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When performing transcriptional analyses, reporter gene-expression vectors are used to insert promoter fragments through the selected use of a multiple cloning site (MCS) located upstream of the reporter gene. The MCS from pBluescript TM has frequently been transferred into reporter plasmids (usually bearing the chloramphenical acetyltransferase reporter gene) and used to subclone various promoter fragments from diverse genes. Analyses in electrophoretic mobility shift assay using this MCS as labeled probe revealed that it specifically binds multiple nuclear proteins from a whole array of widely used cell types. Moreover, the presence of the MCS sequence dramatically altered promoter activity in a totally unpredictable fashion that depends on the distance between the MCS and the basal promoter start site of the gene, leading to severe misinterpretation of the transfection data. Finally, we provide evidence that the BamHI/SmaI/PstI restriction site combination is likely one of the major binding site for nuclear proteins on the pBluescript TM MCS, therefore suggesting that this particular combination of restriction sites should be avoided in the MCS from plasmids that are to be used in promoter studies.