14.4 Introduction to the cloning interface


Tools for Restriction Cloning, Golden Gate and Gibson Assembly can be found under the Cloning menu. These tools share a common interface which differs only in options specific to each cloning tool. Input sequences may be selected prior to launching the operation, or may be selected or removed from within the cloning options.


Figure 14.4: A Restriction Cloning options dialog. The Construct Layout Panel shows the backbone sequence shaded blue to the left and an insert to the right. The Detailed View Panel shows the selected annotated sequence.

Use this option to choose a sequence that will serve as the vector in your cloning reaction. When the cloning dialog is first opened, this will show Use Leftmost, which refers to the sequence represented in by the leftmost tag in the Construct Layout panel. Geneious will automatically choose the longest (preferably circular) sequence as the backbone. The backbone can be manually changed either by using the dropdown to choose from selected sequences or recently used backbones, by selecting a backbone with the Choose button, or by dragging and dropping the tags within the Construct Layout panel to the desired position. The sequence chosen as the backbone is always in the leftmost position in the Construct Layout panel and has blue background shading. If a backbone is selected it will always result in a Circular Product. Nucleotides in the final construct will be numbered beginning with the origin of the backbone sequence.
Enzyme or Exonuclease:
Set the enzyme(s) to consider for your reaction. Geneious will automatically try to use the best reaction setup with the specified enzyme(s), and you can manually adjust the reaction setup in the Construct Layout Panel. The enzyme options differ between the individual cloning operations - information specific to each cloning tool can be found in the relevant sections of this manual.
Circular Product:
Select this option to create a circular product when no Backbone is specified.
Construct Layout Panel:
Each sequence or sequence list (where available, these are shown in gold), is represented as a sequence tag in the upper Construct Layout Panel, in the order in which they will be ligated. You can rearrange inserts by dragging and dropping the sequence tags, remove them by clicking on the Õ in the top corner, or select additional inserts using the Add Inserts button. If you have designated a sequence as the Backbone (vector), it will appear in green to the left of any other sequences.
show the following information:
Sequence Name
(highlighted in blue if reverse complemented)
Reaction Type
Additional Information
such as extracted region, length, or number of sequences
for sequences in an error state, shown in red. Additional details about these warnings can be found below the Detailed View Panel.
Dropdown Arrow
to adjust individual reactions or modify the sequence
Detailed View Panel:
Click on a sequence tag in the Construct Layout Panel to display the annotated sequence for that tag in the Detailed View Panel. The region of your sequence that will be included in the final construct will be highlighted by a thick blue line. You can adjust the zoom level in the detail view using the keyboard and mouse, as described in section  5.2.1 . It is not possible to edit sequences in the Detailed View Panel – if you wish to edit your sequence, you will need to close the cloning dialog and edit the sequence directly in the sequence viewer.
Save used primers
(where applicable): Creates a primer document for each of the primers used in the operation. For batch cloning reactions, the set of primers for each ligation product will be saved in a sequence list.
Save intermediate products:
Creates a primer document for each of the primers used in the operation. For batch cloning reactions, the set of primers for each ligation product will be saved in a sequence list.
Save in sub-folder:
Create a new sub-folder within the current directory to save result documents to. If another folder with the specified name already exists, a new folder with that name and an incrementing number will be created.