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pSCANS Vector

Sequence:   fasta   GenBank

Closeup of multiple cloning region
Gel showing nested deletion time course
Sample Alignment of nested deleteion clones
pSCANS Cookbook
A new plasmid vector, pSCANS, has been constructed which allows rapid generation of an ordered set of nested deletions from either strand of a cloned DNA fragment. pSCANS is based on the low-copy F replicon. The size of the vector DNA has been reduced to the 4.4-kbp range by removing the 2.5-kbp sop (stability of plasmid genes) region. The resulting plasmid has the low copy number typical of F plasmids and it remains stable enough to be easily maintained by growth in the presence of kanamycin, the selective antibiotic. DNA in amounts convenient for sequencing is readily obtained by amplification from an IPTG-inducible P1 lytic replicon. The vector's multiple cloning region (MCR), which has several unique sites for both shotgun and directional cloning, is flanked on one side by recognition sequences for the extremely rare cutting intron encoded nucleases I-CeuI and I-SceI, and on the other side by a recognition sequence for another intron encoded enzyme, PI-PspI and a nicking site for the phage f1 protein, gpII, that initiates f1 rolling circle DNA replication. Cleavage with the intron encoded enzymes leaves four-base 3' overhangs that are resistant to digestion with E. coli ExoIII. Between these sites and the MCR are recognition sites for several rare 8-base cutters that leave ExoIII sensitive termini. Double cutting with one intron encoded enzyme and an adjacent rare cutting restriction endonuclease allows for unidirectional 3' to 5' digestion across the insert with ExoIII. Alternatively, plasmid linearized with I-SceI can be blunt ended to produce an ExoIII sensitive end and then cut with I-CeuI to generate an ExoIII resistant end on one side of an insert. The f1 nicking site can be used for ExoIII digestion of the other strand of the insert or for producing single-stranded plasmid circles for library normalization or subtraction. After ExoIII digestion, the resulting single-stranded regions are digested with S1 nuclease, and the ends are repaired and ligated with T4 DNA polymerase and ligase. Pooling samples from several different ExoIII digestion time points before subsequent S1 treatment generates a good distribution of deletion clones following electroporation. Deletion clones are sized and sequenced using vector specific forward and reverse primers.


Last updated 12:07 pm, Mar 27, 2002
Maintained by Sean R. McCorkle (mccorkle@bnl.gov).

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