pEC3079 (p7INT-Pveg_lacI)
(Plasmid #218505)

• Purpose: integrative plasmid enabling constitutive expression of lacI in Streptococcus, 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): 5599
• Modifications to backbone: The MCS of p7INT (Plac_lacZa) was replaced with the constitutive Pveg promoter from B. subtilis and the lacI repressor gene from pPEPY-PF6-lacI. See associated publication for detailed construction 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: lacI
• Species: Escherichia coli
• Insert Size (bp): 1312
• Entrez Gene: lacI (a.k.a. b0345, ECK0342)
• Promoter: Pveg

Cloning Information

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

Resource Information

• Supplemental Documents:
  • p7INT-Pveg_lacI.gb

Terms and Licenses

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

Depositor Comments

This plasmid enables the constitutive expression of LacI in S. pyogenes. It can be used for inducible gene expression in combination with the Plac promoter variants described in the associated publication.

Resource information: The Pveg promoter originates from the Bacillus subtilis PY79 genome. The lacI repressor is derived from pPEPY-PF6_lacI (Plasmid #85589) and originally codon-optimised for Streptococcus pneumoniae. However, codon usage is quite similar between S. pyogenes and S. pneumoniae.
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 file linked below to obtain a well annotated map of the plasmid.