px458_Conc3
(Plasmid #134450)

• Purpose: (Empty Backbone) Empty concatemer vector in which 3 sgRNAs can be inserted; with Cas9 + GFP
• Depositing Lab: Daniel Stange
• Publication: Gaebler et al J Vis Exp. 2020 Feb 18;(156). doi: 10.3791/60704.

Backbone

• Vector backbone: pSpCas9(BB)-2A-GFP (PX458)
• Backbone manufacturer: Feng Zhang
• Modifications to backbone: The Backbone vector was PciI/XbaI digested and an amplified region from 3 gRNA concatemer vector (#84880) was inserted, which encodes for three U6 promotors.
• Vector type: Mammalian Expression, CRISPR
• Promoter: U6
• Selectable markers: GFP

Growth in Bacteria

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

Cloning Information

• Cloning method: Restriction Enzyme

Resource Information

• Supplemental Documents:
  • px458_Conc3.gbk
  • Sequences of the customized overhangs for golden gate cloning into

Terms and Licenses

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

Depositor Comments

The sgRNAs can get introduced by golden gate cloning like described in Andersson-Rolf A et al.: Simultaneous paralogue knockout using a CRISPR-concatemer in mouse small intestinal organoids. Dev Biol. 2016. doi: 10.1016/j.ydbio.2016.10.016. For more information see protocol.