pEC2735 (pSpy0C2)
(Plasmid #220279)

• Purpose: (Empty Backbone) integrative plasmid for heterologous gene expression in S. pyogenes SF370, integrates into the amyA 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: pSpy0C2
• Backbone size (bp): 5618
• 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. Successful integrations into the streptococcal genome can be detected on starch agar plates as integration into amyA will result in the inability to degrade starch.
• Copy number: High Copy

Cloning Information

• Cloning method: Golden Gate
• 5′ sequencing primer: GTAAAACGACGGCCAGTC
• 3′ sequencing primer: CAGGAAACAGCTATGAC

Resource Information

• Supplemental Documents:
  • pSpy0C2.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 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 amyA were amplified from the S. pyogenes SF370 genome.
Linearise the plasmid with the restriction enzyme Alw44I before transformation into S. pyogenes to favour double crossover recombination of the plasmid into the amyA locus. Positive transformants will show loss in starch degradation activity on starch agar plates.

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