pEC3135 (pERASE)
(Plasmid #220306)

• Purpose: (Empty Backbone) Enables scarless gene deletions in Streptococcus pyogenes using the counterselection marker pheS*
• 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: pERASE
• Backbone size (bp): 4457
• Modifications to backbone: See associated publication for detailed construction information.
• Vector type: Synthetic Biology
• Selectable markers: pheS*

Growth in Bacteria

• Bacterial Resistance(s): Erythromycin, 200 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For E. coli, add 300 µg/mL erythromycin in LB medium. For S. pyogenes, add 3 µg/mL erythromycin in THY medium. To select for plasmid loss after recombination in S. pyogenes, streak colonies on THY agar plates containing 5 mM PCPA (4-chlor-DL-phenylalanine).
• Copy number: High Copy

Cloning Information

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

Resource Information

• Supplemental Documents:
  • pERASE.gb

Terms and Licenses

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

Depositor Comments

This plasmid allows to obtain scarless gene deletions in S. pyogenes and potentially other Gram-positive bacteria. The multiple cloning site (EcoRI, XbaI, SpeI, PstI) contains a Plac_mrfp cassette, allowing for red-white screening of clones in E. coli (DH5a). When the Plac_mrfp cassette is replaced with the insert of interest, colonies will appear white on LB agar plates. See the associated publication for more information.

Resource Information: The pUC origin for replication in E. coli was taken from pUC19 (Plasmid #50005). The erythromycin resistance cassette was taken from p7INT. The MCS containing the mrfp cassette was taken from pSpy0C4. The coding sequence for the counterselection marker pheS* was taken from the native pheS coding sequence found in S. pyogenes SF370. This sequence was adapted to avoid recombination events of the plasmid at the phis locus due to high DNA sequence homology. Two mutations were introduced into the adapted pheS sequence to facilitate incorporation of the toxic phenylalanine analogue PCPA (4-chlor-DL-phenylalanine) into proteins, leading to bacterial cell death.

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.