Subcloning of one insert into an expression vector.

<p>(A) Maps of entry clone pE-GFP, expression cloning vector pX-lacZ and expression construct pX-GFP. Black arrows show the position of the restriction sites for the enzymes BseRI and HindIII, and the numbers next to these indicate the sizes of the restriction fragments obtained. The grey triangle represents a <i>Streptomyces</i> phage C31 attB recombination site (this site is not used for the cloning procedure described here). Z, LacZ alpha fragment; N, Viral 3′ Non-translated region; T, Nos terminator; RB/LB, T-DNA right/left borders; S1–S2, selectable markers 1 and 2 (resistance to carbenicillin and kanamycin, respectively). (B) Plasmid DNA from 48 white colonies and vector digested by BseRI and HindIII and run on a 1% agarose gel. The upper and lower panels show minipreps obtained from cloning performed using ligation buffer or NEB buffer 3, respectively. DNA from all white colonies has the restriction pattern of pX-GFP (the 119 bp fragment is too faint to be visible on the picture). M: GeneRuler 1kb DNA Ladder Plus from Fermentas. V, vector pX-lacZ.