Fig. 3. Schematic diagram of the constructs used to make transgenic Xenopus tadpoles for the characterization of Xrx1A regulatory sequences. Transgene construct 1 was made by cloning the Xrx1A regulatory sequences (SstI-PstI fragment) in front of GFP in pBS-GFP. The full-length promoter construct 1 was digested with NotI and SalI, AvaI, BamHI, BglII, and BanI, respectively, to release the transgene constructs 2, 3, 4, 5 and 6. Transgene construct 9 was obtained by inserting a heat shock protein promoter (hsp) into the SmaI site of pBS-GFP. The BglII-BanI fragment from the Xrx1A promoter was subcloned into the EcoRV site of construct 9 and pBS-GFP to generate transgene constructs 7 and 8, respectively. To make transgene constructs 12 and 13, the SstI-BamHI fragment from the Xrx1A promoter was subcloned into pBS-GFP first, then the hsp promoter (blunt-ended HindIII-NcoI fragment of phs3LSN) or the BanI-PstI fragment from the Xrx1A promoter were inserted into the EcoRV site between the Xrx1A early enhancer and GFP. Construct 10, containing nucleotides (nt) -857 to 0 of the Xrx1A regulatory sequence, was prepared by PCR of construct 1, with a GFP-specific primer (see below) and the Xrx1A promoter specific primer: 5'-GATCGGATCCCTTCCAGCAATCATATCCTA-3' (-857 to -838). The resulting product was digested with PstI (3'-end of the Xrx1A regulatory region) and BamHI (included in the Xrx1A-specific primer) and subcloned into pBS-GFP. Construct 11, including nt -986 to -838 of the Xrx1A regulatory region was prepared by PCR of construct 1 using the following primers: 5'-GATCAGATCTTAGGATATGATTGCTGGAAG-3' (the complement of the previous primer encompassing nt -857 to -838, but with a BglII site at the end); and 5'-GATCGGATCCGATCTGTTATCTGGAAAACCCC-3' (nt -986 to-965 of the Xrx1A regulatory sequence and a BamHI site). The PCR product was digested with BamHI and BglII and subcloned into the BamHI site of construct 9.