Two tandem copies of the 260bp twi proximal element (PE) (located between -440 and -180 relative to the twi promoter), followed by twi sequence from -180 to +160bp relative to the transcription start site, are fused upstream of lacZ. Both copies of the PE have been mutated; the more distal copy contains i. inactivating mutations in the core sequences of the low affinity TD4 and TD5 dl binding sites and ii. two copies of an E box from the rho promoter (with core sequence CATATG) inserted just upstream of the TD4 and TD5 sites, while in the proximal copy, the core sequences of the low affinity TD4 and TD5 dl binding sites have been mutated to the optimal sequence to create high affinity sites. This results in the E boxes and the functional dl sites being separated by approximately 260bp.