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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