Synthesis of long repetitive genes

Expression Vector Construction

A unique Sfi I site is inserted which after digestion with Sfi I create N-terminal Pfl MI compatible end and C-terminal BglI compatible end. See : 1783 pT7A31

Recursive directional ligation (concatemerization)

clone repetitive element into pEC. This is the parental vector

digest donor (repetitive element) in pEC (parental vector) with PflM1 + BglI

Digest destination vector (also parental vector if it is first round) with PflMI, dephosphorylate with CIP (1 unit of CIAP per pmole of vector for 1 hour at 50°C)
and then purify using a  spin column.

ligate and transform

Screen by colony PCR and digestion

Verify by sequencing

Subcloning

Subclone repetitive gene from final repetitive contruct in pEC into pT7A31 using

donor: PflM1 + BglI

pT7A31 vector: Sfi1, CIP treated vector

Dephosphorylate the linearized vector (e.g., using 1 unit of CIAP per pmole of vector for 1 hour at 50°C). Heat-inactivate the CIAP for 10 minutes at 75°C.
Gel purify the linearized, dephosphorylated vector

Other vectors can be modified with one of these enzymes: AlwNI, BglI, BslI, BstAPI, DraIII, MwoI, PflMI, and SfiI, although it must be unique

ligation and transformation

Verification

Short repetitive gene: PCR to identify positive colonies, then digestion and sequencing

Long repetitive gene:  digestion and sequencing