Pcmv int

The ФC31 integrase expression plasmid (pCMV-Int) and reporter pBCPB⁺ were obtained from Addgene (Cambridge, MA, USA). The reporter donor pEGFP-N1-attB was constructed as previously described [10 (link)]. The reporter plasmid pT2Kmin-XIpGbR was kindly provided by Dr. James A. Lister (Virginia Commonwealth University, Richmond, VA, USA). TA cloning plasmid was purchased from Takara (Dalian, China). Competent E. coli DH5α cells were prepared according to standard protocols. PureLink^® HiPure Plasmid Filter Purification Kits were used for midi and maxi preparation of all plasmid DNA (Invitrogen, Carlsbad, CA, USA). A P-class nanophotometer was used to measure the quality and quantity of DNA (Implen, Munich, Germany).

The antibiotic markers KanMX and hph expressed under the TEF promoter and terminator were amplified with the primers Fw-A1-attP-tefpr and Rv-A2-attB-teft and cloned into pJET1,2 (Thermo Scientific, Belgium) following the protocol of the kit. The resulting plasmids were called pJET1,2-attB-KanMX-attP and pJET1,2-attB-hph-attP respectively. The PhiC31 integrase was amplified from pCMVInt (Addgene, Cambridge, Massachusetts, USA) using the primers Fw-PstI-PhiC31 and Rv-BamHI-PhiC31 and cloned into the PstI and BamHI sites of pBEVY-Nat giving the pBEVY-Nat-PhiC31.