pEC2488 (pSpy3K)
(Plasmid #218498)

• Purpose: replicative E. coli-S. pyogenes shuttle plasmid for heterologous gene expression
• 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: pBAV1K-T5-gfp
• Total vector size (bp): 3741
• Modifications to backbone: The engineered pWV01* replicon and kanamycin resistance cassette were amplified from pBAV1K-T5-gfp and combined with a new MCS featuring the Plac_mrfp cassette from pBS3Clux.
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For E. coli, use 50 µg/mL kanamycin in LB medium. For S. pyogenes, use 300 µg/mL kanamycin in THY medium. The plasmid has medium copy numbers in E. coli and high copy numbers in S. pyogenes.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: mrfp
• Species: Discosoma sp.
• Insert Size (bp): 678
• GenBank ID: CAH64892
• Promoter: Plac

Cloning Information

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

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• 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. When the Plac_mrfp cassette is replaced with the insert of interest, colonies will appear white.

Resource Information: The backbone of pBAV1K-T5-gfp featuring the engineered pWV01* replicon and the kanamycin resistance cassette was taken from pBAV1K-T5-gfp (Plasmid #26702). The Plac_mrfp cassette as part of the MCS was taken from pBS3Clux (Plasmid #55172).

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