Construction and properties of the vehicle for cloning the promoter containing dna fragments the cloning of escherichia coli and phage t 7 promoters
Serpinskii, O.I.; Karginova, E.A.; Mikryukov, N.N.; Kravchenko, V.V.; Zaichikov, E.F.; Maksimova, T.G.; Onikienko, A.I.; Pletnev, A.G.; Mitina, Y.L.
Bioorganicheskaya Khimiya 8(6): 840-847
A 15 base pair nucleotide AluI fragment was removed from the region of the tetracycline promoter (pTc) of plasmid pBR322. This deletion interferes with the in vivo function of the pTc promoter.sbd.the plasmid obtained (named pSK) fails to provide the E. coli cells with resistance to tetracycline. This ability may be recovered if a promoter fragment is introduced into pSK using the EcoRI site in such a way that transcription proceeds towards the tet gene. This property of the pSK plasmid was exploited for cloning the BspI, MspI and AluI fragments, which contained the lacUV5 promoter of E. coli and the A2 promoter of phage T7. In all cases introduction of the promoter fragments was accomplished in such a way that the GAATTC sequence was recovered at the points of ligation. The promoter fragments were easily released from pSK after EcoRI digestion.