pKT0140
(Plasmid #8732)

• 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): 4894
• Vector type: Yeast Expression
• Selectable markers: KanR

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: yECitrine
• Alt name: Codon-optimized GFP variant
• Alt name: pKT140 (pFA6a–link–yECitrine–Kan)
• Mutation: yEGFP with S65G, V68L, Q69M, S72A, T203Y
• 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-yECitrine-KanR. yECitrine 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. The G418 resistance marker (KanR) was introduced into the plasmid by subcloning the BglII/EcoRI fragment of pDH3 (Hailey et al., 2002) into the plasmid, in place of SpHIS5.

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).