pFUGW-GFP-2-cut reporter
(Plasmid
#131669)
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PurposeLentiviral plasmid that expresses a GFP-based reporter for monitoring CRISPR based precise end-joining.
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 131669 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepFUGW
- Backbone size w/o insert (bp) 8700
- Total vector size (bp) 9600
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Modifications to backboneThe GFP-2-cut reporter was constructed by removing Cas9 from the lenti-SpCas9 blast plasmid (Addgene #104997) by XbaI and BamHI digestion. The plasmid was re-circularized using complementary oligonucleotides containing unique restriction sites. The resulting plasmid, which was was then digested with AgeI and BamHI and the GFP-2-cut reporter cassette (ordered as a gBlock from IDT) was cloned using Gibson assembly. The GFP-2-cut reporter contains a 192 bp sequence inserted into the GFP sequence that was generated using a random DNA sequence generator (http://www.faculty.ucr.edu/~mmaduro/random.htm). STOP codons were removed manually from the randomly generated sequence and PAM motifs for Cas9 targeting were added at the junctions with the GFP sequence.
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Vector typeMammalian Expression, Lentiviral
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Selectable markersBlasticidin
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 numberUnknown
Gene/Insert
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Gene/Insert nameGFP-2-cut reporter
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SpeciesSynthetic
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Insert Size (bp)915
- Promoter EFS-NS
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Tag
/ Fusion Protein
- P2A (C terminal on insert)
Cloning Information
- Cloning method Restriction Enzyme
- 5′ cloning site XbaI (destroyed during cloning)
- 3′ cloning site BamHI (unknown if destroyed)
- 5′ sequencing primer pLLU-F (5'-gggacagcagagatccagtt-3')
- 3′ sequencing primer blast-R (5'-gctctttcaatgagggtgga-3') (Common Sequencing Primers)
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:
pFUGW-GFP-2-cut reporter was a gift from Alberto Ciccia (Addgene plasmid # 131669 ; http://n2t.net/addgene:131669 ; RRID:Addgene_131669) -
For your References section:
Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant. Nambiar TS, Billon P, Diedenhofen G, Hayward SB, Taglialatela A, Cai K, Huang JW, Leuzzi G, Cuella-Martin R, Palacios A, Gupta A, Egli D, Ciccia A. Nat Commun. 2019 Jul 30;10(1):3395. doi: 10.1038/s41467-019-11105-z. 10.1038/s41467-019-11105-z PubMed 31363085