eSpCas9(1.1) vector (V012346)

Basic Vector Information

      • Vector Name:
      • eSpCas9(1.1)
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 8506 bp
      • Type:
      • CRISPR Plasmids
      • Source/Author:
      • Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F.
      • Copy Number:
      • High copy number

eSpCas9(1.1) vector Vector Map

eSpCas9(1.1)8506 bp4008001200160020002400280032003600400044004800520056006000640068007200760080008400U6 promotergRNA scaffoldpolIII terminatorCMV enhancerchicken beta-actin promoterhybrid intronATG3xFLAGSV40 NLSeSpCas9(1.1)nucleoplasmin NLSbGH poly(A) signalAAV2 ITRf1 oriAmpR promoterAmpRori

Plasmid Resuspension Protocol:

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5.Store the plasmid at -20 ℃.

eSpCas9(1.1) vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                8506 bp ds-DNA    circular SYN 03-2-2016
DEFINITION  Zhang lab vector for expressing a single guide RNA (sgRNA) together 
            with an S. pyogenes Cas9 variant with improved targeting 
            specificity.
ACCESSION   .
VERSION     .
KEYWORDS    eSpCas9(1.1)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8506)
  AUTHORS   Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F.
  TITLE     Rationally engineered Cas9 nucleases with improved specificity.
  JOURNAL   Science 2016;351:84-8.
  PUBMED    26628643
REFERENCE   2  (bases 1 to 8506)
  AUTHORS   Zhang Lab / Addgene #71814
  TITLE     Direct Submission
COMMENT     An error in codon 848 was corrected based on sequencing results from
            Addgene.
FEATURES             Location/Qualifiers
     source          1..8506
                     /organism="synthetic DNA construct"
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
     promoter        1..241
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        268..343
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      344..349
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     enhancer        440..725
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /note="chicken beta-actin promoter"
     intron          1005..1233
                     /note="hybrid intron"
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1251..1253
                     /codon_start=1
                     /product="start codon"
                     /note="ATG"
                     /translation="M"
     CDS             1254..1319
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /note="3xFLAG"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1326..1346
                     /codon_start=1
                     /locus_tag="
                     "
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /note="
                     "
                     /protein_id="
                     "
                     /translation="PKKKRKV"
     CDS             1371..5471
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /note="eSpCas9(1.1)"
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5472..5519
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5553..5760
                     /note="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5769..5909
                     /note="AAV2 ITR"
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5984..6439
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        6721..6825
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             6826..7686
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7857..8445
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"

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