pEC3115 (pSpy0K6-P23_mKate2~*ssrA)
(Plasmid #218509)

• Purpose: integrative plasmid enabling constitutive expression of mKate2 in Streptococcus pyogenes, integrates into the transcriptionally silent locus Spy_1078 via homologous recombination
• 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: pSpy0K6
• Total vector size (bp): 5268
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For E. coli, use 50 µg/mL kanamycin in LB medium. For Streptococcus pyogenes, use 300 µg/mL kanamycin in THY medium.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: mKate2
• Species: Entacmaea quadricolor
• Insert Size (bp): 699
• Mutation: Deletion of Plac_mrfp cassette, introduction of a stop codon at the 3' end of the mNeongreen coding sequence to uncouple the ssrA degradation tag
• Promoter: P23
• Tag / Fusion Protein:
  • ssrA degradation tag (C terminal on insert)

Cloning Information

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

Resource Information

• Supplemental Documents:
  • pSpy0K6-P23_mKate2.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Evrogen Limited Use Label License for FPs
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This integrative plasmid enables stable expression of the fluorescence reporter mKate2 and therefore allows for fluorescence labelling of S. pyogenes cells. The pSpy0K6 plasmid integrates into the gene SPy_1078 in S. pyogenes SF370, which was found to be transcriptionally inactive. Due to an introduced stop codon upstream of the ssrA degradation tag sequence, the SsrA tag sequence will not be fused to mKate2 during translation. Consequently, the mKate2 protein, and therefore, the fluorescence signal is highly stable over time. See associated publication for more details.

Resource Information: The mKate2 gene was codon-optimised for expression in S. pyogenes and obtained by gene synthesis. The lactococcal P23 promoter was taken from pLZ12Km2-P23R:TA:ffluc (Plasmid # 88900).

Please download the file linked below to obtain a well annotated map of the plasmid.