Changing between Circular and Linear Topology
If you would like to change a sequence from its current topology to another, navigate to the “Edit” tab in the top banner and find the “Topology” dropdown. You can select the dropdown and the current topology will be indicated via a checkmark next to the word “Circular” or “Linear”.
In order to change the topology, select the alternative option. This will change the sequence to be linear or circular. A linear sequence can be used without linearization in molecular cloning workflows under the “Cloning” tab. You will see that the map view changes according to your selection, and the checkmark will be displayed accordingly in the “Topology” dropdown in the “Edit” menu.
Linearizing Plasmids via PCR or Restriction Digest
Plasmids can also be linearized systematically via PCR or restriction digestion, this allows you to simulate these processes in-silico and design PCR primers. To do this navigate to the “Edit” menu in the top banner and find the “Linearize” button. Selecting it will take you into the linearization modal box. Here you can select your method of linearization, either PCR or Restriction digest, to do this select the “Linearize by” dropdown menu at the top left (note that by default it is set to PCR).
For PCR you have the option to select a given region to amplify by selecting the “Create primers from selection” button, this will automatically generate primers based on the target annealing temperature set (by default the Tm is 60C) and the selection of a blunt end or 3’ A/G overhang polymerase. The other option for PCR is to chose a single location at which to linearize via PCR, to do this place your cursor on the sequence and select a location, then use the button “Create primers to linearize at cursor” to automatically generate primers based on set parameters. It is also possible to select primer modifications to add 5’ phosphorylation or 5’ enzymatic phosphorylation via the select modification dropdown. If you would like to add a 5’ restriction site to the primer you can do so with the “Select enzyme” dropdown.
Lastly it is also possible to perform PCR with your own primers. To do this either paste the primer in 5’ to 3’ orientation in the box under “Primer 1” or select the search icon next to the box with the vector name (by default this is set as Vector.For) this will give you a dropdown selection with all the primers associated to this sequence file already.
The next option for linearization is via restriction digest. Once you have selected this method from the “Linearize by” dropdown you can select which enzyme(s) to use and which 5’ modifications to include. You can also digest with multiple restriction enzymes using the “+ Add enzyme” button. Next you will be able to “Select region” where you can choose which digest fragment to save as the new linearized file. If you would like to simulate the gel electrophoresis for this digestion there is a “Simulate digest on gel” which will allow you to visualize and export this. Finally you con decide if you want to include primers and features in the final product (note it is also possible to make a selection to keep some via the checkbox selections).
Once you have finalized your selection for linearization either by PCR or by restriction digest you can preview the result by selecting “PCR result” or “Digest result” above the plasmid map. If the result is correct you can choose to save the file linearized sequence as a new file by clicking save at the bottom right (note you may need to scroll down to see the save button depending on your screen size). After selecting save you will see the saving modal where you can select a new name for the file (by default the word linearized is appended to the previous file name) and select a location for it to be saved.
