Hypothetical question. A new biotech company intends to clone the gene encoding the ABA 8-hydroylase enzyme, an important protein regulating plant hormone degradation that controls seed dormancy and environmental stress responses. The protein is known to belong to a class of enzymes known as p450s of which there are 500 different genes in Arabidopsis, the model plant system. The company isolates a full length cDNA for each gene using PCR with primerscontaining attB sequences and then each individually cloned into pDONR/Zeo (from Invitrogen) via a BP reaction for all 500 different p450 genes from Arabidopsis (the cDNAs vary in length from 1.5-2.5 kb). Next the company proposes to express each gene in yeast, and then carry out an ABA-8 hydroxylase reaction in vivo to determine which cDNA encodes ABA 8-hydroxylase activity.

Outline a cloning strategy(s) (experimental procedure) the company could take to express all these genes in yeast - a simple flow diagram detailing the steps is all that is needed. Design a vector(s) that would be central in this strategy (can just draw by hand) and list all the important features that would be required (i.e what cloning sites or antibiotic resistance genes would be required if any) but not any features that would be excess to what is required.