pEC3104 (pSpy1C-PgyrA(Sag)_ermBL-ermB'~ffluc)
(Plasmid #218513)

• Purpose: replicative E. coli-S. pyogenes shuttle plasmid for erythromycin-inducible expression of the Firefly luciferase
• Depositing Lab: Emmanuelle Charpentier
• Publication: Lautenschlager et al Front Bioeng Biotechnol. 2024 Jun 7;12:1395659. doi: 10.3389/fbioe.2024.1395659. eCollection 2024.

Backbone

• Vector backbone: pSpy1C
• Total vector size (bp): 5880
• Modifications to backbone: The MCS with the mrfp cassette was replaced with the erythromycin-inducible riboswitch (PgyrA_ermBL-ermB') and the ffluc reporter gene. See the associated publication for detailed construction information.
• Vector type: Bacterial Expression, Luciferase

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For E. coli, use 20 µg/mL chloramphenicol in LB medium. For S. pyogenes, use 20 µg/mL chloramphenicol in THY medium. Expression of ffluc reporter in the presence of erythromycin. See associated publication for details.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: ffluc
• Species: Photinus pyralis
• Insert Size (bp): 1653
• Mutation: deletion of the Plac_mrfp cassette
• GenBank ID: XM_031473197
• Promoter: PgyrA_ermBL-ermB'

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: GTAAAACGACGGCCAGTC
• 3′ sequencing primer: CAGGAAACAGCTATGAC

Resource Information

• Supplemental Documents:
  • pSpy1C-PgyrA_ermBL-ermB'~ffluc.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Luciferase Limited Use Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This high-copy replicative shuttle plasmid features an erythromycin-inducible riboswitch driving expression of the Firefly luciferase in the presence of erythromycin. Luminescence can be detected by adding luciferin to the culture. See associated publication for more details.

Resource Information: The erythromycin-inducible riboswitch was taken from pLZ12Km2-TA:PgyrA(Sag)-ermBL-ermB':A2I:ffluc-tt3, which was used in the following publication:
Dynamics of diversified A-to-I editing in Streptococcus pyogenes is governed by changes in mRNA stability
Thomas F. Wulff, Karin Hahnke, Anne-Laure Lécrivain, Katja Schmidt, Rina Ahmed-Begrich, Knut Finstermeier, Emmanuelle Charpentier
bioRxiv 2023.09.19.555891; doi: https://doi.org/10.1101/2023.09.19.555891
The ffluc reporter gene was taken from the original pLZ12Km2-P23R:TA:ffluc plasmid (Plasmid #88900).

Please download the detailed plasmid map using the file linked below