<p style="text-align: justify;">Event MON 15985 was developed by biolistic transformation of cotton meristems with purified DNA containing the <i>cry2Ab2</i> and <i>uidA</i> (GUS) expression cassettes. The parental variety used in the transformation, DP50B, was derived from a conventional cross between DP50 and the transgenic Bollgard&reg; cotton line 531. Therefore, the parental line already contained the <i>cry1Ac</i> gene as well as two selectable marker genes, the <i>nptII</i> gene encoding neomycin phosphotransferase II (NPTII) and the <i>aad</i> gene encoding aminoglycoside adenyltransferase (AAD).</p>

<p style="text-align: justify;">The DNA used for transformation was an approximately 6 kb fragment containing the expression cassettes for <i>cry2Ab2</i> and <i>uidA</i>, and was derived from plasmid PF-GHBK11 following restriction endonuclease digestion and high pressure liquid chromatography (HPLC) purification. The purified DNA fragment did not contain any other plasmid-derived sequences, such as the bacterial origin of replication site or antibiotic resistance marker genes.</p>

<p style="text-align: justify;">The <i>cry2Ab2</i> construct consisted of a synthetic copy of the <i>cry2Ab2</i> gene originally isolated from <i>B. thuringiensis</i> subsp. <i>kurstaki</i> (<i>Btk</i>) under the regulatory control of the doubly enhanced cauliflower mosaic virus 35S promoter (e35S) and a polyadenylation signal isolated from the 3&rsquo;-terminal untranslated region of the nopaline synthase (NOS) gene from <i>Agrobacterium tumefaciens</i>. Transcriptional activation was modulated by inclusion of the 5&rsquo;-terminal untranslated leader sequence from the petunia heat shock 70 protein (PetHSP70). Targeting of the expressed protein to chloroplasts was accomplished by fusing the sequence encoding the chloroplast transit peptide, isolated from the <i>Arabidopsis thaliana</i> 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene, to the 5&rsquo;-terminus of the <i>cry2Ab2</i> gene sequence.</p>

<p style="text-align: justify;">The <i>cry2Ab2</i> gene was completely re-synthesized to incorporate plant-preferred codons, and the expressed protein was 88% identical (97% similarity with conservative substitutions) to the native Cry2Ab2 protein expressed in <i>B. thuringiensis</i>.</p>

<p style="text-align: justify;">The <i>uidA</i> (synonym <i>gus</i>) gene, isolated from <i>E. coli</i> strain K12, encodes the enzyme &szlig;-D-glucuronidase (GUS), and was included as a scorable marker allowing colorimetric identification of transformed plant tissue following histochemical staining. Gene expression was regulated using the same e35S promoter and NOS 3&rsquo; sequences as used for the <i>cry2Ab</i> gene construct.</p>
