pMP108 Nanog-Venus-2a-mCherry
(Plasmid #159743)

• Purpose: Nanog targeting vector to insert fluorescent reporter of protein and gene expression levels from endogenous locus in mouse embryonic stem cells. Please see depositor comments for more detail.
• Depositing Lab: Sergi Regot
• Publication: Pokrass et al Dev Cell. 2020 Nov 9;55(3):328-340.e5. doi: 10.1016/j.devcel.2020.09.013. Epub 2020 Oct 21.

Backbone

• Vector backbone: pENTR1a
• Backbone size w/o insert (bp): 2229
• Total vector size (bp): 7407
• Vector type: Mouse Targeting
• Selectable markers: Neomycin (select with G418)

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: Nanog homeobox
• Alt name: Nanog
• Alt name: ENK
• Alt name: 2410002E02Rik
• Species: M. musculus (mouse)
• Insert Size (bp): 3170
• Entrez Gene: Nanog (a.k.a. 2410002E02Rik, ENK, Stm1, ecat4)
• Tag / Fusion Protein:
  • Venus-2a-mCherry (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: ATTL1
• 3′ sequencing primer: ATTL2

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Insert was cloned and modified from Addgene plasmid 59995

Terms and Licenses

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

Depositor Comments

Please note that this plasmid runs as a multimer (>14kb). While this may reduce DNA yield, the plasmid still functions as expected for generating ES cells.

From original plasmid (Addgene 59995), 0.5kb of 5' and 3' homology arms were amplified with 2a-mCherry insert and cloned into pENTR backbone. A linker-venus sequence was inserted between the Nanog coding sequence and the 2a-mCherry cassette.

To target Nanog, we used a "double cut donor" strategy (see DOI 10.1186/s13059-017-1164-8). In brief, we designed an sgRNA sequence to make a double strand break near the stop codon of the Nanog ORF. We then inserted flanking sgRNA recognition sequences at the 5' and 3' ends of the Nanog targeting sequence in the repair template. Cells were co-transfected with pMP108 and the Cas9/sgRNA expression plasmid.