Size (kb): 12.0349998474121100
Vector: pDO17 (cosmid)
Construction: pDO2, pBR322
Construct size (kb): 12.03499984741211
Features: marker(s): ampR
other: lambda cos site
repressor gene: cro
coding sequence: CII
coding sequence: O
coding sequence: P
coding sequence: ren
vector containing primer sites useful for sequencing
vector for generating nested deletions of cloned inserts
vector permitting visual detection of recombinants
Restriction digests of the clone give the following sizes (kb): BglI--7.8, 2.5, 1.6, 0.78; EcoRI--12.5; PstI--9.0, 2.5, 1.1.
Plasmid contains the following restriction sites (approximate kb from nt 1): BamHI--6.98; BglI--6.61, 7.54, 7.77, 10.9, 11.63; EcoRI--3.89; HindIII--2.31; PstI--0.65, 1.78, 5.62, 10.22; PvuI--10.34; SalI--7.26; SmaI--0.99, 4.61; XhoI--0.89.
A series of overlapping deletion segments can be generated by partial digestion with a restriction enzyme not found in the vector, followed by complete digestion with lambda terminase.
Resulting fragments are blunted and circularized by ligation, and transformants are selected for either ampicillin or kanamycin resistance.
The following restriction sites do not cut pDO184: AatII AflII ApaI AscI AvrII Bpu1102I Bsp120I BsrGI Bsu36I HpaI MunI NotI NsiI PacI PaeR7I PmeI PmlI Ppu10I SacII SfiI SnaBI SpeI SrfI StuI SwaI.
IMPORTANT: To prevent amplification of a rearranged and/or deleted cosmid, we recommend streaking on LB + amp plates at 30C and picking small colonies for liquid culture.
Cosmid vector containing two compatible origins of replication, for generating bidirectional deletions of large inserts to facilitate sequencing.
AmpR transformants can be sequenced in the clockwise direction using the cosL primer 5'-TCATAAATAGCGAAAACC-3'. KanR transformants can be sequenced in the counterclockwise direction using the cosR primer 5'-ACTTTACGGGTCCTTTCCG-3'.
ATCC Medium 1065 (see below) plus ampicillin (50.0 mcg/ml) plus kanamycin (20.0 mcg/ml)
ATCC Medium 1065:
Tryptone (Difco 0123), 10.0 g
Yeast Extract (Difco 0127), 5.0 g
NaCl, 10.0 g
Distilled water, 1.0 L
Ahmed A. Double-origin vectors for isolating bidirectional deletions useful in DNA sequence analysis. Gene 141: 71-73, 1994. PubMed: 8163177