Sorry for my ignorance, but I am having difficulties to understand why in the plasmid pHSN6A01 (https://www.addgene.org/browse/sequence/258465/), between the pol III promoter (AtU6-26) and the gRNA scaffold, there is a sequence which carries antibiotic resistance (SmR). I can not find the sense of it as the Pol III could not transcribe the resistance gene and all of the space would be negative for the sgRNA generation.
Hi Andres,
The SmR is used as a stuffer. You need to digest the plasmid with BsaI (double cutter) to insert your sgRNA. The SmR is only useful for propagation (ensure that the cloning cassette is not lost) and counter-selection (+ clones should not grow on Spec). Details for the cloning method are found in the original publication, this is a common cloning strategy for sgRNAs using annealed oligos cloning.
Hope it helps
Francesco
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