pEC2796 (pSpy0C4)
(Plasmid #220280)

• Purpose: (Empty Backbone) integrative plasmid for heterologous gene expression in S. pyogenes SF370, integrates into the sagB locus 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: pSpy0C4
• Backbone size (bp): 5256
• Modifications to backbone: See associated publication for detailed construction information.
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For E. coli, use 20 µg/mL chloramphenicol in LB medium. For S. pyogenes, also use 20 µg/mL chloramphenicol in THY medium. Integrations into the streptococcal genome can be screened on TSA blood agar plates as disruption of sagB leads to a non-hemolytic phenotype.
• Copy number: High Copy

Cloning Information

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

Resource Information

• Supplemental Documents:
  • pSpy0C4.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

The multiple cloning site (EcoRI, XbaI, SpeI and PstI) contains the Plac_mrfp cassette. The mrfp reporter allows for red-white screening during sub-cloning in E. coli (DH5a). When the Plac_mrfp cassette is replaced with the insert of interest, colonies will appear white.

Resource Information: The MCS featuring the Plac_mrfp cassette and the chloramphenicol resistance cassette were taken from pSpy1C. The pUC origin for replication in E. coli was taken from pUC19-mKate2∼ssrA. Up- and downstream homologous regions of sagB were amplified from the S. pyogenes SF370 genome.
Linearise the plasmid with the restriction enzyme Alw44I before transformation in S. pyogenes to favour double crossover recombination of the plasmid into the sagB locus. Positive transformants will show loss of hemolysis on TSA blood agar plates.

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