LOCUS       Exported                7194 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Provides the human codon optimized CDS of Cas9 protein with mutated 
            (D10A, H840A) and inactivated catalytic domains as a level 0 
            GoldenBraid part for C-terminal fusions.
ACCESSION   .
VERSION     .
KEYWORDS    pdCas9 (GB1079)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7194)
  AUTHORS   Vazquez-Vilar M, Bernabe-Orts JM, Fernandez-Del-Carmen A, Ziarsolo 
            P, Blanca J, Granell A, Orzaez D
  TITLE     A modular toolbox for gRNA-Cas9 genome engineering in plants based 
            on the GoldenBraid standard.
  JOURNAL   Plant Methods. 2016 Feb 1;12:10. doi: 10.1186/s13007-016-0101-2. 
            eCollection 2016.
  PUBMED    26839579
REFERENCE   2  (bases 1 to 7194)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7194
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             9..29
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             30..4130
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     promoter        complement(4244..4262)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4245..4262)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4280..4296)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4280..4296)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4293..4315)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4304..4320
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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(4328..4358)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4373..4394
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4511..4528)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4682..5270)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4762..4781)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5441..6301)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6064..6083
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6302..6406)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6484..6939)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6621..6642)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6833..6852
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7065..7087
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7079..7096
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7080..7096
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7103..7122
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7103..7121
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      7152..7161
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      7156..7165
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
ORIGIN
        1 ctcgaatgcc caagaagaag aggaaggtgg acaagaagta ctccattggg ctcgctatcg
       61 gcacaaacag cgtcggctgg gccgtcatta cggacgagta caaggtgccg agcaaaaaat
      121 tcaaagttct gggcaatacc gatcgccaca gcataaagaa gaacctcatt ggcgccctcc
      181 tgttcgactc cggggaaacg gccgaagcca cgcggctcaa aagaacagca cggcgcagat
      241 atacccgcag aaagaatcgg atctgctacc tgcaggagat ctttagtaat gagatggcta
      301 aggtggatga ctctttcttc cataggctgg aggagtcctt tttggtggag gaggataaaa
      361 agcacgagcg ccacccaatc tttggcaata tcgtggacga ggtggcgtac catgaaaagt
      421 acccaaccat atatcatctg aggaagaagc ttgtagacag tactgataag gctgacttgc
      481 ggttgatcta tctcgcgctg gcgcatatga tcaaatttcg gggacacttc ctcatcgagg
      541 gggacctgaa cccagacaac agcgatgtcg acaaactctt tatccaactg gttcagactt
      601 acaatcagct tttcgaagag aacccgatca acgcatccgg agttgacgcc aaagcaatcc
      661 tgagcgctag gctgtccaaa tcccggcggc tcgaaaacct catcgcacag ctccctgggg
      721 agaagaagaa cggcctgttt ggtaatctta tcgccctgtc actcgggctg acccccaact
      781 ttaaatctaa cttcgacctg gccgaagatg ccaagcttca actgagcaaa gacacctacg
      841 atgatgatct cgacaatctg ctggcccaga tcggcgacca gtacgcagac ctttttttgg
      901 cggcaaagaa cctgtcagac gccattctgc tgagtgatat tctgcgagtg aacacggaga
      961 tcaccaaagc tccgctgagc gctagtatga tcaagcgcta tgatgagcac caccaagact
     1021 tgactttgct gaaggccctt gtcagacagc aactgcctga gaagtacaag gaaattttct
     1081 tcgatcagtc taaaaatggc tacgccggat acattgacgg cggagcaagc caggaggaat
     1141 tttacaaatt tattaagccc atcttggaaa aaatggacgg caccgaggag ctgctggtaa
     1201 agcttaacag agaagatctg ttgcgcaaac agcgcacttt cgacaatgga agcatccccc
     1261 accagattca cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat ttctacccct
     1321 ttttgaaaga taacagggaa aagattgaga aaatcctcac atttcggata ccctactatg
     1381 taggccccct cgcccgggga aattccagat tcgcgtggat gactcgcaaa tcagaagaga
     1441 ctatcactcc ctggaacttc gaggaagtcg tggataaggg ggcctctgcc cagtccttca
     1501 tcgaaaggat gactaacttt gataaaaatc tgcctaacga aaaggtgctt cctaaacact
     1561 ctctgctgta cgagtacttc acagtttata acgagctcac caaggtcaaa tacgtcacag
     1621 aagggatgag aaagccagca ttcctgtctg gagagcagaa gaaagctatc gtggacctcc
     1681 tcttcaagac gaaccggaaa gttaccgtga aacagctcaa agaagattat ttcaaaaaga
     1741 ttgaatgttt cgactctgtt gaaatcagcg gagtggagga tcgcttcaac gcatccctgg
     1801 gaacgtatca cgatctcctg aaaatcatta aagacaagga cttcctggac aatgaggaga
     1861 acgaggacat tcttgaggac attgtcctca cccttacgtt gtttgaagat agggagatga
     1921 ttgaagaacg cttgaaaact tacgctcatc tcttcgacga caaagtcatg aaacagctca
     1981 agaggcgccg atatacagga tgggggcggc tgtcaagaaa actgatcaat gggatccgag
     2041 acaagcagag tggaaagaca atcctggatt ttcttaagtc cgatggattt gccaaccgga
     2101 acttcatgca gttgatccat gatgactctc tcacctttaa ggaggacatc cagaaagcac
     2161 aagtttctgg ccagggggac agtctccacg agcacatcgc taatcttgca ggtagcccag
