This protocol describes standard methods for constructing a yeast single gene expression clone (or Transcription Unit) using the yeast pathway kit.

The first step is to prepare the backbone plasmid vector. This vector must provide a selectable marker that functions in S. cerevisiae and must be able to replicate. Further, at least a partial E. coli CRP locus is necessary for recombination. Example of such vectors are pYPKpw or pTA1.

Use a standard miniprep kit if available, but the [low cost protocol](super snik) yields sufficiently pure DNA for this purpose.

pYPKpw can be linearized with blunt enzymes ZraI, FspAI or EcoRV/Eco32I. This may vary with the plasmid used. It is always a good idea to perform an in-silico cloning before

The digestion below is enough for ten constructs:

• 6 µL water
• 1 µL backbone plasmid miniprep (ex pYPKpw or pTA1)
• 1 µL 10X Buffer EcoRV
• 1 µL EcoRV (5U/µL)
• 1 µL SAP, FastAP or some other alkaline phosphatase

Inactivate ZraI, EcoRV or Eco32I at 80°C for 20 min. It is practical to do the above digestion/inactivation in a PCR thermal cycler in available. Put the digestion on ice immediately after the digestion has finished.

The digested vector can be saved at -20°C at this point.