pSB2K0

Description

Warning, this plasmid does not have a proper BioBrick cloning site.

Notes

"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 an
ent generates a good distribution of deletion clones following electroporation.
Deletion clones are sized and sequenced using vector specific forward and
reverse primers.
"