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. 2014 May 30;9(5):e96693. doi: 10.1371/journal.pone.0096693

Figure 1. Integration of a GOI into the pBASE backbone vector.

Figure 1

(A) PCR amplification of the GOI using uracil-containing primers and a uracil compatible DNA polymerase (e.g. PfuX7). (B) The PCR fragment of B and a vector backbone fragment obtained by PacI/Nt.BbvCI digest (see Figure S1) is mixed. Addition of the USER™ enzyme mix catalyzes uracil-excision and generates compatible overhangs on the PCR fragment for directed vector assembly. Subsequently, the cloning mix is directly transformed into E. coli cells, where the fragments are ligated together.