A KpnI/SacI double digest releases the hsp70 transcription module, for subsequent ligation into other plasmids such as those containing Drosophila P elements. KpnI BglII NotI XhoI-promoter-MCS-terminator-SpeI XbaI NotI EagI BstXI SacII SacI. Restriction digests of the clone give the following sizes (kb): PstI--3.5; HindIII--3.4; BamHI--3.4. The transcription module is flanked by restriction sites (facilitating its removal), T7 and T3 promoters (for in vitro RNA synthesis), and primers for Sanger sequencing. There is also an M13 origin of replication. One of a series (ATCC 37642-37645) of plasmid vectors for expressing genes under the control of the Drosophila hsp70 promoter and terminator. Major Drosophila hsp70 regulator elements are: 2 heat shock consensus elements (-85 to -72 & -62 to -49), a TATA box (-33 to -26) & transcription initiation site (+1) in the 5' segment; the termination triplet and polyA site in the 3' segment. The KpnI/SacI fragment of pHSREM1 (ATCC 37642) containing the hsp70 transcription module was ligated into KpnI/SacI digested Bluescript SK+. |
