LOCUS       40924_26186        7689 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses SaKKH-BE3 in mammalian cells [Edit].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7689)
  AUTHORS   Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR
  TITLE     Increasing the genome-targeting scope and precision of base editing 
            with engineered Cas9-cytidine deaminase fusions.
  JOURNAL   Nat Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3803.
  PUBMED    28191901
REFERENCE   2  (bases 1 to 7689)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7689)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2017 Feb 13. doi: 10.1038/nbt.3803."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        179..558
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        559..762
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        804..822
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             836..1522
                     /codon_start=1
                     /label=APOBEC-1
                     /note="cytidine deaminase (C to U editing enzyme) from rat"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             1574..4729
                     /codon_start=1
                     /label=SaCas9
                     /note="Cas9 endonuclease from the Staphylococcus aureus
                     Type II CRISPR/Cas system"
                     /translation="KRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             4736..4756
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4763..4828
                     /codon_start=1
                     /label=3xFLAG
                     /note="three tandem FLAG(R) epitope tags, followed by an 
                     enterokinase cleavage site"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4841..5089
                     /codon_start=1
                     /label=UGI
                     /note="uracil-DNA glycosylase inhibitor from a Bacillus
                     subtilis bacteriophage (Mol et al., 1995)"
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5102..5122
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             5131..5148
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     polyA_signal    5177..5401
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5472..5488)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5496..5512)
                     /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(5520..5550)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5565..5586)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(5703..5720)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5874..6462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6636..7493)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7494..7598)
                     /label=AmpR promoter
ORIGIN
        1 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
       61 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      121 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      181 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      241 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      301 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      361 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      421 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      481 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      541 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      601 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      661 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      721 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
      781 atccgctaga gatccgcggc cgctaatacg actcactata gggagagccg ccaccatgag
      841 ctcagagact ggcccagtgg ctgtggaccc cacattgaga cggcggatcg agccccatga
      901 gtttgaggta ttcttcgatc cgagagagct ccgcaaggag acctgcctgc tttacgaaat
      961 taattggggg ggccggcact ccatttggcg acatacatca cagaacacta acaagcacgt
     1021 cgaagtcaac ttcatcgaga agttcacgac agaaagatat ttctgtccga acacaaggtg
     1081 cagcattacc tggtttctca gctggagccc atgcggcgaa tgtagtaggg ccatcactga
     1141 attcctgtca aggtatcccc acgtcactct gtttatttac atcgcaaggc tgtaccacca
     1201 cgctgacccc cgcaatcgac aaggcctgcg ggatttgatc tcttcaggtg tgactatcca
     1261 aattatgact gagcaggagt caggatactg ctggagaaac tttgtgaatt atagcccgag
     1321 taatgaagcc cactggccta ggtatcccca tctgtgggta cgactgtacg ttcttgaact
     1381 gtactgcatc atactgggcc tgcctccttg tctcaacatt ctgagaagga agcagccaca
     1441 gctgacattc tttaccatcg ctcttcagtc ttgtcattac cagcgactgc ccccacacat
     1501 tctctgggcc accgggttga aaagcggcag cgagactccc gggacctcag agtccgccac
     1561 acccgaaagt gggaaacgga actacatcct ggggcttgcc attgggataa ccagcgttgg
     1621 ctacggaatt attgattatg agacacgcga tgtgattgac gccggggtta ggctgttcaa
     1681 agaggccaac gttgaaaaca acgagggaag acggagtaag cgcggagcaa gaagactcaa
     1741 gcgcagacgg agacatcgga ttcagagggt gaaaaagctg ctcttcgatt acaatctcct
     1801 gaccgatcat agtgagctga gcggaatcaa cccctacgag gcgcgagtga aagggctttc
     1861 ccagaagctg tccgaagagg agttctccgc cgcgttgctg cacctggcca aacggagggg
     1921 ggttcacaat gtaaacgaag tggaggagga cacgggcaat gaacttagta cgaaagaaca
     1981 gatcagtagg aactctaagg ctctcgaaga gaaatacgtc gctgagttgc agcttgagag
     2041 actgaaaaaa gacggcgaag tacgcggatc tattaatagg ttcaagactt cagattacgt
     2101 aaaggaagcc aagcagctcc tgaaagtaca gaaagcgtac catcagctcg atcagagctt
     2161 catcgatacc tacatagatt tgctggagac acggaggaca tactacgagg gcccagggga
     2221 aggatctcct tttgggtgga aggacatcaa ggaatggtac gagatgctta tgggacattg
     2281 tacatatttt ccggaggagc tcaggagcgt caagtacgcc tacaatgccg acctgtacaa
     2341 tgccctcaat gacctcaata acctcgtgat taccagggac gagaacgaga agctggagta
     2401 ctatgaaaag ttccagatta tcgagaatgt gtttaagcag aagaagaagc cgacacttaa
     2461 gcagattgca aaggaaatcc tcgtgaatga ggaagatatc aagggataca gagtgacaag
