pCas

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  • Model: PVT10640
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pCas


Catalog No. PVT10640
Packing 2ug

 

pCas Information

Promoter: RNR2 promoter

Plasmid size: 8761bp

Prokaryotic resistance: kanamycin Kan

Cloning strains: Escherichia coli such as DH5alpha

Culture conditions: 37 C, aerobic, LB

 

pCas Description

Expresses S. pyogenes Cas9 plus an HDV ribozyme-sgRNA for genome editing in yeast.

 

pCas Sequence

LOCUS       Exported                8761 bp ds-DNA     circular SYN 11-OCT-2017
DEFINITION  synthetic circular DNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8761)
  AUTHORS   .
  TITLE     Direct Submission
            
FEATURES             Location/Qualifiers
     source          1..8761
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_RNA        225..300
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        434..777
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             778..1587
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      1593..1790
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    1855..2558
                     /label=RNR2 promoter
     CDS             2559..6662
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6672..6692
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6708..6731
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      6735..6924
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(6987..7575)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    complement(8566..8613)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
        1 gtatatgtgt tatgtagtat actctttctt caacaattaa atactctcgg tagccaagtt
       61 ggtttaaggc gcaagactgt aatttatcac tacgaaatct tgagatcggg cgttcgactc
      121 gcccccggga gagatggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca
      181 ccttcgggtg gcgaatggga ctttcataat aacgtccaat aaatgtttta gagctagaaa
      241 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
      301 tttttttatt ttttgtcact attgttatgt aaaatgccac ctctgacagt atggaacgca
      361 aacttctgtc tagtggatat aggtctagag atctgtttag cttgcctcgt ccccgccggg
      421 tcacccggcc agcgacatgg aggcccagaa taccctcctt gacagtcttg acgtgcgcag
      481 ctcaggggca tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc
      541 atttgcatcc atacattttg atggccgcac ggcgcgaagc aaaaattacg gctcctcgct
      601 gcagacctgc gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc
      661 gcccctgtag agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact
      721 tccttttaaa atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg
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