In this exercise we will use the Geneious Golden Gate tool to assemble six sequences and recombine them with a vector "backbone" to create a circular construct.
This exercise has been devised to demonstrate how the rules outlined in the introductory section of this tutorial are implemented, and also, to demonstrate the general procedure for use of the Golden Gate tool.
The six sequences for assembly comprise portions of the green fluorescent protein (GFP) coding sequence (CDS). The various sequences have fusion boundaries defined by existing type IIS sites, primer_bind annotations (with and without extensions) and by blunt ends.
If the assembly goes to plan, Golden Gate-mediated recombination of the six sequences will regenerate a complete CDS encoding the GFP and insert it into a vector "backbone".
In this exercise we will be using the vector pGoldenGate-SE7. This vector contains two BsaI sites with unique overhangs, that will be used to "receive" our six-Part insert. This vector sequence is provided with this tutorial, click on the above link to select and view the vector sequence.
The six insert fragments, labelled GFP1 to GFP6 are also provided with this tutorial. Click this link to select and view the six sequences.
Stage 1 - Checking the sequences
With the six insert sequences selected and visible in the sequence viewer, select the Annotations and Tracks tab and ensure ORF, primer_bind and Restriction Site annotations are displayed.
You will see each fragment is annotated with an ORF annotation which defines a region corresponding to a portion of the GFP CDS. The ORF annotations correspond precisely to the regions we wish to assemble. Preexisting BsaI sites are also visible as well as a number of primer_bind annotations.
You will notice that the first sequence, GFP1, is a blunt fragment with a central primer_bind annotation named A Primer. We wish to assemble this entire sequence. However, Geneious will interpret the primer_bind annotation as a fusion boundary (as per Rules 3-6 described in the Tutorial Introduction). We will take steps, described in the next section to ensure Geneious does not use this primer_bind annotation as a boundary.
At this point we should also confirm that none of the sequences contain unwanted internal BsaI sites. Click on the Restriction Analysis tab, select the Type IIS subet of enzymes, then click on Advanced and select BsaI and double check there are no unexpected internal BsaI sites in the six sequences.
Go to Stage 2: Performing the Assembly