pSK9065
(Plasmid #84345)

• Purpose: (Empty Backbone) tightly controlled expression of mRFP fusion proteins in S. aureus
• Depositing Lab: Neville Firth
• Publication: Brzoska et al Appl Environ Microbiol. 2013 May;79(9):3133-6. doi: 10.1128/AEM.00144-13. Epub 2013 Mar 1.

Backbone

• Vector backbone: constructed by Firth lab
• Modifications to backbone: See supplemental information from associated article for full details.
• Vector type: Bacterial Expression
• Promoter: Pxyl/tet
• Selectable markers: Neomycin (select with G418)
• Tag / Fusion Protein:
  • mRFPmars (C terminal on backbone)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Cloning Information

• Cloning method: Restriction Enzyme

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

In S. aureus, pSK9065 replicates via the origin of replication from the staphylococcal multiresistance plasmid pSK1, whereas in pSK9067, the compatible origin from the conjugative S. aureus multiresistance plasmid pSK41 is used. These low-copy-number theta-replicating staphylococcal origins (5 to 10 copies per cell) were employed to minimize the structural instability that can arise in recombinant plasmids that utilize rolling circle replication. pSK9065 contains the aadD gene, which encodes neomycin resistance in S. aureus. pSK9065 contains the Pxyl/tet which has been modified to enable tight regulation of the expression of rfp gene fusions. The tetR promoter contains an optimized −10 box. These vectors facilitate tightly controlled expression of fusion proteins in S. aureus to enable visualization in the presence of natively expressed wild-type counterparts.
The excitation and emission spectra for mRFPmars is 585 and 602 nm, respectively.