This tool helps you to design a crRNA-insert for pX334a. The oligos contain overhangs which fit to the BbsI-overhangs by pX334a.

For repression of gene transcription by targeting the coding sequence it´s crucial to target the non template (= coding) DNA strand.
Therefore the oligos must be designed as follows:

1. Search at your desired target sequence for a CCN (reverse complement of the PAM sequence) at the coding strand.
2. Extract the following (3') 30 nucleotides.
3. Extract the reverse complement.
4. Add AAAC at the 5' end and GT at the 3' end. This will be your fist oligo.
5. Take the sequence from step 2 and add TAAAAC at the 5' end. This will be your second oligo.