pmRNA-iEMCV-EGFP-A120
(Plasmid #225905)

• Purpose: In vitro transcription of cap-independently (EMCV IRES) translated mRNA encoding EGFP.
• Depositing Lab: Tom Brown
• Publication: Golojuch et al Nature Communications volume 16, Article number: 9205 (2025)

Backbone

• Vector backbone: pCI
• Backbone manufacturer: Promega
• Vector type: Mammalian Expression, Synthetic Biology ; mRNA preparation

Growth in Bacteria

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

Gene/Insert 1

• Gene/Insert name: A-inserted T7 class III Φ6.5 promoter
• Species: Synthetic
• Insert Size (bp): 20

Gene/Insert 2

• Gene/Insert name: EMCV IRES
• Alt name: iEMCV
• Alt name: Encephalomyocarditis virus internal ribosome entry site
• Species: Synthetic
• Insert Size (bp): 578

Gene/Insert 3

• Gene/Insert name: EGFP
• Alt name: Enhanced green fluorescent protein
• Species: Synthetic
• Insert Size (bp): 720

Gene/Insert 4

• Gene/Insert name: 2x β-globin UTR
• Species: Synthetic
• Insert Size (bp): 270

Gene/Insert 5

• Gene/Insert name: poly(A) tail
• Species: Synthetic
• Insert Size (bp): 120

Resource Information

• Supplemental Documents:
  • pmRNA-iEMCV-EGFP-A120.gbk
• A portion of this plasmid was derived from a plasmid made by: Stephen Ikeda (pRNA2-(A)128, Addgene plasmid #174006; RRID:Addgene_174006). Elizabeth J. Robertson (pCAG-EOMES-EGFP-IRES-Puro).

Terms and Licenses

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

Depositor Comments

The A120 sequence can undergo recombination, resulting in its shortening to ~A80 or less. The likelihood of this recombination is significantly reduced when the plasmid is maintained in NEB® Stable Competent E. coli at 30 °C with 30 μg/mL kanamycin. Nevertheless, we recommend picking several colonies and verifying the poly(A) tail length. The length of the A120 sequence can be assessed through a double restriction digest using BsaI-HF®v2 and PmeI in rCutSmart™ Buffer, followed by analysis on a 3.0% agarose (1x TBE) gel. We do not recommend relying on sequencing to assess the poly(A) tail length, as long homopolymer sequences are often misread and appear shorter than they actually are.

A DNA template for in vitro transcription can be prepared from this plasmid using BsaI-HF®v2 restriction digest.

The presence of the A-inserted T7 class III Φ6.5 promoter facilitates highly efficient in vitro transcription priming with primers, such as CleaN3, containing AG dinucleotide sequence that base-pairs with the promoter.