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URL: http://www.addgene.org/220306

Proper Citation: RRID:Addgene_220306

Bacterial Resistance: Erythromycin

Defining Citation: PMID:38911550

Vector Backbone Description: Backbone Size:4457; Vector Backbone:pERASE; Vector Types:Synthetic Biology; Bacterial Resistance:Erythromycin

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.

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