A cassette with seven unique restriction sites, including octanucleotide sequences: extension of multiple-cloning-site plasmids.
A 27-bp synthetic DNA cassette was constructed which contains the restriction sites of the two rare-cutter enzymes NotI and SfiI and, in an overlapping arrangement, those of five enzymes with 6-bp recognition sequences: ApaI, BalI, NdeI, SacII, XmaIII. The protruding termini of the fragment allow its insertion into any EcoRI-cut DNA creating a new EcoRI site at one side of the cassette only. This fragment was integrated into the pUC18-like multiple-cloning-site (MCS) plasmids pTZ18R and pTZ19R, producing a set of vectors which carry seven additional unique restriction sites (giving a total of 17) within their MCS. They still provide the capabilities of simple recombinant selection by blue/white coloured colonies, creation of single-stranded DNA in the presence of a helper phage, and in vitro transcription of cloned DNA using T7 RNA-polymerase. Plasmids with two copies of the DNA cassette inserted into their MCS were also constructed. Beside the advantages they provide in some cloning procedures, these latter plasmids, which carry a tandem repeat, are valuable sources of related 27-bp fragments, with features similar to the original but with different cloning termini.
Hoheisel
J D
JD
Fakultät für Biologie, Universität Konstanz, F.R.G.
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005822:Genetic Vectors; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics; D010957:Plasmids
D009841:Oligonucleotides; D004247:DNA; D004262:DNA Restriction Enzymes; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases