pFUGW-GFP-2-cut reporter
(Plasmid #131669)

• Purpose: Lentiviral plasmid that expresses a GFP-based reporter for monitoring CRISPR based precise end-joining.
• Depositing Lab: Alberto Ciccia
• Publication: Nambiar et al Nat Commun. 2019 Jul 30;10(1):3395. doi: 10.1038/s41467-019-11105-z.

Backbone

• Vector backbone: pFUGW
• Backbone size w/o insert (bp): 8700
• Total vector size (bp): 9600
• Modifications to backbone: The 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.
• Vector type: Mammalian Expression, Lentiviral
• Selectable markers: Blasticidin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: GFP-2-cut reporter
• Species: Synthetic
• Insert Size (bp): 915
• Promoter: EFS-NS
• 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'​)

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry