Hello everyone,
I'm currerntly working with a Cas12a vector, which contains a cutting site for Eco31i and a 19 bp direct repeat, which is necessary for the Cas12a to function.
After the GoldenGate-Assembly, I sequenced the vector and noticed, that the 19 bp fragment is missing and therefore the vector is unusable. I already checked the sequence and the vector does not contain another restriction site, that would cause the 19 bp fragment to also be cut off.
Does anybody have an idea as to what causes such a problem? I am out of ideas...