Here at MacVector we always try to listen to our end users when deciding what functionality to add to new versions. The more users that request a new feature, the more likely it is to get to the top of the list. One item that recently reached the top of our list was support for the Topo and Gateway cloning technologies from Invitrogen. MacVector 11.0.4 has a number of new additions that make it extremely easy to simulate Topo and Gateway cloning constructions with a few mouse clicks.
First, we added pseudo-Restriction Enzyme sites representing the TOPO and att recognition sequences to the Common Enzymes file. This is the file containing the default set of enzymes that are automatically displayed in the Map tab of all open sequences.
Second, we added a large number of Invitrogen vectors that are installed in the /Applications/MacVector 11/Common Vectors/Invitrogen/ folder. These include a selection of “Entry” and “Destination” vectors. Others can be downloaded from the Invitrogen web site – you can use MacVector’s Auto-Annotation function to quickly annotate additional vectors with common TOPO and Gateway features.
A typical Gateway cloning vector with att and TOPO sites highlighted.
Its then very easy to simulate a TOPO or Gateway cloning experiment to create a constructed molecule with the identical sequence at the junctions to the one you would get in the lab. To clone a PCR fragment, simply select the region in a source molecule (or from an external text editor) and copy it to the clipboard, then select the TopoTA/BLNT site in the vector and click on the Ligate button. A Ligation dialog opens showing you the compatible blunt ends.
This gives you the option of flipping the source fragment if you wish. Note that this also works for TA cloning as well as ZERO-BLUNT cloning manipulations, even though the dialog does not show the overhanging T-A ends.
When Ligate is clicked, the fragment gets inserted into the vector. To perform a Gateway cloning, you can select the two att sites (attL1 and attL2) and copy the fragment to the clipboard.
Then open a suitable target/destination vector – these typically have attR1 and attR2 sites.
Now when you click on the Ligate button you’ll see the core recombination sequences of the att sites shown as overhanging ends.
Note that there is a single residue difference between the core sequences of the attL1/attR1 and attL2/attR2 sites;
The single T-C mismatch between the two core sequences ensures that the att recombination can only occur in one orientation. MacVector understands this and will automatically flip the source fragment if needed so that it becomes inserted in the biologically correct orientation. Finally, clicking Ligate inserts the fragment into the target vector.
For a more in-depth discussion of the new Gateway and TOPO cloning functionality in MacVector, download the Gateway and TOPO Cloning Tutorial from our web site.
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