PCR targeting system in Streptomyces coelicolor ... - Streptomyces UK
PCR targeting system in Streptomyces coelicolor ... - Streptomyces UK
PCR targeting system in Streptomyces coelicolor ... - Streptomyces UK
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Template plasmids<br />
Eco RI<br />
FRT<br />
prim<strong>in</strong>g site<br />
amp<br />
pIJ773<br />
Sst I*<br />
aac(3)IV<br />
Xho I*<br />
4334 bps<br />
oriT<br />
FRT<br />
prim<strong>in</strong>g site<br />
Sst I*<br />
H<strong>in</strong> dIII<br />
Fig. 5: Template plasmid pIJ773 conta<strong>in</strong><strong>in</strong>g the apramyc<strong>in</strong> resistance gene aac(3)IV<br />
(AC=X99313) and the oriT of plasmid RP4 (=RK2) (AC=L27758), flanked by FRT sites<br />
(FLP recognition targets, see Datsenko and Wanner, 2000). The disruption cassette was<br />
cloned <strong>in</strong>to the EcoRV site of pBluescript KS (+) allow<strong>in</strong>g its isolation as a 1382 bp<br />
EcoRI/H<strong>in</strong>dIII fragment.<br />
* <strong>in</strong>dicates suitable restriction sites for verification of mutagenised cosmid DNA by<br />
restriction analysis (for example: SstI generates a 751 bp <strong>in</strong>ternal fragment with<strong>in</strong> the<br />
disruption cassette).<br />
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