B-HypaR-SpCas9
(Plasmid
#126764)
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PurposeExpression plasmid for human codon-opt. increased fidelity Blackjack-Hypa2SpCas9 (w/o U6-sgRNA). Cleaves both 20nt and 5'-extended 21nt spacer containing sgRNAs with higher fidelity than Hypa2SpCas9.
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 126764 | 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-Hypa2SpCas9
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Alt nameBlackjack-Hypa2SpCas9
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SpeciesS. pyogenes
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Insert Size (bp)4251
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MutationR661A; N692A; M694A; Q695A; H698A; amino 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-Hypa2SpCas9-NLS (without U6-sgRNA coding sequence, with silent mutations).
Blackjack variants cleave both 20nt and 5'-extended 21nt spacer containing sgRNAs. B-Hypa2SpCas9 shows higher fidelity and target selectivity than its parental increased fidelity variant, Hypa2SpCas9.
The lack of sgRNA expression cassette allows for easy testing of various SpCas9 variants with the same sgRNA expressed from a separate plasmid (e.g. pmCherry_gRNA, Addgene# #80457).
For detailed information and plasmid usage, please see the associated publication. Please visit https://doi.org/10.1101/2022.08.16.504097 for bioRxiv preprint.
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-HypaR-SpCas9 was a gift from Ervin Welker (Addgene plasmid # 126764 ; http://n2t.net/addgene:126764 ; RRID:Addgene_126764) -
For your References section:
A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets. Kulcsar PI, Talas A, Ligeti Z, Toth E, Rakvacs Z, Bartos Z, Krausz SL, Welker A, Vegi VL, Huszar K, Welker E. Nat Commun. 2023 Sep 16;14(1):5746. doi: 10.1038/s41467-023-41393-5. 10.1038/s41467-023-41393-5 PubMed 37717069