In order to produce the specific sticky ends of each gene to be assembled, unique plasmids were developed and named pAB and pBA. These plasmids were designed and constructed from pUC19 and contain the pMB1 high copy origin. Unique cassettes were designed containing PstI, XbaI, and primer annealing regions complementary to the ‘A’ and ‘B’ ends. The cassette was synthesized and inserted using two restriction sites (EcoRI and NsiI). This left an EcoRI site on the final plasmid as well a PstI scar site. The primer annealing regions in pAB are reverse to that in pBA so that compatible sticky ends can be produced in either plasmid. Genes can be inserted using XbaI and PstI (or an enzyme which produces a compatible sticky end). The plasmids were originally designed for a previous but now obsolete system, where the sticky ends were generated via nicking enzymes.

Shown are the AB and BA cassettes of pAB and pBA respectively. “ORF” is the open reading frame; the gene to be edited. “RBS” is the ribosome binding site. The primer binding sites are highlighted in blue/green and the complementary - primers are aligned to the plasmid. The bolded red ‘U’s” denote the uracils that the USER™ system acts on. USER^TM mix removes the uracil and creates a single stranded break in its place. The resulting short oligonucleotides can be purified away from the PCR product to produce a Byte.