321 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7381 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7441 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7501 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7561 tgccacctga cgtcgacgga tcgggagatc gatctcccga tcccctaggg tcgactctca
     7621 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     7681 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     7741 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtac
//
