pEC2935 (p7INT-P23_mNeongreen~ssrA (LDD variant))
(Plasmid #218508)

• Purpose: integrative plasmid enabling constitutive expression of a destabilized mNeongreen reporter in Streptococcus pyogenes, targets the attP site of phage T12 in several S. pyogenes strains via an integrase
• 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: p7INT
• Total vector size (bp): 5391
• Modifications to backbone: The MCS of p7INT (Plac_lacZa) was replaced with the lactococcal Prmoter P23 and a codon-optimised (S. pyogenes) mNeongreen reporter that has a C-terminal ssrA degradation tag sequence (translational fusion). The last three amino acids encoded by the ssrA degradation tag were mutated from LAA to LDD to render the mNeongreen protein less stable. See associated publication for more detailed information.
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Erythromycin, 200 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For E. coli, use 300 µg/mL erythromycin in LB medium. For S. pyogenes, use 3 µg/mL erythromycin in THY medium.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: mNeongreen
• Alt name: mNG, mNeonGreen
• Species: Synthetic; Branchiostoma lanceolatum
• Insert Size (bp): 711
• Mutation: deletion of MCS containing Plac_lacZa, introduction of a mutation to the last three amino acids of the ssrA degradation tag
• GenBank ID: AGG56535
• Promoter: P23
• Tag / Fusion Protein:
  • modified ssrA degradation tag (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: gtaaaacgacggccag
• 3′ sequencing primer: gtcatagctgtttcctg

Resource Information

• Supplemental Documents:
  • p7INT-P23_mNeongreen-ssrA(LDD).gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • mNeonGreen Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This integrative plasmid enables expression of the fluorescence reporter mNeongreen in S. pyogenes. Due to a mutation in the SsrA degradation tag sequence (LAA to LDD), that is translationally fused to the mNeongreen protein, the reporter is destabilised and shows a reduced half-life compare to the untagged mNeongreen protein. The destabilised reporter is advantageous when gene expression is monitored over time as dynamic changes in gene expression (e.g. downregulation) can be observed. See the associated publication for more details.

Resource Information: The mNeongreen 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).
p7INT was a gift from Prof. Michael Federle (University of Illinois Chicago, USA). The p7INT plasmid integrates into the 3' end of the tmRNA locus in several S. pyogenes strains. It was originally described in:
McShan, W.M., McLaughlin, R.E., Nordstrand, A. et al. Vectors containing streptococcal bacteriophage integrases for site-specific gene insertion. Methods Cell Sci 20, 51–57 (1998). https://doi.org/10.1023/A:1009773309163

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