Restriction digests of the clone give the following sizes (kb): PvuI--3.6, 3.0; SalI--6.6; EcoRI--6.6; SmaI--6.6; BamHI--6.6. Expression is inducible by galactose. YX-type expression shuttle vector permitting visual detection of recombinants and production of ssDNA in E. coli. Contains promoters for in vitro RNA synthesis and encodes the lacZ alpha (lacZ') peptide. Transcription from the GAL10 promoter (ATG is included in the sequence) permits the synthesis of a fusion protein (starting with 26 aa of GAL10) through the polylinker in the same frame as lacZ'. pRSS56, constructed by ligating a PvuI fragment (bp 498-2412) of pBluescript KS+ to a PvuI fragment (bp 2850-730) of pBS(+), contains the KS MCS from pBluescript KS+ and the unique NdeI and AatII sites between bla and f1 origin of pBS(+). A fragment containing the TRP1 gene was inserted into the NdeI site, a DraIII/EcoRI fragment containing the GAL10 promoter was inserted into the Asp718 site and a URA3 fragment was inserted between the SpeI and NotI sites of the MCS. The order of the major features in this plasmid is: TRP1 - URA 3 - f1 ori (NaeI) - T7 promoter - lacZ'/MCS - T3 promoter - GAL10 promoter - GAL1 promoter - pMB1 ori - bla - CEN - ARS1. |
