For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pelB sequence and following the termination codon.
Conclusion: The broad-host-range vector for marker-free allelic exchange described here, pCM433, has the advantages of a high copy, general Escherichia coli replicon for easy cloning, an IncP oriT enabling conjugal transfer, an extensive set of restriction sites in its polylinker, three antibiotic markers, and sacB (encoding levansucrase) for negative selection upon sucrose plates.
pELMO and pGEM-T Easy cloning efficiency regarding low, medium and large sized inserts.
Plasmid identity was confirmed by sequencing, using the ccdBsec-F/ccdBsec-R and SP6/T7 primer sets; these were then aligned with the corresponding pELMO and pGEM-T Easy cloning site flanking sequences.
Every Gateway vector possesses att sites necessary for recombination and easy cloning of cDNA.
PCR products were cloned using the pGEM-T Easy cloning system (Clontech) and sequenced with Applied Biosystems Big dye terminator kit v1.1 and the ABCB7 forward primer.
The amplified fragment was cloned into pGEM-T easy cloning vector (Promega), sequenced.
Products were ligated into pGEM-T Easy Cloning Vector (Promega, Southampton, United Kingdom).
The amplified silencing fragments were cloned into the pGEM-T vector (pGEM-T Easy cloning kit; Promega, UK).
The amplified sequences were cloned into pGEM-T easy cloning system (Promega) and transformed into Escherichia coli DH5α cells.
