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A versatile prokaryotic cloning vector with six dual restriction enzyme sites in the polylinker facilitates efficient subcloning into vectors with unique cloning sites. 1998

D R Sage, and A C Chillemi, and J D Fingeroth
Division of Infectious Diseases, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

In large and complex vectors a single restriction enzyme recognition site may be available for introduction of additional DNA requiring the development of linker fragments to create compatible insertion sites. This technology can be time consuming and costly. We describe the construction of a simple phagemid, pSFI, with a polylinker that contains six pairs of dual, rare-cutting, restriction enzyme recognition sites (NotI, SpeI, EcoRV, PstI, SacII, EagI) with multiple unique sites between each pair. This has permitted rapid subcloning of DNA with creation of single flanking restriction enzyme sites. pSFI was used to expedite transfer of viral genes to a LacZ-inducible expression vector and to an adenovirus expression cassette for production of replication-defective virus. The use of this phagemid has facilitated complex vector manipulations and is a valuable adjunct to the family of multifunctional cloning vectors.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011387 Prokaryotic Cells Cells lacking a nuclear membrane so that the nuclear material is either scattered in the cytoplasm or collected in a nucleoid region. Cell, Prokaryotic,Cells, Prokaryotic,Prokaryotic Cell
D004262 DNA Restriction Enzymes Enzymes that are part of the restriction-modification systems. They catalyze the endonucleolytic cleavage of DNA sequences which lack the species-specific methylation pattern in the host cell's DNA. Cleavage yields random or specific double-stranded fragments with terminal 5'-phosphates. The function of restriction enzymes is to destroy any foreign DNA that invades the host cell. Most have been studied in bacterial systems, but a few have been found in eukaryotic organisms. They are also used as tools for the systematic dissection and mapping of chromosomes, in the determination of base sequences of DNAs, and have made it possible to splice and recombine genes from one organism into the genome of another. EC 3.21.1. Restriction Endonucleases,DNA Restriction Enzyme,Restriction Endonuclease,Endonuclease, Restriction,Endonucleases, Restriction,Enzymes, DNA Restriction,Restriction Enzyme, DNA,Restriction Enzymes, DNA
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D005822 Genetic Vectors DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition. Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D001435 Bacteriophages Viruses whose hosts are bacterial cells. Phages,Bacteriophage,Phage
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining

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