pmRNA-iSyn-EGFP
(Plasmid #225907)

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

Backbone

• Vector backbone: pRC
• 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: PABP motif
• Species: Synthetic
• Insert Size (bp): 50

Gene/Insert 2

• Gene/Insert name: HRV-B3-derived synthetic IRES
• Species: Synthetic
• Insert Size (bp): 666

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: HBA1 UTR
• Species: Synthetic
• Insert Size (bp): 101

Resource Information

• Supplemental Documents:
  • pmRNA-iSyn-EGFP.gbk
• A portion of this plasmid was derived from a plasmid made by: Howard Chang (circRNA-synIRES-R25-mNeonGreen, Addgene plasmid #188115; RRID:Addgene_188115). Stephen Ikeda (pRNA2-(A)128, Addgene plasmid #174006; RRID:Addgene_174006).

Terms and Licenses

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

Depositor Comments

The original version of this plasmid, containing mNeonGreen insert, has been described in the following article: Chen, R., Wang, S.K., Belk, J.A. et al. Engineering circular RNA for enhanced protein production. Nat Biotechnol 41, 262–272 (2023). https://doi.org/10.1038/s41587-022-01393-0, and deposited to Addgene (circRNA-synIRES-R25-mNeonGreen, Addgene plasmid #188115; http://n2t.net/addgene:188115; RRID:Addgene_188115).

pmRNA-iSyn-EGFP plasmid contains homopolymeric regions, which are often misread by sequencing and appear shorter than they actually are.

DNA templates for in vitro transcription can be prepared from this plasmid by PCR using the primers below.

linear mRNA:
mRNA F w/ A-inserted T7 class III Φ6.5 promoter (5′ GACTTAATACGACTCACTATAAGGCGTCATATTCGGCG 3′)
mRNA R w/ A120 tail (5′ TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCATCTACTGAGTCGGC 3′)

circular RNA:
circRNA F w/ T7 class III Φ6.5 promoter (5′ AAAAAAAAAAAAAAAAAAAAAAAAAAAGGCCAGTGAATTGTAATACGACTCACTATAGGG 3′)
circRNA R (5′ TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAAAAAACCCCTCAAGACCCGTTTAGAGGC 3′)

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