Vectors with the fd replicon for in vivo cloning and analysis of genes.
We have constructed two new mini-Mu derivatives, pMRfP and pBEf, that combine the properties of known mini-Mu vectors and the advantages of the replication origin (orifd) of filamentous phage fd. Mini-Mu pMRfP consists of the left (850 bp) and the right (216 bp) ends of the Mu genome, orifd, packaging signal of fd, and the gene conferring resistance to chloramphenicol. The second mini-Mu, termed pBEf, carries the left end of Mu (1001 bp), which contains the so-called internal activation sequence (enhancer of transposition), required for a higher frequency of transposition, the right end (116 bp) and the gene conferring resistance to kanamycin. These new mini-Mu vectors are suitable for in vivo cloning with the ability of single-stranded DNA preparation using one of the helper phages (M13K07, rv1, IR1, R408) and with a large cloning capacity (the size of the cloned fragment can be up to 35 kb). They can also be used as the hoppers (a transposable ori that can be turned on or off depending on the presence of the fd gene 2 product). Thus, these mini-Mu derivatives can be employed as vectors for in vivo cloning, and as regulated transposons or mobile replicons.
Osuský
M
M
Department of Biochemistry, Comenius University, Bratislava, Slovakia.
Stuchlík
S
S
Zámocký
M
M
Dubaová
M
M
Janitorová
V
V
Turna
J
J
D003001:Cloning, Molecular / Q000379:methods*; D003090:Coliphages / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D005838:Genotype; D017912:Inovirus / Q000235:genetics*; D007613:Kanamycin Resistance / Q000235:genetics; D010641:Phenotype; D010957:Plasmids; D018741:Replication Origin; D012093:Replicon; D015183:Restriction Mapping; D017384:Sequence Deletion