Cloning Tool

The Cloning Tool lets you simulate cloning experiments and produce new projects/maps with minimal user effort. Start the Cloning Tool, select two sequences (project files) and enzymes for digestion. The two tables with resulting fragments are updated automatically when enzymes are selected/deselected or project files are replaced. Then select one fragment from each table and click the Ligate button to create the new construct. Continue to make more constructs, or close the window when finished. A new construct is given the name "New subclone" and opened in a new sequence tab in the main window, from which it may be edited and saved to file.

The ligation process is based on sequence overhangs. Any pair of compatible ends can be ligated.

A fragment containing an internal zero position from its ancestor sequence is considered being a vector, and this zero position is conserved in the new construct, given it becomes circular. A 'vector' fragment can be selected in both panes (left, right, or both). If the fragment selected in the left pane is 'vector' it will have priority when setting the plus strand and zero position in the new construct (unless the construct becomes linear and the other fragment is joined to the 5' end).

If the fragment selected in the right pane is 'vector', while the left-pane fragment is not, the right-pane fragment will set the plus strand and zero position in the new construct.

If the "Gateway" mode is selected, the Cloning tool will operate in Gateway att-site recombination mode. The function supports the basic gateway technology: attB1-attB2 x attP1-attP2 to produce attL1-attL2 entry clones, and attL1-attL2 x attR1-attR2 to produce attB1-attB2 expression clones. For easy operation, leave all Gateway recombination sites selected.

New projects generated by the Cloning Tool are stripped for annotations. Features of interest must be remapped and annotated by running analysis tools. This will ensure that annotations are correctly implemented.

Manual cloning procedure

When editing constructs and sequences by manual copy/paste operations, existing annotations may be preserved. This may be convenient in many situations, while it also poses a risk for producing broken or incorrect annotations. It's recommended to run the Checkup and Cleanup functions after such edits, and to run the analyses supposed to detect the annotated features to verify correctness.

The procedure below exemplifies how to make a new subclone map, and shows the basic steps in manual editing.

Create PlasmidTools projects for the sequences involved.
Open the target and source projects in different tabs.
Run restriction site analysis on both sequences and annotate the enzymes to be used in cloning. This will make it easier to cut and paste in correct positions.
Select the source sequence contained between the two cut sites (annotations) in question and select Copy (right-click menu).
In the target sequence, select any sequence contained between the corresponding cloning sites (annotations) and select Paste (right-click menu) to replace selection.
Run the Checkup tool and verify that annotation syntax is correct (no need to bother about sequence characters at his point).
Having confirmed that annotation syntax is correct, run the Cleanup tool to remove non-sequence characters and punctuation.
Enter a new name (in the name text box) and click "Save as.." to save the project with a new filename.
Run restriction enzyme analysis to verify integrity of cloning sites.
Run other analyses and verify that annotated features still exist, and with correct start and end positions.