pKT0174
(Plasmid #8720)

• Depositing Lab: Kurt Thorn
• Publication: Sheff et al Yeast 2004 Jun;21(8):661-70.

Backbone

• Vector backbone: pFA6a-link
• Backbone size w/o insert (bp): 4900
• Vector type: Yeast Expression
• Selectable markers: URA3 ; Candida albicans

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: yECFP
• Alt name: Codon-optimized GFP variant
• Alt name: pKT174 (pFA6a–link–yECFP–CaURA3)
• Mutation: yEGFP with F64L, S65T, Y66W, N144I, M153T, V163A
• Tag / Fusion Protein:
  • Codon optimized linker (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: PacI (not destroyed)
• 3′ cloning site: AscI (not destroyed)
• 5′ sequencing primer: SP6

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Full name of this plasmid is pFA6a-link-yECFP-CaURA3. yECFP is created by mutagenesis from a codon-optimized green fluorescent protein - yEGFP1 (Cormack et al. 1997). This yEGFP variant is cloned into pDH5, replacing YFP. An codon-optimized linker was added into the yEGFP variant to improve expression level. C. albicans URA3 (CaURA3) was introduced by subcloning the BglII/SacI fragment of pAG60 (Goldstein et al., 1999) into the plasmid, in place of SpHIS5.

Note that yECFP in this plasmid contains N144I mutation relative to yEGFP. The associated publication mentions a N146I mutation, but N146 is present in the yECFP in this plasmid. The yeast GFP used to create this plasmid contained a point mutation M233I that is present in all GFP variants derived from this original yEGFP. The mutation had no effect on fluorescence (see associated publication for more details).