LOCUS       Exported                7599 bp DNA     circular SYN 22-MAR-2022
DEFINITION  Mammalian expression of nuclease inactive 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        11..222
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     regulatory      242..251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     CDS             251..316
                     /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 316"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             323..343
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     CDS             368..1054
                     /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             1103..5203
                     /codon_start=1
                     /product="nuclease inative mutant of the Cas9 endonuclease 
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=dSuperFi-Cas9
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /note="This feature has 18 segments:
                         1: 1103 .. 1126 / #993366
                         2: 1127 .. 1129 / #ffcc99 / D10A
                         3: 1130 .. 3616 / #993366
                         4: 3617 .. 3619 / #ffcc99 / H840A
                         5: 3620 .. 4126 / #993366
                         6: 4127 .. 4129 / #ff99cc / Y1010D
                         7: 4130 .. 4135 / #993366
                         8: 4136 .. 4138 / #ff99cc / Y1013D
                         9: 4139 .. 4144 / #993366
                        10: 4145 .. 4147 / #ff99cc / Y1016D
                        11: 4148 .. 4150 / #993366
                        12: 4151 .. 4153 / #ff99cc / V1018D
                        13: 4154 .. 4156 / #ff99cc / R1019D
                        14: 4157 .. 4177 / #993366
                        15: 4178 .. 4180 / #ff99cc / Q1027D
                        16: 4181 .. 4189 / #993366
                        17: 4190 .. 4192 / #ff99cc / K1031D
                        18: 4193 .. 5203 / #993366"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVDGDDKVDDDDKMIAKSEDEIGDATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5216..5464
                     /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             5477..5497
                     /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    5531..5738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      complement(5803..6391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(6562..7422)
                     /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: 6562 .. 7353 / #ccffcc
                        2: 7354 .. 7422 / #ccffcc / signal sequence
                     Cleavage site after base 7353"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7423..7527)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
ORIGIN
        1 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
       61 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
      121 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
      181 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc
      241 cgccaccatg gactataagg accacgacgg agactacaag gatcatgata ttgattacaa
      301 agacgatgac gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc
      361 agcagccatg agctcagaga ctggcccagt ggctgtggac cccacattga gacggcggat
      421 cgagccccat gagtttgagg tattcttcga tccgagagag ctccgcaagg agacctgcct
      481 gctttacgaa attaattggg ggggccggca ctccatttgg cgacatacat cacagaacac
      541 taacaagcac gtcgaagtca acttcatcga gaagttcacg acagaaagat atttctgtcc
      601 gaacacaagg tgcagcatta cctggtttct cagctggagc ccatgcggcg aatgtagtag
      661 ggccatcact gaattcctgt caaggtatcc ccacgtcact ctgtttattt acatcgcaag
      721 gctgtaccac cacgctgacc cccgcaatcg acaaggcctg cgggatttga tctcttcagg
      781 tgtgactatc caaattatga ctgagcagga gtcaggatac tgctggagaa actttgtgaa
      841 ttatagcccg agtaatgaag cccactggcc taggtatccc catctgtggg tacgactgta
      901 cgttcttgaa ctgtactgca tcatactggg cctgcctcct tgtctcaaca ttctgagaag
      961 gaagcagcca cagctgacat tctttaccat cgctcttcag tcttgtcatt accagcgact
     1021 gcccccacac attctctggg ccaccgggtt gaaaagcggc agcgagactc ccgggacctc
     1081 agagtccgcc acacccgaaa gtgacaagaa gtacagcatc ggcctggcca tcggcaccaa
     1141 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt
     1201 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga
     1261 cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag
     1321 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga
     1381 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga
     1441 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac
     1501 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat
     1561 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct
     1621 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca
     1681 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc
     1741 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa
     1801 gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca acttcaagag
     1861 caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga
     1921 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa
     1981 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa
     2041 ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct
     2101 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca
     2161 gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa
     2221 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa
     2281 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat
     2341 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa
     2401 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc
     2461 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac
     2521 cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg
     2581 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct
     2641 gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat
     2701 gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa
     2761 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg
     2821 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata
     2881 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga
     2941 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga
     3001 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg
     3061 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca
     3121 gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat
     3181 gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc
     3241 cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa
     3301 gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca
     3361 caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca
     3421 gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca
     3481 gatcctgaaa gaacacccag tggaaaacac ccagctgcag aacgagaagc tgtacctgta
     3541 ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc
     3601 cgactacgat gtggacgcca tcgtgcctca gagctttctg aaggacgact ccatcgacaa
     3661 caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga
     3721 ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca
     3781 gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc
     3841 cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat
     3901 cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa
     3961 agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa
     4021 agtgcgcgag atcaacaact accaccacgc ccacgacgcg tacctgaacg ccgtcgtggg
     4081 aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtggacg gcgacgacaa
     4141 ggtggacgac gacgacaaga tgatcgccaa gagcgaagac gaaatcggcg acgctaccgc
     4201 caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa
     4261 cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg
     4321 ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat
     4381 cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag
     4441 gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt
     4501 cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc
     4561 caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt
     4621 cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct
     4681 gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct
     4741 ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa
     4801 cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca
     4861 gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag
     4921 cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta
     4981 caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac
     5041 cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa
     5101 gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg
     5161 cctgtacgag acacggatcg acctgtctca gctgggaggc gattcaggcg gatctactaa
     5221 tctgtcagat attattgaaa aggagaccgg taagcaactg gttatccagg aatccatcct
     5281 catgctccca gaggaggtgg aagaagtcat tgggaacaag ccggaaagcg atatactcgt
     5341 gcacaccgcc tacgacgaga gcaccgacga gaatgtcatg cttctgacta gcgacgcccc
     5401 tgaatacaag ccttgggctc tggtcataca ggatagcaac ggtgagaaca agattaagat
     5461 gctctctggt ggttctccca agaagaagag gaaagtctga catgtcctag agctcgctga
     5521 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     5581 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     5641 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     5701 ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgctgag caaaaggcca
     5761 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5821 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5881 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5941 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6001 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6061 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6121 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6181 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6241 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6301 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6361 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6421 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6481 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6541 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6601 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6661 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6721 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6781 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6841 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6901 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6961 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7021 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7081 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7141 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7201 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7261 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7321 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7381 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7441 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7501 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcca
     7561 gcagagatcc actttggcgc cggctcgagt ggctccggt
//
