1. First, select one of the "Cloning Products" from C113, C115, C116, or C117.

2. In the "Vector Linearization Method" radio button, select the appropriate method for vector linearization.

3. Enter the vector sequence according to the linearization method and then select the restriction enzyme cleavage site.

A. If the linearization method is‘Single Restriction Enzyme Digestion’, please enter the complete sequence of the vector or the sequence near cloning site into the first text box. Then select the linearization cleavage site from the restriction site selection list.

B. If the linearization method is‘Double Restriction Enzymes Digestion’, please enter the complete sequence of the vector or the sequence near cloning site into the first text box. Then select the linearization cleavage site (from the restriction site selection list) at both 5' and 3' ends of fragment.

C. If the linearization method is‘PCR Amplification’, please enter the upstream sequence of the vector cloning site into the first text box, and the downstream sequence of the vector cloning site into the second left text box.

4. Select the number of inserts in the‘Num of Inserts’radio button.

5. Enter the complete sequence of each insert in the text box below according the 5' to 3' cloning direction, and the sequence direction is also from 5' to 3'.

6. Click the‘Generate Primers’button. The complete forward and reverse amplification primers of the insert are showed in the lower text box. Each primer contains a recombinant sequence (lowercase) and a specific amplification sequence (uppercase). If the linearization method of the vector is‘PCR Amplification’, the amplification primers sequence of corresponding vector will be showed in the bottom text box.

7. Click the‘Empty’button to continue designing the CE MultiS cloning primers for other inserts.

Note:

1. If the linearization method is‘Single Restriction Enzyme Digestion’and the cleavage site is not unique in the input vector sequence, the sequence between the two outermost cleavage sites will be excised during cloning in the software default.

2. If the linearization method is‘Double Restriction Enzymes Digestion’, the sequence between the two restriction cleavage sites of the input sequence will be excised during cloning in the software default.

3. If the linearization method is‘PCR Amplification’, the inserts will be cloned into the two input sequences in the software default.