pCrU6.4-SaCas9 cloning (pPH156)
From Andre Greiner, Peter Hegemann lab, Humboldt University-Berlin October 2017
Cloning vector for Staphylococcus aureus Cas9 (SaCas9) guide RNA transcription, controlled by the U6 snRNA promoter #4 (3’ of Cre15.g640800). The 20 bp target site can be inserted in a cut-ligation reaction as annealed oligos into an Esp3I cloning site following the protocol of Ann Ran (Ran et al. 2013). The immediate 4-bp sequence upstream of the transcriptional start site was changed to ACTT in all U6 constructs to simplify the cloning procedures.
HindIII and KpnI restriction sites can be used for C. reinhardtii antibiotic resistance gene insertion (aphVII, aphVIII, ble).