pEM040 [Multiome Perturb-seq sgRNA expression vector]
(Plasmid #224898)

• Purpose: Lentiviral CROP-seq vector with lineage barcode cassette and T7 promoter for ID amplification from cDNA, engineered for high-efficiency ID assignment in Multiome Perturb-seq experiments
• 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 SIRLOIN nuclear retention elements, added 10x TSO binding sequence, 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:
  • annotated_pem040-final_multiomeperturbseq_vector.gb

Terms and Licenses

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

Depositor Comments

Recommended vector for Multiome Perturb-seq experiments; CROP-seq transcripts are retained in nucleus by SIRLOIN nuclear retention elements, and 10x TSO binding sequence assists capture. Preprint and T7-assisted ID amplification protocol available at https://www.biorxiv.org/content/10.1101/2024.07.26.605307v1.