Fusion-based cloning in CLC tutorial

Go to parent Exercises for the Cloning tutorial

Open the circular view of vector pAcGFP1-C1. To do something similar as in SnapGene we will clone atp8a1 into the vector as a C-terminal fusion to the AcGFP1 gene.

Restriction analysis of the vector

Show all blunt cutters that cut only once in the vector.

To visualize all blunt cutters click the Manage enzymes button in the side panel: Remove all enzymes that are shown in the view (right panel; red) by using the left pointing arrow. In the Filter of the enzyme list on the left panel type blunt (green). Select all blunt cutters (by holding the Shift key during selection) and visualize them by sending them to the right panel using the right pointing arrow (red). Click Finish (green). The blunt cutters are now visualized on the map but both blunt cutters that cut only once and blunt cutters that cut multiple times are shown. Therefore in the side panel click the Sort enzymes by number of restriction sites button (red). Deselect all non-cutters, double cutters and multiple cutters so that Single cutters are the only selected cutters (green).

Now we need a blunt cutter to make a C-terminal fusion of AcGFP1 and Atp8a1.

Find a blunt cutter at the 3' end of the AcGFP1 gene.
Go to the sequence view by clicking the Show Sequence button in the bottom toolbar. Scroll to the 3' end of the AcGFP1 CDS. The blunt cutter that cuts closest to the 3' end of AcGFP1 is SciI.

To be able to design a good primer for amplifying the Atp8a1 CDS we need to know in which reading frame the enzyme cuts. To know this we need to visualize the translation on the sequence.

Visualize the translation.
Go to the side panel and expand Nucleotide info (red) select to Show the Translation (green). Set the Frame to +1 (blue). You can see if this is the correct frame by checking the translation of AcGFP1.

SciI cuts in +1 so if we want to fuse Atp8a1 in frame we have to add 2 nucleotides at its 5' end.

Generating the fragment to insert

To generate the fragment we need to design primers to amplify Atp8a1. In contrast to SnapGene that designs the primers automatically, CLC expects you to do it yourself. We are not going to explain this here. Consult the [Primer design in CLC tutorial] to see how to design primers in CLC.

Theoretically what you need is the following
For the forward primer:

• 18-30 nucleotides spanning the start of the Atp8a1 CDS
• 2 nucleotides to get Atp8a1 in the correct frame
• 15 nucleotides complementary to the vector sequence

For the reverse primer:

• 18-30 nucleotides downstream of Atp8a1
• 15 nucleotides complementary to the vector sequence

However, CLC will not allow you to use the overlaps for the cloning. So for the in silico cloning in CLC it's better not to include them in the primers. For the real cloning, you do need them of course.

This is why we will simply clone the Atp8a1 mRNA into the vector, just to show you how it works.

Open the cloning editor

Open the cloning editor
Expand Toolbox in the top menu, select Cloning and Restriction Sites and then Cloning. Select atp8a1 mRNA and the pAcGFP1-C1 cloning vector. Click Next. Ask to Open the result and click Finish.

This opens the cloning editor with the Atp8a1 fragment in a linear view.

Prepare the vector sequence

Indicate the part of the vector that you are going to use

Switch to the vector sequence. At this moment the vector sequence is annotated as a fragment not as a vector sequence. Annotate it as a vector sequence by clicking the Change to Current button at the top of the Clone editor. Visualize the SciI site by adding it to the view (side panel -> Manage Enzymes -> Filter on SciI -> right pointing arrow). Select SciI on the map. At the bottom of the view you can now see that the vector will be cut open.

Prepare the insert and do the cloning

Next step is to cut the fragment.

Indicate the fragment that is going to be inserted.
At the top of the view switch to the fragment. Click the Add as Fragment button and click the Clone button. Click 'Finish to generate the construct. Save the resulting construct.

The vector now contains the complete Atp8a1 mRNA fragment. You see that the process for using a single restriction site in the vector is exactly the same as with two restriction sites.