B-SpCas9
(Plasmid
#126760)
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PurposeExpression plasmid for human codon-optimized Blackjack-SpCas9 (w/o U6-sgRNA). Cleaves both 20nt and 5'-extended 21nt spacer containing sgRNAs with higher fidelity than that of WT SpCas9.
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 126760 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepX330-like (without U6-sgRNA coding sequence)
- Total vector size (bp) 8056
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Vector typeMammalian Expression, CRISPR
Growth in Bacteria
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Bacterial Resistance(s)Ampicillin, 100 μg/mL
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Growth Temperature37°C
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Growth Strain(s)NEB Stable
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Copy numberHigh Copy
Gene/Insert
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Gene/Insert nameB-SpCas9
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Alt nameBlackjack-SpCas9
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SpeciesS. pyogenes
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Insert Size (bp)4251
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Mutationamino acids 1005-1013 replaced with two glycine
- Promoter CBh
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Tags
/ Fusion Proteins
- 3xFLAG (N terminal on insert)
- NLS (N terminal on insert)
- NLS (C terminal on insert)
Cloning Information
- Cloning method Restriction Enzyme
- 5′ cloning site MluI (not destroyed)
- 3′ cloning site Pfl23II (destroyed during cloning)
- 5′ sequencing primer GAACATCGTGATCGAAATG
- 3′ sequencing primer TGTCGAAGCCGCCGTAC (Common Sequencing Primers)
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
Depositor Comments
pCbh-3xFLAG-NLS-Blackjack-SpCas9-NLS (without U6-sgRNA coding sequence, with silent mutations).
B-SpCas9 shows higher fidelity than WT SpCas9.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
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For your Materials & Methods section:
B-SpCas9 was a gift from Ervin Welker (Addgene plasmid # 126760 ; http://n2t.net/addgene:126760 ; RRID:Addgene_126760) -
For your References section:
Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5'G-extended sgRNAs. Kulcsar PI, Talas A, Toth E, Nyeste A, Ligeti Z, Welker Z, Welker E. Nat Commun. 2020 Mar 6;11(1):1223. doi: 10.1038/s41467-020-15021-5. 10.1038/s41467-020-15021-5 PubMed 32144253