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PurposeCas9 from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA (V2.0)
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 62988 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 | |
Cloning Grade DNA | 62988-DNA.cg | 2 µg of cloning grade DNA in Tris buffer | 1 | $105 |
Backbone
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Vector backbonePX459
- Total vector size (bp) 9200
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Vector typeMammalian Expression, CRISPR
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Selectable markersPuromycin
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)Stbl3
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Copy numberUnknown
Gene/Insert
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Gene/Insert namehSpCas9-2A-Puro V2.0
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Alt nameSpCas9-2A-Puro V2.0
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Alt nameCas9
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Alt namePX459 V2.0
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SpeciesSynthetic
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Insert Size (bp)6000
- Promoter Cbh
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Tags
/ Fusion Proteins
- 3XFLAG (N terminal on insert)
- 2A-Puro (C terminal on insert)
Cloning Information
- Cloning method Restriction Enzyme
- 5′ cloning site AgeI (not destroyed)
- 3′ cloning site EcoRI (not destroyed)
- 5′ sequencing primer TTTATGGCGAGGCGGCGG
- 3′ sequencing primer puro-variant-R (5'-gtgggcttgtactcggtcat-3') (Common Sequencing Primers)
Resource Information
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Supplemental Documents
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Addgene Notes
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Articles 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.
Depositor Comments
NOTE: This is an improved version of Addgene Plasmid #48139. It has improved puromycin selection. The EcoRI site is not unique and flanks the puromycin cassette.
For more information on Zhang Lab CRISPR Plasmids please refer to: http://www.addgene.org/crispr/zhang/
The guide sequence is cloned into this plasmid using BbsI sites. The hU6-F primer (5'-GAGGGCCTATTTCCCATGATT-3') can be used to confirm the gRNA sequence after cloning into the plasmid. Please see the cloning protocol provided by the Zhang lab for more details.
Information for Cloning Grade DNA (Catalog # 62988-DNA.cg) ( Back to top)
Purpose
Cloning grade DNA is suitable for use in PCR, cloning reactions, or transformation into E. coli. The purity and amount is not suitable for direct transfections.
Delivery
- Amount 2 µg
- Guaranteed Concentration 100 ng/µl +/- 5 ng/µl
- Pricing $105 USD
- Storage DNA can be stored at 4℃ (short term) or -20℃ (long term).
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Quality Control
Addgene has verified this plasmid using Next Generation Sequencing. Results are available here
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:
pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988 ; http://n2t.net/addgene:62988 ; RRID:Addgene_62988) -
For your References section:
Genome engineering using the CRISPR-Cas9 system. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. 10.1038/nprot.2013.143 PubMed 24157548