pEC2485 (pSpy1K)
(Plasmid #220249)

• Purpose: (Empty Backbone) 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: pSpy1C
• Backbone size (bp): 4693
• Modifications to backbone: Replacement of the chloramphenicol resistance cassette with a kanamycin resistance cassette. See associated article for detailed construction information.
• 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.
• Copy number: High Copy

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 (DH5a). When the Plac_mrfp cassette is replaced with the insert of interest, colonies will appear white.

Resource Information: The rolling circle replicon pSH71 and MCS featuring the Plac_mrfp cassette were taken from Spy1C. The kanamycin resistance cassette was taken from pIB184-Km (Plasmid #90195).

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