pGADT7 AD is a yeast expression vector that is designed to express a protein of interest
fused to a GAL4 activation domain (AD; amino acids 768–881). Transcription of the GAL4 AD
fusion is driven by the constitutively active ADH1 promoter (PADH1), and is terminated at the
ADH1 transcription termination signal (TADH1). The GAL4 AD fusion contains an N-terminal
SV40 nuclear localization signal (SV40 NLS; 1) that targets the protein to the yeast nucleus,
and a hemagglutinin epitope tag (HA Tag), located between the GAL4 AD and the protein
of interest, that allows the protein to be easily detected with HA-tag antibodies.
The T7 promoter (PT7), located just upstream of the HA tag sequence, allows in vitro transcription
and translation of the HA-tagged protein of interest (without the GAL4 AD and
the SV40 NLS). pGADT7 AD replicates autonomously in both E. coli and S. cerevisiae from
the pUC and 2 ori, respectively. The vector also contains an ampicillin resistance gene
(Ampr) for selection in E. coli and a LEU2 nutritional marker for selection in yeast.