PurposeVector for expression of SpCas9 in yeast after assembly with PID fragments: TEF1p-SpCas9::KanR-1xNLS-CYC1t (KanR cassette in place of the PID)
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||165615||Standard format: Plasmid sent in bacteria as agar stab||1||$85|
This material is available to academics and nonprofits only.
Backbone manufacturerMarcus Noyes Lab
Vector typeYeast Expression, CRISPR
Growth in Bacteria
Bacterial Resistance(s)Ampicillin and Kanamycin, 100 & 50 μg/mL
Growth Strain(s)XL1 Blue
Copy numberHigh Copy
Gene/Insert nameTEF1p-SpCas9::KanR(in place of PID)-1xNLS-CYC1t (S. cerevisiae TEF promoter driving SpCas9 with KanR cassette replacing PID)
MutationCorrected homology relative to wild type SpCas9 (immediately adjacent to the NheI site upstream of KanR cassette)
- Promoter TEF1
/ Fusion Protein
- SV40 NLS (C terminal on insert)
- Cloning method Gibson Cloning
- 5′ sequencing primer AAATAGGCAAGGCCACCGCTAAG
- 3′ sequencing primer CTAGACTTCAGGTTGTCTAACTC (Common Sequencing Primers)
Terms and Licenses
- Not Available to Industry
- 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.
For your Materials & Methods section:pGG431 was a gift from Marcus Noyes (Addgene plasmid # 165615 ; http://n2t.net/addgene:165615 ; RRID:Addgene_165615)
For your References section:Engineered dual selection for directed evolution of SpCas9 PAM specificity. Goldberg GW, Spencer JM, Giganti DO, Camellato BR, Agmon N, Ichikawa DM, Boeke JD, Noyes MB. Nat Commun. 2021 Jan 13;12(1):349. doi: 10.1038/s41467-020-20650-x. 10.1038/s41467-020-20650-x PubMed 33441553