pIM572
(Plasmid #74640)

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

Backbone

• Vector backbone: pFM571
• Backbone manufacturer: Marian Farkasovsky
• Backbone size w/o insert (bp): 4677
• Modifications to backbone: Replacement of mCherry with yTagRFP-T
• Vector type: Yeast Expression
• 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: yTagRFP-T

Resource Information

• A portion of this plasmid was derived from a plasmid made by: M. Kaern, University Ottawa, Canada

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Evrogen Limited Use Label License for FPs
• 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 obtained from Euroscarf 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 result. In the originally 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.