Part 3: Confirming the CDS in-frame fusion

As mentioned in the introduction to this tutorial, the input PCR product for this demonstration was designed to generate an N-terminal and C-Terminal in-frame fusion between the mature xynA CDS and the CDS that spans the MCS of pET26B.

To check that the in-frame fusions have occurred correctly:

1.  Select the pET26B-XynA_PCR_Product document created by the cloning operation. The concatenation annotations (purple) show you where the PCR product was inserted into the vector.

2.  Locate and select the CDS (yellow) annotation called MCS CDS PelB-MCS-His-tag.  If required, click on the Annotation tab and select it from the Annotation list.  This should select two intervals generated due to insertion of xynA. . Option (ALT)-click on the annotation to zoom in on this region. 

3.  Go menu Edit -> Go to Base, then click Go to select the CDS intervals and the intervening sequence (5,072 to 6,175). Click on the Add Annotation button, and add a new annotation called xynA fusion, of type CDS and go OK.  Note that you will get a warning that the document has an Actively linked parent. Continue with the editing.

4. A new CDS will appear.  Hover over the new CDS to see the automatic translation and confirm the CDS translates correctly over the entire CDS range. Save the document, and choose to save a copy.

5. Select and delete the preexisting CDS's and delete them so only the XynA fusion CDS remains.  Turn on Translation in the General tab, select the Annotations tab and turn on display of Ligation annotations, then zoom in to view the translation at the fusion sites and confirm the gene product is correctly translated through the ligation sites.

Click the link below to go to Part 4: Troubleshooting.

Part 4: Troubleshooting