The sequence of pds in pPlat–pds was derived from H. pluvialis strain NIES-144. While pds of transformed strains showed a sequence similar to that of the pPlat–pds, the WT strain (1844 em. Wille K-0084 (SCCAP)) pds (GenBank accession number: KP826910) and a spontaneous norflurazon-resistant strain showed a different sequence (Fig. 5B). Moreover, sequencing of partial PsaD 5' UTR, full ble CDS, and 3' UTR of PsaD verified insertion of PsaD-ble cassette in all tested colonies (data not shown). In addition, Southern blot analysis using a ble specific probe (corresponding to the full ble CDS and the 3′ UTR region of the PsaD–ble cassette) was performed using isolated gDNA of transformed colonies digested by BamHI and BglII. At least one clear ble copy was detected in the gDNA of six out of eight colonies tested (in three of them, additional copy was detected); WT and empty vector negative control colonies did not display a ble copy, as expected (data not shown). All together these results confirm the successful introduction of the pPlat–pds derived mutated gene into the genome. This confirms the usefulness of the pBS–pds transformation vectors for stably inserting transgenes added both 3′ and 5′ to the selection marker into the genome of H. pluvialis.