Min Overlap Tm (48☌) is the melting temperature of the complementary sequences. In our case we’re ligating one sequence into a vector and because primers are only created for insert sequences the insert primer will have the full 18 bp as extension. When ligating two inserts, each will have half of this length as primer extension. This is the length of the annealing bases, the complementary sequence part that two neighboring sequences will have in common after the operation. The Min Overlap Length is set to a recommended value of 18 bp. Now click Primer Options to check the primer settings. If you are using a related method that uses a different exonuclease (such as SLIC or In-Fusion cloning), you can choose this here. Instead, they can be selected from within the setup options by clicking the Choose button.Ĭheck that the Exonuclease is set on 5′ Exonuclease. Note that in Geneious 8.1 and above, the inserts and vector do not have to be pre-selected before opening the Gibson Assembly tool. Click the tags to see a preview of the sequences that will be used in the assembly. After the vector is selected as the backbone, the tag representing this sequence in the Construct Layout panel should turn green, and the insert tag should be grey, as in the screenshot below. In the Backbone dropdown select the digested vector if it is not already selected. Bring up the Gibson Assembly options ( Cloning → Gibson Assembly…). Now select both the extracted DCN CDS and the digested vector sequences. Click on Extract in the Sequence Viewer and chose an appropriate name, such as “DCN CDS”. We’re going to extract this without the use of primers, as they will be generated later by Geneious during the Gibson operation. Translation should still be turned on, select the CDS bases except for the last TAA (for example by clicking on the CDS annotation, then holding Shift+click just before the ‘TAA’). We want only the CDS to be inserted before GFP, without the stop-codon. We’re finished with the vector, so let’s head over to DCN. Here we don’t have to do anything as we’re not interested in this sequence part as long as it doesn’t block transcription. Scrolling to the very end of the Vector you will notice the other CATG NcoI overhang.
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