pGEmCherry2
(Plasmid #118413)

• Purpose: Red fluorescent reporter for assessing CRISPR gRNA activity
• Depositing Lab: Mark Denham
• Publication: Hojland Knudsen et al Front Cell Dev Biol. 2018 May 15;6:54. doi: 10.3389/fcell.2018.00054. eCollection 2018.

Backbone

• Vector backbone: pmCerulean3- N1
• Vector type: Mammalian Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: mCherry
• Species: Synthetic

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: unknown (unknown if destroyed)
• 3′ cloning site: unknown (unknown if destroyed)
• 5′ sequencing primer: unknown

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

How to Design Genomic recognition sequence for GEmCherry2:
Genomic sequence require 20nt recognition sequence and PAM sequence.
In addition Oligos require overhangs to ligate into Xbal and BamHI sites.
Bottom strand requires BgIII site insead of BamHI to destroy site for subsequent restriction analysis. Inserted sequence may require additional nucleotide to be out of frame.

TOP Oligo 5` CTAGANNNNNNNNNNNNNNNNNNNN(PAM)C 3`
Xbal site 20nt sequence and PAM sequence and cytosine to destroy BamHI sequence.

BOTTOM Oligo 5'GATCG(MAP)NNNNNNNNNNNNNNNNNNNNT 3'

doi: 10.3389/fcell.2018.00054