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PurposeExpression of Cas9 and gRNA cassette in S. cerevisiae; Nourseothricin Resistance
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 81192 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepRS42N
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Backbone manufacturerEuroscarf
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Vector typeYeast Expression, CRISPR
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Selectable markersNourseothricin
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)DH5alpha
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Copy numberHigh Copy
Gene/Insert 1
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Gene/Insert nameCas9
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SpeciesS. pyogenes
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MutationWT - codon optimized
- Promoter ROX3
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Tag
/ Fusion Protein
- NTS (C terminal on backbone)
Cloning Information for Gene/Insert 1
- Cloning method Gibson Cloning
- 5′ sequencing primer unknown (Common Sequencing Primers)
Gene/Insert 2
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Gene/Insert nameempty gRNA cassette
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SpeciesSynthetic
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MutationPlease see depositor comments below.
- Promoter SNP52p
Cloning Information for Gene/Insert 2
- Cloning method Gibson Cloning
- 5′ sequencing primer unknown (Common Sequencing Primers)
Resource Information
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Supplemental Documents
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
Please note: Addgene’s quality control sequences found the guanine nucleotide at the start of the structural guide-RNA sequence was missing. The Boles Lab has indicated that this is a “virtual guanine”. If you sequence this plasmid, you will notice this missing guanine. The Boles lab omitted the guanine in order to work with the empty vector in yeast, since once the structural gRNA has this G, it is active and could create problems in the cell. However, when primers are designed to create the CRISPR/Cas9 vector for a specific target, this G should be in the primer, so that the gRNA becomes functional again. If you do not include this G in your primer sequence, the system will not work.
Plasmid contains a gRNA cassette together with the Cas9 overexpression cassette.
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:
pRCC-N was a gift from Eckhard Boles (Addgene plasmid # 81192 ; http://n2t.net/addgene:81192 ; RRID:Addgene_81192) -
For your References section:
Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae. Generoso WC, Gottardi M, Oreb M, Boles E. J Microbiol Methods. 2016 Aug;127:203-5. doi: 10.1016/j.mimet.2016.06.020. Epub 2016 Jun 17. 10.1016/j.mimet.2016.06.020 PubMed 27327211