"Our RFC"

We made a new standard and we built three parts based on that this year.

The BbsI restriction enzyme recognize the sequence 5’ GAAGAC3’ and cut downstream two bases to six bases. If we make two reverse directions based on BbsI site, we can cut a part and insert another one with seamless. (Mechanism shown below)

Let us make the above sequence as an example. If you want to insert a coding sequence between promoter and polyA, the prefix of forward primer you design must contain GAAGACNNtaaaNNNN and reverse must contain GAAGACNNagttNNNN. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into promoter and polyA seamless. The PCR product must be such sequence, including the protect bases, shows below.

<groupparts>iGEM014 SUSTC-Shenzhen</groupparts>