Virtual Cloning Tutorial - Cloning Vector

Open the cloning vector and you will notice a couple of key genes. First is the GFP gene - our aim is to insert our gene of interest at the 5` end of this (at approximately the 1400 bp point). This will enable us to make a 3` fusion protein (i.e. where the protein is followed by the GFP marker). Some other features are common to all plasmids; rep origin is the origin of replication which allow the plasmid to be propagated in bacteria. The bla gene is an antibiotic resistance gene (ampicillin). Lastly the human elongation factor 1 promoter is a region of DNA that will allow our GFP-fusion protein to be expressed in cells.

We will be using restriction site cloning to insert our gene of interest, so the first step is to locate a useful restriction site. In the sequence viewer, locate the start of the GFP gene and select this region (1,300 to 1,400).

Go to Cloning→Find Restriction Sites, select the commonly used enzymes set and the Must only cut between option. You may need to click the refresh button to ensure the selection is reflected in the boxes.

Now only enzymes which cut in this region will be offered as potential annotations. Click the Apply button to add the restriction sites to the sequence and Save the document.

One of the sites found should be NcoI, which overlaps the beginning of the GFP gene. Zoom in and note how many bases there are between the cut point (the down arrow inside the restriction site annotation) and the beginning of the GFP CDS (1 base).


Exercise 2: Human Proinsulin
Exercise 3: Cloning Primer Design
Exercise 4: Introduce Cut Site using PCR
Exercise 5: Insert into Vector