Figure 1

Outline of the Tet System and the Ptet promoters. (A) The tet-responsive transactivator (here tTA, i.e. Tet-Off system) is constitutively expressed in the cell. tTA homodimers bind to the heptameric tet operator sequence (tetO7) in the absence of Dox (a tetracycline derivative). They dissociate from their recognition sites after Dox addition. The Dox response would be reversed for "Tet-On" type transactivator lines also used in this study (C = CMV minimal promoter). (B) Comparison of Ptet-1, the originally described Ptet promoter and the commercially available Ptet-14 ("Ptight"). Both consist of tet operator heptamers created by monomer ligation via compatible restriction sites (Xho/Sal), but with differently spaced operator centers. The CMV-minimal promoters differ in size, but both contain the authentic TATA-box and initiator sequence (Inr). (C) The newly designed Ptet promoters are shown, consisting of a tet operator heptamer with 36nt spacing, and randomized fusion points between all the operators. The CMV derived minimal promoters of the T2-T7 Ptet promoters all have a consensus ("c") TATA-box and TFIIB binding site but differ in the composition of the 5' UTR as outlined in the text. Point mutations are indicated by (*), deletions by dashed lines. Nucleotide positions are given relative to the transcriptional start site (+1). The initiator (Inr) and downstream promoter element (DPE) is indicated.