How to build new BioBrick vectors. Assembly strategy for a new BioBrick vector using the BioBrick base vector BBa_I51020. (A) The replication origin and antibiotic resistance cassette should each be BioBrick standard biological parts. (B) Assemble the desired replication origin and antibiotic resistance cassette via BioBrick standard assembly to construct a composite origin and antibiotic resistance cassette. (C) Digest the resulting BioBrick composite part with XbaI and SpeI. (D) To excise the ampicillin resistance marker, digest the base vector with NheI. XbaI, SpeI, and NheI all generate compatible cohesive DNA ends that, when ligated with a DNA end from a one of the other enzymes, produce a non-palindromic sequence that cannot be cut by any of the three enzymes. Finally, ligate the digested composite origin and resistance marker to the digested base vector. (E) The result is the new BioBrick vector pSB4K5-I52002.