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.
12 Comments
So waht about the BP cloning step in the gateway system? My attB sites are not recognized. Adding them manually, the ligate option is not available if I mark them together with the auto-annotated attP sites in my pDONR.
Thanks for your help.
Hi Nils, thanks for the comment. So far we’ve concentrated on the next stages in the Gateway system. However, we should be able to add this. Would you email “support@macvector.com” with your email address so we could contact you? Otherwise wait until the next release of MacVector.
Cheers
Chris
Hi Nils,
there’s an updated “Gateway” file available for download.
Get this then place it in your “Restriction Enzyme” folder.
Hi Chris,
Is there an easy way to integrate this updated Gateway file to the common enzymes file? Doing it one by one would be tedious.
Thanks
Hi Nils,
here you go.
Incidentally you can copy and paste single sites between RE files as long as they are unlocked.
You can select multiple sites for copying by drag-selecting, you just can’t command-select or shift-select in MacVector 12 and earlier. As it happens, we’ve rewritten the Restriction Editor window for our upcoming MacVector 12.5 release and it now uses a standard OS X Cocoa TableView, so you get all the usual selection behavior you’d expect in a normal list.
Hello,
Thanks for the quick response.
I downloaded the file and placed it in the “Restriction Enyzme” folder. While it is possible to make a Restriction Enzyme analysis using the updated file, the files is grayed out when I try to select it in the Automatic RE Analysis feature under Map View. The same is true with the other file you provided previously. Any tricks that I can try?
Thanks
Hi Emre,
it sounds like that file has lost its resource fork. Try using this file.
I am having problems with MacVector not recognizing attB2 sites all the time. If cut this sequence:
GGGGACCACTTTGTACAAGAAAGCTGGGTN
it shows an attB2 site. If I then “reverse complement” the same sequence, it no longer shows an attB2 site.
Any suggestons
Hi Jason. Thanks for your comment. There was a bug in the file that caused the reverse complement site to not be recognised. That’s now fixed. I’ll upload the file shortly
Here’s an updated Gateway file
Dear Chris,
Neither any of the files that you uploaded recently nor the original RE files that come wtih MacVector has attP sequences on them. attB 1-4 are there but not the attP except for attP5. And attL4 is not there either. Is there a specific reason for this?
Thanks
Emre