Journal: PLoS ONE
Article Title: Novel gateway binary vectors for rapid tripartite DNA assembly and promoter analysis with various reporters and tags in the liverwort Marchantia polymorpha
Figure Lengend Snippet: Outline of cloning procedure using the R4pMpGWB system. Promoter entry clones are constructed by a BP reaction between pDONR P4-P1R and an attB4 -promoter- attB1 fragment. The cDNA entry clones are constructed by the BP reaction using pDONR221 and the attB1 -cDNA- attB2 fragment. The libraries of promoters and cDNAs can be used as resources for construction of chimeric fusions. Promoter and cDNA entry clones and R4pMpGWB vectors are used in a tripartite LR reaction to form a C-terminal fusion of cDNA-encoded protein and a reporter or tag. Note: as pDONR P4-P1R has been discontinued, pENTR 5′-TOPO (Thermo Fisher Scientific) can be used as an alternative for promoter entry clone production. Arrowheads, T-DNA border sequences; B1 , att B 1 ; B2 , att B2; B4 , att B4; P4 , att P4; P1R , att P1R; L1 , att L1; L2 , att L2; L4 , att L4; R1 , att R1; R2 , att R2; Km r , kanamycin-resistant marker; Cm r , chloramphenicol-resistant marker; Spec r , spectinomycin-resistant marker; ccdB , negative selection marker used in bacteria.
Article Snippet: However, as pDONR P4-P1R has been discontinued, pENTR 5′-TOPO (Thermo Fisher Scientific) can be used as an alternative for promoter entry clone production.
Techniques: Clone Assay, Construct, Marker, Selection