Vectors and cloning strategy. (A) pAZ677 CmR. (B) Construction of pENTR/Zeo by BP recombination with pDONR™/Zeo. The resulting vector has attL1, attL2 sites and two SfiI sites bordering the CmR fragment. (C) The E. coli ORFs of pCA24N were then transferred by SfiI digestion, gel-fractionated and ligated into SfiI-digested pENTR/Zeo vector. The positive clones are selected for Zeocin resistance and Chloramphenicol sensitivity, and are validated by PCR and DNA sequencing (see methods).
Rajagopala et al. BMC Genomics 2010 11:470 doi:10.1186/1471-2164-11-470