     2221 ctatcaaaaa gggaatactg cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg
     2281 gaaggcataa gcccgagaat atcgttatcg agatggcccg agagaaccaa actacccaga
     2341 agggacagaa gaacagtagg gaaaggatga agaggattga agagggtata aaagaactgg
     2401 ggtcccaaat ccttaaggaa cacccagttg aaaacaccca gcttcagaat gagaagctct
     2461 acctgtacta cctgcagaac ggcagggaca tgtacgtgga tcaggaactg gacatcaatc
     2521 ggctctccga ctacgacgtg gatgccatcg tgccccagtc ttttctcaaa gatgattcta
     2581 ttgataataa agtgttgaca agatccgata aaaatagagg gaagagtgat aacgtcccct
     2641 cagaagaagt tgtcaagaaa atgaaaaatt attggcggca gctgctgaac gccaaactga
     2701 tcacacaacg gaagttcgat aatctgacta aggctgaacg aggtggcctg tctgagttgg
     2761 ataaagccgg cttcatcaaa aggcagcttg ttgagacacg ccagatcacc aagcacgtgg
     2821 cccaaattct cgattcacgc atgaacacca agtacgatga aaatgacaaa ctgattcgag
     2881 aggtgaaagt tattactctg aagtctaagc tggtttcaga tttcagaaag gactttcagt
     2941 tttataaggt gagagagatc aacaattacc accatgcgca tgatgcctac ctgaatgcag
     3001 tggtaggcac tgcacttatc aaaaaatatc ccaagcttga atctgaattt gtttacggag
     3061 actataaagt gtacgatgtt aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg
     3121 ccaccgctaa gtacttcttt tacagcaata ttatgaattt tttcaagacc gagattacac
     3181 tggccaatgg agagattcgg aagcgaccac ttatcgaaac aaacggagaa acaggagaaa
     3241 tcgtgtggga caagggtagg gatttcgcga cagtccggaa ggtcctgtcc atgccgcagg
     3301 tgaacatcgt taaaaagacc gaagtacaga ccggaggctt ctccaaggaa agtatcctcc
     3361 cgaaaaggaa cagcgacaag ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg
     3421 gcggattcga ttctcctaca gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag
     3481 ggaagtctaa aaaactcaaa agcgtcaagg aactgctggg catcacaatc atggagcgat
     3541 caagcttcga aaaaaacccc atcgactttc tcgaggcgaa aggatataaa gaggtcaaaa
     3601 aagacctcat cattaagctt cccaagtact ctctctttga gcttgaaaac ggccggaaac
     3661 gaatgctcgc tagtgcgggc gagctgcaga aaggtaacga gctggcactg ccctctaaat
     3721 acgttaattt cttgtatctg gccagccact atgaaaagct caaaggatct cccgaagata
     3781 atgagcagaa gcagctgttc gtggaacaac acaaacacta ccttgatgag atcatcgagc
     3841 aaataagcga attctccaaa agagtgatcc tcgccgacgc taacctcgat aaggtgcttt
     3901 ctgcttacaa taagcacagg gataagccca tcagggagca ggcagaaaac attatccact
     3961 tgtttactct gaccaacttg ggcgcgcctg cagccttcaa gtacttcgac accaccatag
     4021 acagaaagcg gtacacctct acaaaggagg tcctggacgc cacactgatt catcagtcaa
     4081 ttacggggct ctatgaaaca agaatcgacc tctctcagct cggtggagac agcagggctg
     4141 attcgcgaga ccactcatcg cccaatcact agtgaattcg cggccgcctg caggtcgacc
     4201 atatgggaga gctcccaacg cgttggatgc atagcttgag tattctatag tgtcacctaa
     4261 atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4321 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4381 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4441 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4501 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4561 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4621 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4681 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4741 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4801 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4861 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4921 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4981 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5041 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5101 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5161 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5221 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5281 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5341 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5401 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5461 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5521 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5581 gcgacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5641 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5701 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5761 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5821 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5881 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5941 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6001 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6061 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6121 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6181 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6241 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6301 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6361 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6421 aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     6481 aggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     6541 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     6601 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     6661 ccaacgtcaa agggcgaaaa accgtctatc agggcgctgg cccactacgt gaaccatcac
     6721 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     6781 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     6841 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     6901 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc cattcgccat tcaggctgcg
     6961 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     7021 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     7081 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gcccgacgtc
     7141 gcatgctccc ggccgccatg gcggccgcgg gaattcgatt gggcgatgag tggt
//