     2521 tacaggcaag cccgagttca caaatctgaa ggtgtaccac gatattaagg acataaccgc
     2581 acgaaaggag ataatcgaaa acgctgagct cctcgatcag atcgcaaaaa ttcttaccat
     2641 ctaccagtct agtgaggaca ttcaggagga actgactaat ctgaacagtg agctcaccca
     2701 agaggaaatt gagcagattt caaacctgaa aggctacacc gggacgcaca atctgagcct
     2761 caaagcaatc aacctcattc tggatgaact ttggcacaca aatgacaacc aaattgccat
     2821 attcaaccgc ctgaaactgg tgccaaaaaa agtggatctg tcacagcaaa aggaaatccc
     2881 tacaaccttg gttgacgatt ttattctgtc ccccgttgtc aagcggagct tcatccagtc
     2941 aatcaaggtg atcaatgcca tcattaaaaa atacggattg ccaaacgata taattatcga
     3001 gcttgcacga gagaagaact caaaggacgc ccagaagatg attaacgaaa tgcagaagcg
     3061 caaccgccag acaaacgaac gcatagagga aattataaga acaaccggca aagagaatgc
     3121 caagtatctg atcgagaaaa tcaagctgca cgacatgcaa gaaggcaagt gcctgtactc
     3181 tctggaagct atcccactcg aagatctgct gaataatcca ttcaattacg aggtggacca
     3241 catcatccct agatccgtaa gctttgacaa ttccttcaat aacaaagttc tggttaaaca
     3301 ggaggaaaat tctaaaaaag ggaaccggac cccgttccag tacctgagct ccagtgacag
     3361 caagattagc tacgagactt ttaagaaaca tattctgaat ctggccaaag gcaaaggcag
     3421 gatcagcaag accaagaagg agtacctcct cgaagaacgc gacattaaca gatttagtgt
     3481 gcagaaagat ttcatcaacc gaaaccttgt cgatactcgg tacgccacga gaggcctgat
     3541 gaatctcctc aggagctact tccgcgtcaa taatctggac gttaaagtca agagcataaa
     3601 tgggggattc accagctttc tgaggagaaa gtggaagttt aagaaggaac gaaacaaagg
     3661 atacaagcac catgctgagg atgctttgat catcgctaac gcggacttta tctttaagga
     3721 atggaaaaag ctggataagg caaagaaagt gatggaaaac cagatgttcg aggagaagca
     3781 ggcagagtca atgcctgaga tcgagacaga gcaggaatac aaggaaattt tcatcacccc
     3841 tcatcagatt aaacacataa aggacttcaa agactataaa tactctcata gggtggacaa
     3901 aaaacccaat cgcaagctca ttaatgacac cctgtactca acacggaagg atgataaagg
     3961 taataccttg attgtgaata atcttaatgg attgtatgac aaagataacg acaagctcaa
     4021 gaagctgatc aacaagtctc cagagaagct ccttatgtat caccacgacc cacagactta
     4081 tcagaaattg aaactgatca tggagcaata cggggatgag aagaacccac tctacaaata
     4141 ttatgaggaa acaggtaatt acctgaccaa gtactccaag aaggataacg gaccagtgat
     4201 caaaaagata aagtactatg gcaacaaact taatgcgcat ttggacataa ctgacgatta
     4261 ccccaattct cgaaacaagg ttgtgaagct ctccctgaag ccttatagat ttgacgtgta
     4321 cctggataat ggggtttata aattcgtcac cgtgaaaaat ctggacgtga tcaaaaagga
     4381 gaactattat gaagtaaact caaagtgcta tgaggaggcg aagaagctga agaagatctc
     4441 caatcaggcc gagttcatcg cttccttcta taagaacgat ctcatcaaga tcaatggaga
     4501 gctttatcgc gtcattggtg tgaacaatga cttgctgaac aggatcgaag tcaatatgat
     4561 agacattacc taccgggagt atctcgaaaa catgaatgat aaacggccgc ctcacatcat
     4621 caagacaatc gcatctaaaa ctcagtcaat aaaaaagtac tctaccgata tcctggggaa
     4681 tctctatgaa gtgaagtcaa agaagcaccc acaaatcatt aaaaaaggtg gatcccccaa
     4741 gaagaagagg aaagtctcga gcgactacaa agaccatgac ggtgattata aagatcatga
     4801 catcgattac aaggatgacg atgacaagtc tggtggttct actaatctgt cagatattat
     4861 tgaaaaggag accggtaagc aactggttat ccaggaatcc atcctcatgc tcccagagga
     4921 ggtggaagaa gtcattggga acaagccgga aagcgatata ctcgtgcaca ccgcctacga
     4981 cgagagcacc gacgagaatg tcatgcttct gactagcgac gcccctgaat acaagccttg
     5041 ggctctggtc atacaggata gcaacggtga gaacaagatt aagatgctct ctggtggttc
     5101 tcccaagaag aagaggaaag tctaaccggt catcatcacc atcaccattg agtttaaacc
     5161 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     5221 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     5281 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     5341 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     5401 gcttctgagg cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg
     5461 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5521 aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc
     5581 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5641 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5701 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5761 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5821 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5881 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5941 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6001 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6061 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6121 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6181 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6241 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6301 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6361 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6421 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6481 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6541 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6601 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6661 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6721 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6781 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6841 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6901 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6961 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7021 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7081 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7141 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7201 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7261 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7321 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7381 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7441 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7501 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7561 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7621 cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca
     7681 atctgctct
//
