Option F – Use restriction enzyme cut sites to create the insert

1. Use File > Open to open the file or SeqBuilder Pro project (.sbd) containing the vector sequence.

2. Use the Enzymes tool ( ) to apply the set of Unique Sites.

3. When you find suitable left and right cut sites, select the region between them by clicking on the label for the restriction site on one end and then Shift+clicking on the label on the other end.

4. Choose Cloning > Copy Restriction Insert or use the keyboard shortcut Ctrl/Cmd+Shift+V.

5. Choose Cloning > Paste Restriction Insert.

The two-tabbed Clone Insert dialog opens, with separate tabs for the Left (5’) End and Right (3’) End. The corresponding ends of the vector sequence are displayed adjacent to insert ends on each panel.

There are several operations you can perform while in this dialog:

• • To simulate modification of overhangs for incompatible ends, use the Trim Blunt or Fill In buttons.
    
    
  • To manipulate the overhangs manually, move the arrows above and below the two strands. This can be used to simulate partial fill or end protection.
    
    
  • To return to the default overhangs, click Revert.
    
    
  • To flip the insert, with respect to the vector, check the Reverse Complement Insert box.
    
    
  • The translation for the reading frame specified in the Translate field is shown between the top and bottom strands for both the vector and insert sequences. As the ends of the vector and insert are adjusted, the translation is updated automatically. To view the translation for a different reading frame, select the desired reading frame from the Translate drop-down menu. Reading frames in this list correspond to the reading frames displayed on the Primer Design, Sequence, and Linear views.

6. Once the vector and insert have been constructed with compatible overhangs, click the Clone button.

7. Choose Cloning >Restriction Enzyme Cloning.

8. The Project window opens with the Clone tab active. The selection from Steps 1-2 is placed in the Inserts folder in the Project tab, as well as in the Fragments section at the bottom of the Clone tab.

Proceed to Step 2: Specify the method and options.