A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

Table 2 Panel of expression vectors encoding fusion proteins

Vector	5′-module	Central module	3′-module
Set 1
pEF5/FRT-DEST-R4-R3_V5-6xHis: PAR2:mKate2	V5-6xHis	PAR-2	mKate2
pEF5/FRT-DEST-R4-R3_V5-6xHis: PAR2: EGFP	V5-6xHis	PAR-2	EGFP
Set 2
pEF5/FRT-DEST-R4-R3_mKate2: SIRT1	mKate2	SIRT1	SV40 polyA
pEF5/FRT-DEST-R4-R3_mKate2: ΔNtermSIRT1^a	mKate2	ΔNtermSIRT1	SV40 polyA
Set 3
pEF5/FRT-DEST-R4-R3_mkate2: RelA:V5-6xHis	mKate2	p65	V5-6xHis
pEF5/FRT-DEST-R4-R3_V5-6xHis:p65:mKate2	V5-6xHis	p65	mKate2

MultiSite Gateway recombination reactions were carried out between the adapted destination expression vector pEF5/FRT-DEST-R4-R3, and plasmids encoding 5′-module, the ORF of interest (central module) and the 3′-module as described. The resulting expression plasmids are listed on the table, with an indication of the functional modules used for making each fusion protein, and are organised in sets, according to the toolkit properties intended to illustrate (see main text). The central module encoding wt SIRT1 is an entry clone obtained from an ORFeome library, used in the MultiSite cloning reaction with no prior modifications. The SV40 polyA module (italics) does not encode for a peptide but contains a polyA signal that precedes the BGH polyA signal provided by the pEF5/FRT-DEST-R4-R3 vector.
^aThe “Δ” symbol stands for “deleted”.

ISSN: 1471-2199