LOCUS       pFH45                   7576 bp    DNA     circular UNA 10-APR-2019
DEFINITION  Level0 ABE7.10 base changer, human codon optimized.
ACCESSION   urn.local...136-abnbxnp
VERSION     urn.local...136-abnbxnp
KEYWORDS    .
SOURCE      
  ORGANISM  
            .
FEATURES             Location/Qualifiers
     CDS             215..1225
                     /label="Sm/Sp no DraIII"
     rep_origin      1385..1999
                     /modified_by="hahnf"
                     /label="rep (pMB1)"
     rep_origin      1984
                     /label="ORI"
     misc_feature    2225..2230
                     /Recognition_pattern="GGTCTC(1/5)"
                     /label="BsaI"
                     /note="Geneious type: restriction site"
     ligation        2232..2235
                     /label="Ligation"
     CDS             2233..2733
                     /created_by="hahnf"
                     /label="ecTadA (wt), monomer 1 of 2"
     Linker          2734..2829
                     /created_by="hahnf"
                     /label="Linker"
     misc_feature    2758..2805
                     /created_by="hahnf"
                     /label="XTEN Linker"
     CDS             2830..3327
                     /created_by="hahnf"
                     /label="ecTadA (evolved), monomer 2 of 2"
     Linker          3328..3423
                     /created_by="hahnf"
                     /label="Linker"
     misc_feature    3352..3399
                     /created_by="hahnf"
                     /label="XTEN linker"
     CDS             3424..7557
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the
                      Streptococcus pyogenes Type II CRISPR/Cas system"
                     /note="generates RNA-guided single strand nicks in DNA due
                      to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE
                     SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM
                     IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKS
                     RRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
                     LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLL
                     KALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL
                     NREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY
                     VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP
                     KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED
                     YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTL
                     FEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL
                     KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTV
                     KVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH
                     PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL
                     TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG
                     FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY
                     KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM
                     PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA
                     KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE
                     LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHK
                     HYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP
                     AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
                     /label="Cas9(D10A)"
     modified_base   3448..3450
                     /created_by="hahnf"
                     /modified_by="hahnf"
                     /label="D10A"
     sig_peptide     7537..7557
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian
                     virus 40) large T antigen"
                     /translation="PKKKRKV"
                     /modified_by="hahnf"
                     /label="SV40 NLS"
     Stop_codon      7558..7560
                     /Stop_codon
                     /created_by="hahnf"
                     /label="Stop"
     ligation        7561..7564
                     /label="Ligation"
     misc_feature    complement(7566..7571)
                     /Recognition_pattern="GGTCTC(1/5)"
                     /label="BsaI"
                     /note="Geneious type: restriction site"
ORIGIN      
        1 gtggctcttc agtggacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
       61 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
      121 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
      181 ataaatgctt caataatatt gaaaaaggaa gagtatgcgc tcacgcaact ggtccagaac
      241 cttgaccgaa cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg
      301 tttttttggg gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg
      361 tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac
      421 aaagttaaac atcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt
      481 agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc
      541 cgcagtggat ggcggcctga agccacacag cgatattgat ttgctggtta cggtgaccgt
      601 aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc
      661 ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat
      721 cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga
      781 cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac
      841 aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc
      901 ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc
      961 gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag
     1021 cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct
     1081 gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga
     1141 agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccattacg taaaaggcga
     1201 gatcaccaag gtagtcggca aataactgtc agaccaagtt tactcatata tactttagat
     1261 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1321 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1381 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1441 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1501 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     1561 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1621 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1681 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1741 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1801 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1861 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1921 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1981 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2041 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2101 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2161 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatcact
     2221 ctgtggtctc aaatgtccga agtcgagttt tcccatgagt actggatgag acacgcattg
     2281 actctcgcaa agagggcttg ggatgaacgc gaggtgcccg tgggggcagt actcgtgcat
     2341 aacaatcgcg taatcggcga aggttggaat aggccgatcg gacgccacga ccccactgca
     2401 catgcggaaa tcatggccct tcgacaggga gggcttgtga tgcagaatta tcgacttatc
     2461 gatgcgacgc tgtacgtcac gcttgaacct tgcgtaatgt gcgcgggagc tatgattcac
     2521 tcccgcattg gacgagttgt attcggtgcc cgcgacgcca agacgggtgc cgcaggttca
     2581 ctgatggacg tgctgcatca cccaggcatg aaccaccggg tagaaatcac agaaggcata
     2641 ttggcggacg aatgtgcggc gctgttgtcc gacttttttc gcatgcggag gcaggagatc
     2701 aaggcccaga aaaaagcaca atcctctact gactctggtg gttcttctgg tggttctagc
     2761 ggcagcgaga ctcccgggac ctcagagtcc gccacacccg aaagttctgg tggttcttct
     2821 ggtggttctt ccgaagtcga gttttcccat gagtactgga tgagacacgc attgactctc
     2881 gcaaagaggg ctcgagatga acgcgaggtg cccgtggggg cagtactcgt gctcaacaat
     2941 cgcgtaatcg gcgaaggttg gaatagggca atcggactcc acgaccccac tgcacatgcg
     3001 gaaatcatgg cccttcgaca gggagggctt gtgatgcaga attatcgact tatcgatgcg
     3061 acgctgtacg tcacgtttga accttgcgta atgtgcgcgg gagctatgat tcactcccgc
     3121 attggacgag ttgtattcgg tgttcgcaac gccaagacgg gtgccgcagg ttcactgatg
     3181 gacgtgctgc attacccagg catgaaccac cgggtagaaa tcacagaagg catattggcg
     3241 gacgaatgtg cggcgctgtt gtgttacttt tttcgcatgc ccaggcaggt ctttaacgcc
     3301 cagaaaaaag cacaatcctc tactgactct ggtggttctt ctggtggttc tagcggcagc
     3361 gagactcccg ggacctcaga gtccgccaca cccgaaagtt ctggtggttc ttctggtggt
     3421 tctgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc
     3481 ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt
     3541 cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag
     3601 gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt
     3661 tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt
     3721 ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga
     3781 aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa
     3841 aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat
     3901 atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat
     3961 gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct
     4021 ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga
     4081 cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac
     4141 cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa
     4201 gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca
     4261 caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc
     4321 ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca
     4381 atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt
     4441 cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca
     4501 ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta
     4561 gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga
     4621 aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat
     4681 gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt
     4741 gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct
     4801 cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa
     4861 gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag
     4921 aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg
     4981 tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta
     5041 agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca
     5101 gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc
     5161 tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata
     5221 attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg
     5281 ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct
     5341 cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga
     5401 cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc
     5461 gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac
     5521 tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg
     5581 cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca
     5641 gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta
     5701 atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg
     5761 atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct
     5821 gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg
     5881 gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac
     5941 attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg
     6001 gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag
     6061 aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta
     6121 actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag
     6181 ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat
     6241 acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca
     6301 aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac
     6361 taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa
     6421 tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag
     6481 atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct
     6541 aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga
     6601 cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc
     6661 gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg
     6721 cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc
     6781 gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc
     6841 tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc
     6901 aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac
     6961 ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag
     7021 tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt
     7081 caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc
     7141 cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag
     7201 cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc
     7261 atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa
     7321 cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct
     7381 ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag
     7441 gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata
     7501 gatttgtcac agcttggggg tgactctggt ggttctccca agaagaagag gaaagtctaa
     7561 gctttgagac cacgaa
//
