How do I simulate NEBuilder Hifi DNA Assembly® in SnapGene?
In this lesson we will "clone" the mEGFP coding sequence (CDS) into the controlled expression vector pET-52B(+). The mEGFP CDS will be positioned in-frame with the vector-based N-terminal Strep-tag II and C-terminal 10xHIS-tag.
Open or Import a Vector Sequence
Import or open a sequence file for the plasmid vector that will be used for NEBuilder HiFi DNA Assembly®.
Select the Vector Region for Insertion or Replacement
Import or Open the vector sequence.
In Map or Sequence view:
- Click within the sequence to select the insertion point, or
- Click on an enzyme site to select it as the insertion point, or
- Select a specific region that will be replaced by the insert, or
- Select a restriction fragment that will be replaced by the insert.
To select a restriction fragment for replacement, click on the first restriction site, then shift click on the second restriction site. Alternatively, click on the first site then drag to the second site.
In the above example, the vector fragment generated by digestion with SmaI and SacI will be replaced by the insert in the NEBuilder® Hifi DNA Assembly reaction.
Start the NEBuilder HiFi DNA Assembly® Tool
Click Actions → NEBuilder HiFi DNA Assembly® → Insert Fragment....
When starting the NEBuilder® Hifi DNA Assembly tool, the DNA sequence selection in the frontmost window will automatically be set as the vector region to be replaced by the insert.
If a vector sequence is not open when you start the NEBuilder Hif DNA Assembly® tool then you can choose and define the vector in the Vector tab.
The NEBuilder HiFi DNA Assembly® tool opens showing the "Vector" tab. The option to "Linearize with restriction enzymes" is automatically selected, and the appropriate vector restriction fragment for replacement selected.
Check the Vector Orientation
In Map View, if required, click the Zoom control to zoom in and view the feature elements at the point of insertion. Alternatively, switch to Sequence view.
If required, reverse the orientation of the vector for the NEBuilder® HiFi DNA Assembly.
In this example, We see the vector-based open reading frame used for recombinant addition of Strep-Tag II and HIS-tags is in the reverse orientation. Therefore, we click the right "Orientation of Vector" button to set the vector to be input in the reverse orientation.
Select an Insert Fragment
Switch to the "Fragment" tab.
Set the "Source of Fragment" sequence by one of the following methods:
- Click the dropdown menu and select from the list of Recent or Open files, or
- Click the dropdown menu and Browse... to locate the insert sequence file on your computer, or
- Click the "Click Here" link to browse to locate the insert sequence file, or
- Drag and drop a sequence file into the window.
Define the Insert Fragment Region
In this example we will manually define the insert region, excluding the mEGFP stop codon to allow in-frame fusion to the vector-based CDS encoding a C-terminal Thrombin cleavage site and 10xHIS-tag.
Switch to Sequence view, locate the start of the insert and click to set the cursor to the start position.
Scroll to the end of the insert and SHIFT-click to select the desired insert region. In this example the mEGFP stop codon is excluded from the selection.
Design Primers to Amplify the Insert Fragment
Switch to the Product tab and click Choose Overlapping PCR Primers.
Set the desired Tm for the primers.
Set the desired overlap length between vector and insert ends.
Set the target Tm for the NEBuilder HiFi DNA assembly®.
Click Choose Primers to design new PCR primers.
In this example we wish to regenerate the partial vector-based SacI restriction site.
Validate the Vector/Insert Fusion Points
In the Product tab, switch to Sequence view.
In this example we see that the vector-based "Strep-Tag II/HRV 3C" features are in-frame with mEGFP CDS insert.
Scroll to the end of the insert fragment.
In this example we confirm the mEGFP insert is in-frame with the vector-based Thrombin/10xHIS tag feature, and confirm the SacI site has been regenerated.
Name the New Primers and Make the Product Sequence
Switch to the Fragment tab.
Enter appropriate names for the new primers.
Enter an appropriate name for the new product.
Click Assemble to create the new NEBuilder Hifi DNA Assembly® product.
Click File → Save to save the new sequence to an appropriate location on your computer.
View History
View the newly created product file, switch to History view to see all steps simulated during the NEBuilder Hifi DNA Assembly® procedure.
Order the PCR primers
Seethe lesson on Exporting Primers for details on how to export and order the new primer sequences.