Restriction digests of the clone give the following sizes (kb): BglII--16.2; SphI--8.0, 6.2, 1.9; BamHI--16.2. The S. achromogenes fragment contains the following restriction sites (approximate kb from one end): MluI--1.7, 2.5, 3.0, 5.4, 5.9, 9.4, 10.2; SphI--2.1, 9.7; BglII--8.0. When grown at 28C in Streptomyces lividans, the plasmid is present at high copy number and permits expression of spectinomycin resistance without amplification. When grown at 39C, the plasmid is cured and streptomycin resistance is lost, except when AUD-Sar 1 sequences become integrated into the host chromosome where they undergo amplification. Curable vector that can be used to insert and amplify cloned sequences into host chromosomal DNA. The 10.4 kb fragment from S. achromogenes contains the complete 8.8 kb amplifiable unit with 0.8 kb direct repeats at its termini. The unique BglII site is at one end of the 1.7 kb SphI/BglII fragment that determines spectinomycin resistance. pMT660 is a temperature sensitive replication mutant of pIJ702. Distributed in aliquots of 200 ng dried. |
