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--3.4; HindIII--3.3; EcoRI--3.4; BamHI--3.3. 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. BamHI/BglII fragment of pCIII1 with terminator plus several upstream sites of Bluescript MCS ligated into BamHI of pCII38 = pCIII8. KpnI/XhoI region of pCIII8 replaced with synthetic adapter with NotI and BglII sites = pHSREM1. XhoI/HindIII fragment of Drosophila hsp70 with nt -89 to +89 (relative to transcription start site) ligated to XhoI/HindIII Bluescript KS+ = pCII38. 248 bp SalI/XhoI fragment with 3' end of hsp70 ligated into SalI of Bluescript KS+ = pCIII1. |
