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PurposeExpresses S. pyogenes CRISPR chimeric RNA element with customizable sgRNA from U6 promoter and puromycin resistance from EF-1a promoter. 3rd generation lentiviral backbone.
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 52963 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 | |
Cloning Grade DNA | 52963-DNA.cg | 2 µg of cloning grade DNA in Tris buffer | 1 | $105 |
Backbone
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Vector backboneCustom
- Total vector size (bp) 10183
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Vector typeMammalian Expression, Lentiviral, 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)NEB Stable
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Growth instructionsUse SapI digest to check for unwanted recombination of lentiviral plasmid. Only amplify in RecA- bacteria (eg. Stbl3 or NEB stable).
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Copy numberHigh Copy
Gene/Insert 1
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Gene/Insert nameS. pyogenes sgRNA cassette
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Alt nameS. pyogenes CRISPR customizable RNA element
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SpeciesSynthetic
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Insert Size (bp)100
- Promoter hU6
Cloning Information for Gene/Insert 1
- Cloning method Restriction Enzyme
- 5′ cloning site BsmBI (not destroyed)
- 3′ cloning site BsmBI (not destroyed)
- 5′ sequencing primer hU6-F
- 3′ sequencing primer hGata4-rev (5'-ATTGTGGATGAATACTGCC-3') (Common Sequencing Primers)
Gene/Insert 2
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Gene/Insert namePuromycin resistance
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Alt namepuromycin N-acetyl-transferase
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Alt namePAC
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Insert Size (bp)600
- Promoter Ef1-a
Cloning Information for Gene/Insert 2
- Cloning method Restriction Enzyme
- 5′ cloning site BsiWI (not destroyed)
- 3′ cloning site MluI (not destroyed)
- 5′ sequencing primer EF-1a-F
- 3′ sequencing primer WPRE-R (Common Sequencing Primers)
Resource Information
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Supplemental Documents
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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 that this plasmid does NOT contain Cas9. It should be used in conjunction with lentiCas9-Blast (Addgene #52962) or otherwise with cell lines already expressing Cas9.
Special note from the Zhang lab: We are constantly improving our CRISPR reagents. Please check https://zlab.bio/ for the most up-to-date information.
Information for Cloning Grade DNA (Catalog # 52963-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:
lentiGuide-Puro was a gift from Feng Zhang (Addgene plasmid # 52963 ; http://n2t.net/addgene:52963 ; RRID:Addgene_52963) -
For your References section:
Improved vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047. 10.1038/nmeth.3047 PubMed 25075903