pEC3105 (pSpy1C-PxylS2_ffluc)
(Plasmid #218512)

• Purpose: replicative E. coli-S. pyogenes shuttle plasmid, constitutive expression of firefly luciferase reporter
• 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): 5706
• Modifications to backbone: The Plac_mrfp cassette was replaced with the synthetic PxylS2 promoter and the ffluc reporter from pLZ12Km2-P23R:TA:ffluc. See associated publication for detailed construction information.
• Vector type: 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, also use 20 µg/mL chloramphenicol in THY medium.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: ffluc
• Species: Photinus pyralis
• Insert Size (bp): 1653
• Mutation: deletion of Plac_mrfp cassette
• GenBank ID: XM_031473197
• Promoter: PxylS2

Cloning Information

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

Resource Information

• Supplemental Documents:
  • pSpy1C-PxylS2_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 the constitutive synthetic PxylS2 promoter from S. pyogenes driving expresses the Firefly luciferase. Luminescence can be detected by adding luciferin to the culture. See associated publication for more details.

Resource Information: The PxylS2 promoter was obtained via gene synthesis. Its sequence was derived from the following publication:
Xie Z, Qi F, Merritt J2013.Development of a Tunable Wide-Range Gene Induction System Useful for the Study of Streptococcal Toxin-Antitoxin Systems. Appl Environ Microbiol79:.https://doi.org/10.1128/AEM.02320-13
The ffluc reporter gene was taken from pLZ12Km2-P23R:TA:ffluc (Addgene plasmid #88900).

Please download the detailed plasmid map using the file linked below.