<p><B>Figure 1</B>: pAB and pBA multiple cloning sites with highlighted primers prA1/B1u and prA2/B2u annealing regions<p>

<p>The new and improved method for Byte production (ie: USER^TM) necessitated a change in anchoring system. Longer sticky ends were also desired to increase the efficiency of recircularization. These factors led to the development of a USER^TM-based anchoring system. An anchoring piece, constructed of two annealed oligomers, is bound to the streptavidin-coated bead via a 5' biotin modification and provides a sticky 3' overhang complementary to an A end. Once the desired number of Bytes is added, a terminator (again, two annealed oligomers) is annealed and ligated to the available end of the final brick (in this case, a B end). The entire construct is then treated with USER^TM enzyme mix. The resulting end product from the digestion of uracil contained within the anchor, anneals to the terminator overhang and can be ligated to form a circular product. The ligation also yields a complete SceI site that can be used to linearize the construct for recombination into the <i>E. coli</i> genome. See <B>Figure 2</B>.</P>

<img src="https://static.igem.org/mediawiki/2009/c/c0/UofA09_Bead_Overview_prA12B12u.png">

<p><B>Figure 2:</B> Showing anchor and terminator fragments and effect of USER^TM treatment. I SceI site and A ends are highlighted<p>