We are almost trying to simulate what the plasmid will look like, the finished product. We don't need the cut site because that will get cut out of the finished product during processing.

Components

Cut Site 1. For restriction enzymes

Used to open up the plasmid during gibson assembly.

Note that gibson assembly will erase the cut site, so we can erase the cut site sequence.

Promoter 2. TF Promoter

Initiates transcription

Place immediately after the cut site.

Kozak Sequence 3.

The protein translation initiation site in most eukaryotic cells.

Your Gene 4.

Probably want introns already removed.

Make sure there are no restriction enzyme binding sites in this gene for the restriction site you selected!

Stop Codon 5.

TGA, TAA, TAG

Terminator Sequence 6.

Insert immediately after the stop codon, make sure you don't cover it up!

Auxotrophic Selection Marker

In case of natural antibiotic resistance in yeast, we need a more thorough selection marker.

A strain of yeast is modified to lose the production of leucine. It's grown in a leucine-enriched medium.

The selection marker will enable the production of leucine so that transformed targets are pro-trophic.

After transformation, the leucine-enriched medium is removed and non-transformed targets die.

Origin of Replication

So the plasmid gets replicated (dna is cloned) inside each cell.

Backbone will have an origin of replication from the source organism (E. coli).

We need to insert in an origin of replication for the target organism; they are different.

• E. coli is prokaryotic and has different machinery than a eukaryotic target (different by species).