LOCUS       Exported                7764 bp DNA     circular SYN 22-MAR-2022
DEFINITION  Mammalian expression of nuclease inactive SuperFi-Cas9 ABE7 base 
            editor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7764)
  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..7764
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     promoter        82..293
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     regulatory      313..322
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     CDS             322..345
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /note="color: #cc99b2"
                     /translation="DYKDDDDK"
     CDS             352..372
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     CDS             397..894
                     /codon_start=1
                     /label=TadA
                     /note="color: #993366"
                     /translation="SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWN
                     RPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFG
                     ARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQS
                     STD"
     CDS             991..1488
                     /codon_start=1
                     /label=TadA
                     /note="color: #993366"
                     /translation="SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWN
                     RAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFG
                     VRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQS
                     STD"
     CDS             1513..1560
                     /codon_start=1
                     /label=x ten
                     /note="color: #993366"
                     /translation="SGSETPGTSESATPES"
     CDS             1585..5685
                     /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: 1585 .. 1608 / #993366
                         2: 1609 .. 1611 / #ffcc99 / D10A
                         3: 1612 .. 4098 / #993366
                         4: 4099 .. 4101 / #ffcc99 / H840A
                         5: 4102 .. 4608 / #993366
                         6: 4609 .. 4611 / #ff99cc / Y1010D
                         7: 4612 .. 4617 / #993366
                         8: 4618 .. 4620 / #ff99cc / Y1013D
                         9: 4621 .. 4626 / #993366
                        10: 4627 .. 4629 / #ff99cc / Y1016D
                        11: 4630 .. 4632 / #993366
                        12: 4633 .. 4635 / #ff99cc / V1018D
                        13: 4636 .. 4638 / #ff99cc / R1019D
                        14: 4639 .. 4659 / #993366
                        15: 4660 .. 4662 / #ff99cc / Q1027D
                        16: 4663 .. 4671 / #993366
                        17: 4672 .. 4674 / #ff99cc / K1031D
                        18: 4675 .. 5685 / #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             5686..5733
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /note="color: #993366"
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5767..5974
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      complement(6039..6627)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(6798..7658)
                     /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: 6798 .. 7589 / #ccffcc
                        2: 7590 .. 7658 / #ccffcc / signal sequence
                     Cleavage site after base 7589"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7659..7763)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
ORIGIN
        1 acatttcccc gaaaagtgcc acctgacgtc cagcagagat ccactttggc gccggctcga
       61 gtggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
      121 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
      181 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
      241 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtgtcgt
      301 gacgcgggat ccgccaccat ggattacaaa gacgatgacg ataagatggc cccaaagaag
      361 aagcggaagg tcggtatcca cggagtccca gcagccagtg aggtcgaatt tagtcatgag
      421 tattggatga gacacgccct gacccttgca aaacgcgcct gggatgaaag ggaagtccct
      481 gtgggggccg tccttgtcca taataatcga gtgattggag agggctggaa tcgccctatt
      541 ggaaggcacg accccactgc acacgcagag attatggctc tccgacaggg tggactggta
      601 atgcagaatt accggctgat cgacgccacc ctctatgtca ctcttgaacc ctgtgtaatg
      661 tgcgctggcg ccatgatcca cagcagaata ggaagagtcg tcttcggcgc tagagatgct
      721 aaaactggag ctgcagggag tttgatggat gtactccacc accccgggat gaatcatcgg
      781 gtggagataa ccgaaggaat cctggctgat gaatgcgctg ctctgttgag cgatttcttt
      841 aggatgagga ggcaggagat taaggcacaa aagaaagctc agagctctac tgacagtggg
      901 gggagttccg gtggatctag tggtagcgag acacccggga cttccgaaag tgctacccca
      961 gaatcatccg gggggagttc aggcggaagt tctgaagtag agttctctca cgagtattgg
     1021 atgcgccacg cactgacact ggctaagcgg gcaagggacg aacgagaagt cccagtcggg
     1081 gctgtcctcg tcttgaataa tagagttatt ggggaggggt ggaaccgagc tattggactg
     1141 catgacccaa ctgcacacgc tgaaattatg gccttgagac agggcggtct cgtaatgcag
     1201 aattatagat tgatagatgc tactttgtat gtgactttcg agccatgcgt catgtgtgcc
     1261 ggggcaatga tccacagcag aattggaagg gttgtattcg gcgtccgaaa cgctaagacc
     1321 ggggctgccg ggtctctcat ggacgtcctt cactatcctg gtatgaatca ccgagtggaa
     1381 attaccgaag gaatcctcgc tgacgaatgc gcagccctcc tctgttattt ctttcggatg
     1441 ccaagacagg tctttaatgc tcagaagaaa gctcagtcct ccactgactc aggtggctcc
     1501 agcggtggaa gctcaggatc tgagacccca ggaacatctg agtcagccac tcctgaatcc
     1561 tcaggtggta gctctggggg gtctgacaag aagtacagca tcggcctggc catcggcacc
     1621 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     1681 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     1741 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     1801 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     1861 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     1921 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     1981 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     2041 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     2101 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     2161 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     2221 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     2281 aagaatggcc tgttcggcaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     2341 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     2401 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     2461 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     2521 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     2581 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     2641 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     2701 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     2761 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     2821 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     2881 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     2941 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     3001 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     3061 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     3121 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     3181 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     3241 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     3301 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     3361 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     3421 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     3481 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     3541 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     3601 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     3661 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     3721 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     3781 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     3841 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     3901 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     3961 cagatcctga aagaacaccc agtggaaaac acccagctgc agaacgagaa gctgtacctg
     4021 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     4081 tccgactacg atgtggacgc catcgtgcct cagagctttc tgaaggacga ctccatcgac
     4141 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     4201 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     4261 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     4321 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     4381 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     4441 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     4501 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     4561 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgga cggcgacgac
     4621 aaggtggacg acgacgacaa gatgatcgcc aagagcgaag acgaaatcgg cgacgctacc
     4681 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
     4741 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
     4801 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
     4861 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
     4921 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
     4981 ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
     5041 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
     5101 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
     5161 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
     5221 ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
     5281 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
     5341 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
     5401 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
     5461 tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt
     5521 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
     5581 aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
     5641 ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaagcg tcctgctgct
     5701 actaagaaag ctggtcaagc taagaaaaag aaatgacatg tcctagagct cgctgatcag
     5761 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     5821 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     5881 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     5941 aggattggga agagaatagc aggcatgctg gggagcggcc gctgagcaaa aggccagcaa
     6001 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6061 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6121 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6181 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6241 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6301 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6361 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6421 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6481 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6541 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6601 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6661 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6721 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6781 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6841 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6901 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6961 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7021 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7081 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7141 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7201 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7261 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7321 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7381 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7441 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7501 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7561 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7621 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7681 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7741 aataaacaaa taggggttcc gcgc
//
