pTet-tTS expresses the tetracycline/doxycycline-controlled transcription silencer (tTS), which is a fusion of the tet repressor protein (TetR) with a KRAB silencing domain. tTS can be used to significantly lower the basal expression from first-generation Tet systems, but is not required for Tet-On 3G or Tet-One systems since the basal expression is typically very low.

In the absence of doxycycline, tTS binds to the tet-operator sequences within TRE promoters, silencing gene expression. Upon addition of doxycycline, tTS undergoes a conformational change that causes its release from the promoter, to be replaced by the Tet-On transactivator.