1. Be sure that 'Cloning' is selected in the "Select Project Type" box in the left column. ('Cloning' will be selected by default upon opening the primer design tool.)
   
   

Use the "Destination Vector" box to input your vector sequence in one of the following ways:

Provide your own vector sequence

1. Click on 'Input full vector sequence'.
2. Paste your nucleotide sequence into the text box as plain text, without nucleotide or line numbering.
   
   

Choose from the preloaded list of vectors

1. Click on the plus sign next to "Select a Takara or Clontech Vector".
2. Choose a vector from the list.
   
   

NOTE: You can delete a vector sequence by hovering the mouse over the sequence in the "Destination Vector" window and clicking on the trash can () icon that appears.

Use the "Linearize by" box to select one of three options for linearizing your destination vector: Restriction Digest, PCR, Already Linearized.

Restriction digest

1. Click on the 'Restriction Digest' option. Available restriction enzyme sites will be displayed on the vector map to the right.
   
   

   

   NOTE: To see enzymes with different numbers of cut sites, or to choose enzymes by name, click on the down arrow icon () in the "Search cut sites" text box.
   

   
   
   
   

2. Choose your restriction site(s) in the vector map by clicking on each desired enzyme. The selected enzyme names will populate at the bottom of the window with their cut sites indicated.
   
   

   

   NOTE: If you want to generate primers that preserve the restriction site(s) in your final construct, click on the 'Include restriction site in final product' checkbox.
   

PCR

1. Click on the 'PCR' option.
2. Click on the vector to indicate the point at which the insert should be placed, or click and drag on the vector to indicate the region which should be replaced by the insert. You can also select the insert site by typing in the appropriate nucleotide numbers in the boxes below the map.
   
   

   

Prelinearized vector

1. To select a prelinearized vector, click the 'Already Linearized' option. The map will display the vector in linear format.
   
   

   

1. Add your first insert sequence to the "Add Inserts" box by either:
   
   • Clicking on 'Input insert sequence' and pasting your nucleotide sequence into the text box
     
     
     
     
   • Clicking on the up arrow icon () to upload a vector file (.fasta or .gb format) into the text box. You can also drag and drop vector files into the text box.
     
     
     
     NOTE: If you are cloning just one insert into your vector, skip to Step 4.
     
     
2. For multiple-insert cloning projects, click "+ Add another insert". A new insert box will appear.
   
    
3. Enter your insert sequence as described in Step 1. Repeat as necessary for each additional insert you want to clone into your vector.
   
   
   
   NOTES:
   
   • If you need to remove an incorrect insert sequence, hover your mouse over the right side of the insert box. Click on the trash can icon that appears. The insert box will remain, but the sequence will be deleted.
     
     
   • You can also rearrange or delete your insert boxes. Hover your mouse above the insert box you wish to move and two new icons will appear. Click and drag the  icon to move the insert box to its correct position. Click the "Remove insert" text icon to remove the insert box completely.
     
     

1. When you have finished entering your insert(s), click on the [Design Primers] button.
   
   

   

2. View your results. The window will switch from the Design Page tab to the Results Page tab. This tab is divided into the following sections: your final vector map and sequence, a sequence view of PCR fragment overlaps, and an output protocol with oligo and PCR information (see Step 3).
   
   

   

   

3. Download your primer information as an Excel file by clicking the [Download Results] button at the bottom of the page. This data can be entered into any nucleotide sequence analysis software tool, such as SnapGene Viewer (available for free online), which allows easy visualization of In-Fusion Cloning primer locations in both sense and antisense strands.
   
   

   

Design your primers now »