Schematic overview of (MultiSite) GatewayTMcloning and properties of the constructed destination vectors. (A) Overview of the GatewayTM cloning procedure. Attachment of attB sites to a DNA segment of choice, often generated by PCR or as synthetic DNA, allows recognition by the BP ClonaseTM and recombination into a donor vector (pDONR) containing attP sites, yielding an entry clone (pENTR) carrying attL sites. The DNA segment in the pENTR clone can then be transferred to a destination vector (pDEST) by recombination between attL and attR sites present on the pDEST vector mediated by the LR ClonaseTM. This yields an expression clone (pEXP) in which the DNA segment again becomes flanked by attB sites. Positive selection of pENTR and pEXP clones on medium containing appropriate antibiotics together with negative selection of starting products and by-products (shown in grey) based on the presence of a negative (ccdB) selection marker between the recombination sites (GatewayTM Cassette) further increases the efficiency of the system. (B) A combination of existing pENTRs, or new pENTRs, are easily assembled in a single MultiSite GatewayTM reaction catalyzed by the LR II ClonaseTM Plus using pMG as destination vector. (C) Schematic representation of the MultiSite GatewayTM compatible vector set (pMG) for transformation of S. cerevisiae. GOI, POI and TOI indicate gene, promoter and tag of interest, respectively. CmR, AmpR and KmR indicate chloramphenicol, ampicillin and kanamycin resistance, respectively. B1-B4, P1-P2, L1-L4 and R1-R4 stand for the respective att sites. CycT indicates the CYC1 terminator on the vector’s backbone.