pU6-(BsaI)_CBh-UC-Cas9-T2A-mCherry
(Plasmid
#135014)
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PurposeExpression vector for sgRNAs cloned into the BsaI sites and for expression of Cas9 with 126aa MRN-recruiting domain from HSV-1 UL12 fused to C-terminus of Cas9, linked to mCherry via a T2A peptide
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 135014 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepU6-(BbsI)_CBh-Cas9-T2A-mCherry (Addgene 64324), modification of pX330 (Addgene 42230)
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Backbone manufacturerKuehn lab
- Total vector size (bp) 9663
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Modifications to backboneSites for cloning sgRNAs changed to BsaI. Sticky ends for cloning sgRNAs are the same as the original parent vectors. 126aa domain from HSV-1 UL12 fused to C-terminus of Flag-Cas9, upstream of the T2A-mCherry
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Vector typeMammalian Expression
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 namehumanized S. pyogenes Cas9
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Alt nameSpCas9
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Alt namehSpCas9
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Specieshumanized S. pyogenes
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Insert Size (bp)4272
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Mutation126aa domain from HSV-1 UL12 fused to the C-terminus of Flag-Cas9, upstream of the T2A-mCherry
- Promoter CBh
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Tags
/ Fusion Proteins
- 3xFLAG (N terminal on insert) (N terminal on insert)
- 3x Flag (N terminal on insert)
- NLS (N terminal on insert)
- NLS (C terminal on insert)
- T2A-mCherry (C terminal on insert)
Cloning Information
- Cloning method Restriction Enzyme
- 5′ cloning site FseI (not destroyed)
- 3′ cloning site HindIII (not destroyed)
- 5′ sequencing primer GCTGGACGCCACCCTGATC (Common Sequencing Primers)
Resource Information
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A portion of this plasmid was derived from a plasmid made byThe UL12 fragment was pcr amplified from the pSAK UL12/12.5 plasmid from Sandra K. Weller's laboratory, University of Connecticut Health Center
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Article Citing this Plasmid
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.
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:
pU6-(BsaI)_CBh-UC-Cas9-T2A-mCherry was a gift from Yosef Shaul (Addgene plasmid # 135014 ; http://n2t.net/addgene:135014 ; RRID:Addgene_135014) -
For your References section:
Recruitment of DNA Repair MRN Complex by Intrinsically Disordered Protein Domain Fused to Cas9 Improves Efficiency of CRISPR-Mediated Genome Editing. Reuven N, Adler J, Broennimann K, Myers N, Shaul Y. Biomolecules. 2019 Oct 8;9(10). pii: biom9100584. doi: 10.3390/biom9100584. 10.3390/biom9100584 PubMed 31597252