LOCUS       Exported                7599 bp DNA     circular SYN 22-MAR-2022
DEFINITION  Mammalian expression of SuperFi-Cas9 CBE base editor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7599)
  AUTHORS   Welker Lab
  TITLE     Direct Submission
  JOURNAL   Exported Mar 22, 2022 from SnapGene 6.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7599
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     promoter        81..292
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     regulatory      312..321
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     CDS             321..386
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /note="color: #cc99b2
                     Cleavage site after base 386"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             393..413
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     CDS             438..1124
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /note="color: #993366"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             1173..5273
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=SuperFi-Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /note="This feature has 16 segments:
                         1: 1173 .. 1196 / #993366
                         2: 1197 .. 1199 / #ffcc99 / D10A
                         3: 1200 .. 4196 / #993366
                         4: 4197 .. 4199 / #ff99cc / Y1010D
                         5: 4200 .. 4205 / #993366
                         6: 4206 .. 4208 / #ff99cc / Y1013D
                         7: 4209 .. 4214 / #993366
                         8: 4215 .. 4217 / #ff99cc / Y1016D
                         9: 4218 .. 4220 / #993366
                        10: 4221 .. 4223 / #ff99cc / V1018D
                        11: 4224 .. 4226 / #ff99cc / R1019D
                        12: 4227 .. 4247 / #993366
                        13: 4248 .. 4250 / #ff99cc / Q1027D
                        14: 4251 .. 4259 / #993366
                        15: 4260 .. 4262 / #ff99cc / K1031D
                        16: 4263 .. 5273 / #993366"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVDGDDKVDDDDKMIAKSEDEIGDATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5286..5534
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /note="color: #993366"
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5547..5567
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     polyA_signal    5601..5808
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      complement(5873..6461)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(6632..7492)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 6632 .. 7423 / #ccffcc
                        2: 7424 .. 7492 / #ccffcc / signal sequence
                     Cleavage site after base 7423"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7493..7597)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
ORIGIN
        1 catttccccg aaaagtgcca cctgacgtcc agcagagatc cactttggcg ccggctcgag
       61 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      121 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      181 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      241 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg
      301 acgcgggatc cgccaccatg gactataagg accacgacgg agactacaag gatcatgata
      361 ttgattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag gtcggtatcc
      421 acggagtccc agcagccatg agctcagaga ctggcccagt ggctgtggac cccacattga
      481 gacggcggat cgagccccat gagtttgagg tattcttcga tccgagagag ctccgcaagg
      541 agacctgcct gctttacgaa attaattggg ggggccggca ctccatttgg cgacatacat
      601 cacagaacac taacaagcac gtcgaagtca acttcatcga gaagttcacg acagaaagat
      661 atttctgtcc gaacacaagg tgcagcatta cctggtttct cagctggagc ccatgcggcg
      721 aatgtagtag ggccatcact gaattcctgt caaggtatcc ccacgtcact ctgtttattt
      781 acatcgcaag gctgtaccac cacgctgacc cccgcaatcg acaaggcctg cgggatttga
      841 tctcttcagg tgtgactatc caaattatga ctgagcagga gtcaggatac tgctggagaa
      901 actttgtgaa ttatagcccg agtaatgaag cccactggcc taggtatccc catctgtggg
      961 tacgactgta cgttcttgaa ctgtactgca tcatactggg cctgcctcct tgtctcaaca
     1021 ttctgagaag gaagcagcca cagctgacat tctttaccat cgctcttcag tcttgtcatt
     1081 accagcgact gcccccacac attctctggg ccaccgggtt gaaaagcggc agcgagactc
     1141 ccgggacctc agagtccgcc acacccgaaa gtgacaagaa gtacagcatc ggcctggcca
     1201 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     1261 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     1321 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     1381 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     1441 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     1501 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     1561 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     1621 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     1681 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     1741 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     1801 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     1861 gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca
     1921 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     1981 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     2041 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     2101 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     2161 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     2221 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     2281 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     2341 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     2401 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     2461 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     2521 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     2581 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     2641 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     2701 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     2761 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     2821 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     2881 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     2941 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     3001 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     3061 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     3121 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     3181 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     3241 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     3301 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     3361 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     3421 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     3481 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     3541 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     3601 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     3661 accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact
     3721 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     3781 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     3841 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     3901 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     3961 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     4021 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     4081 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     4141 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtggacg
     4201 gcgacgacaa ggtggacgac gacgacaaga tgatcgccaa gagcgaagac gaaatcggcg
     4261 acgctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     4321 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     4381 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     4441 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     4501 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     4561 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     4621 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     4681 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     4741 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     4801 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     4861 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     4921 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     4981 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     5041 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     5101 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     5161 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     5221 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gattcaggcg
     5281 gatctactaa tctgtcagat attattgaaa aggagaccgg taagcaactg gttatccagg
     5341 aatccatcct catgctccca gaggaggtgg aagaagtcat tgggaacaag ccggaaagcg
     5401 atatactcgt gcacaccgcc tacgacgaga gcaccgacga gaatgtcatg cttctgacta
     5461 gcgacgcccc tgaatacaag ccttgggctc tggtcataca ggatagcaac ggtgagaaca
     5521 agattaagat gctctctggt ggttctccca agaagaagag gaaagtctga catgtcctag
     5581 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     5641 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     5701 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     5761 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgctgag
     5821 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5881 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5941 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6001 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6061 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6121 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6181 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6241 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6301 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6361 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6421 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6481 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6541 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6601 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6661 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6721 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6781 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6841 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6901 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6961 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7021 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7081 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7141 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7201 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7261 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7321 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7381 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7441 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7501 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7561 aatgtattta gaaaaataaa caaatagggg ttccgcgca
//
