Restriction digests of the clone give the following sizes (kb): EcoRI--6.6; BamHI--6.6; SalI--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 URA3 gene was inserted into the NdeI site and a DraIII/EcoRI fragment containing the GAL1 and GAL10 promoters was inserted into the Asp718 site of the MCS of pRSS56. All ends were blunted. The order of the major features in this plasmid is: URA3 - f1 ori (NaeI) - T7 promoter - lacZ'/MCS - T3 promoter - GAL10 promoter - GAL1 promoter - pMB1 ori - bla - CEN - ARS1. |
