pFM571
(Plasmid #74634)

• Purpose: alias pFA6a-mCherry-kanMX6 - vector for C-terminal tagging of yeast genes with mCherry and G418 selection marker
• Depositing Lab: Jiří Hašek
• Publication: Malcova et al FEMS Yeast Res. 2016 Mar 17. pii: fow027.

Backbone

• Vector backbone: pYM27
• Backbone manufacturer: Janke et al., (2004) Yeast 21:947-962
• Backbone size w/o insert (bp): 4940
• Total vector size (bp): 4677
• Modifications to backbone: replacement of EGFP with mCherry
• Selectable markers: Kanamycin - G418

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: mCherry
• Alt name: fluorescent tag
• Promoter: none

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: SalI (not destroyed)
• 3′ cloning site: BamHI (not destroyed)
• 5′ sequencing primer: CCGTACATGAACTGAGGG
• 3′ sequencing primer: GCAGCGAGGAGCCGTAAT

Resource Information

• A portion of this plasmid was derived from a plasmid made by: R. Tsien, San Diego, USA

Terms and Licenses

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

Depositor Comments

During verification of the constructed cassettes by restriction analysis and sequencing we found out that there are 254 bp missing after the BamHI site in between the fluorophore and the Ashbya gossypii TEF promotor region regulating expression of the selectable marker in pFM571, pIM572 and pIM573. We checked the original pYM27 plasmid (Janke, et al., 2004, Yeast 21:947-962 ) we had in our hands and compared the obtained sequence to the published one. We also checked by restriction analysis pYM27 from an independent source (but also obtained from Euroscarf) and got the same results. In the published pYM27 sequence, there is a sequence AGATCC resembling BamHI site (GGATCC) at position 1044-1049 bp. Likely due to a BamHI star activity that led to cutting this BamHI-like site in parallel with the true BamHI site, the nucleotides between these two sites got lost during construction of pYM27 or its predecessors and are missing in pYM27 and thus also in our pYM27-based modules - pFM571, pIM572 and pIM573. It seems, however, that the functionality of the vector and of the selection marker is not disturbed since transformants were readily obtained with all the concerned cassettes when selecting on geneticin.