pEM047 [T7-assisted CROPseq sgRNA expression vector]
(Plasmid #224899)

• Purpose: Lentiviral CROP-seq vector with lineage barcode cassette and T7 promoter for ID amplification from cDNA
• Depositing Lab: Thomas Norman
• Publication: Metzner et al bioRxiv [Preprint]. 2024 Jul 27:2024.07.26.605307. doi: 10.1101/2024.07.26.605307.

Backbone

• Vector backbone: pBA950
• Backbone manufacturer: Addgene #122239
• Modifications to backbone: Switched BFP selectable marker to eGFP, removed WPRE, added T7 promoter, added lineage barcode cassette in 3' UTR
• Vector type: Mammalian Expression, Lentiviral, CRISPR
• Selectable markers: eGFP

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): NEB Stable
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: eGFP
• Species: Synthetic
• Insert Size (bp): 720
• Promoter: EF-1a

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: N/A whole plasmid sequencing

Gene/Insert 2

• Gene/Insert name: LacZ fragment (removed by digestion with BstXI/BlpI for sgRNA cloning)
• Insert Size (bp): 1119

Resource Information

• Supplemental Documents:
  • pem047-multiome_-sirlointso.gb

Terms and Licenses

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

Depositor Comments

Recommended for whole-cell CROPseq assays with Gene Expression readout. In our hands, T7-assisted ID amplification is easier to perform than traditional CROP-seq amplification and improves assignment even in whole cells. This vector would yield lower assignment in Multiome assays due to the lack of SIRLOIN nuclear retention elements and TSO capture sequence. Preprint and T7-assisted ID amplification protocol available at https://www.biorxiv.org/content/10.1101/2024.07.26.605307v1.