Stage 3 - Checking the Assembly
Select the newly created sequence named pGoldenGate-SE7 - GFP1 - GFP2 and 4 other sequences and view it in the Sequence View panel. In the Annotations and Tracks tab ensure the Concatenated Sequences annotation type is checked. To reduce the screen clutter turn off display of Primer Bind and Ligation annotations.
To confirm that our six fragments have recombined precisely to reform the GFP CDS We will define a CDS annotation for the region and examine the gene product.
To do this, select the Ligation of GFP1 - GFP2 - GFP3 - GFP4 - GFP5 - GFP6 into pGoldenGate-SE7 file.
Select the GFP1 ORF annotation (in orange), then hold down shift and select the GFP6 ORF annotation (in orange). This should select the range 2679 to 3395. Hit the "Zoom to Selection" button to zoom in on this region.
Then, click Add Annotation, and define a new annotation for this range called GFP Reassembled of Type: CDS, and click ok.
A new yellow CDS annotation should appear. Click on the yellow GFP reassembled annotation and hover over it to bring up a yellow tool tip showing the details of the Annotation. You should see the CDS translates to the complete GFP gene product starting MRK..., ending ...LYK*, with no internal stop codons.
If you wish, you can extract the CDS to a file and perform a pairwise alignment between the GFP reassembled sequence, and the original GFP sequence , which has also been provided with this tutorial. The alignment will show you that the reassembled sequence is identical to the original GFP sequence.
That concludes this tutorial.