Q.pBR322, the well-known cloning vector of E. coli, is represented as a circular DNA map. Recognition sites for several restriction enzymes are marked around the circle: EcoR I, Cla I and Hind III are clustered close together at the top; Pvu I lies on the upper-left; Sal I lies on the right; and Pvu II lies at the bottom. Three functional regions are already named on the map: the origin of replication ('ori') and the copy-number control region ('rop') lie together near the bottom, and a selectable-marker gene ('tetR') lies along the right-hand arc. Three other regions carry only letter labels and must be identified: region A is the right-hand arc that also bears the tetR marker and the Sal I site; region C is the upper-left arc that bears the Pvu I site; and region B is the short segment on the lower-left lying immediately next to the origin of replication. Name the regions marked A, B and C.

What pBR322 contains

pBR322 is the classic E. coli cloning vector. Its essential parts are:

• ori – the origin of replication, the sequence at which replication of the plasmid DNA begins; it also controls how many copies of the plasmid a cell keeps.
• rop – a gene whose product helps regulate the copy number of the plasmid.
• Two antibiotic-resistance genes that serve as selectable markers: ampR (ampicillin resistance) and tetR (tetracycline resistance). A cell carrying the plasmid survives on medium containing the antibiotic, so these genes let us select transformed cells.
• Recognition sites for restriction enzymes (EcoR I, Cla I, Hind III, BamH I, Sal I, Pvu I, Pvu II, and others). Several of these sites lie within the resistance genes, which is what makes insertional inactivation possible.

Identifying A, B and C

• Region A is the right-hand arc, the one carrying the tetR marker and the Sal I site. Sal I cuts inside the tetracycline-resistance gene, confirming the identity of this arc. Therefore A is the tetracycline-resistance gene (tetR).
• Region C is the upper-left arc, carrying the Pvu I site. Pvu I cuts inside the ampicillin-resistance gene. The two large arcs are the plasmid's two resistance genes; since the right arc is tetR, the left arc must be the other marker. Therefore C is the ampicillin-resistance gene (ampR). …

Method 2 -- use the numbered bp-coordinate map instead of qualitative clue-matching

pBR322's features have fixed, well-known positions on its standard circular map (traditionally drawn with position 0 at the EcoRI site, numbering clockwise to about 4,361 bp):

• ampR (ampicillin resistance) spans roughly bp 3,300-4,150 -- the upper-left arc, and it is the gene into which Pvu I cuts.
• tetR (tetracycline resistance) spans roughly bp 90-1,300 -- the right-hand arc, and it is the gene into which Sal I (and BamHI, HindIII) cut.
• ori/rop sit together in the lower portion, around bp 2,200-2,600, next to the EcoRI/ClaI/HindIII cluster's opposite side of the circle. …