<p style="text-align: justify;">The cotton lines 1445 and 1698 were produced by <i>Agrobacterium</i>-mediated transformation of the cotton (<i>Gossypium hirsutum</i> L. ) cultivar Coker C312, with plasmid PV-GHGT07. The T-DNA region of the <i>Agrobacterium</i> Ti plasmid used to develop line 1445 contained sequences encoding CP4 EPSPS, glyphosate oxidase (GOX), NPTII, and AAD. The plasmid used to produce 1698 did not include the GOX encoding gene.<br />
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Plasmid PV-GHGT07 is a single border vector that has only a right border containing the following elements: the 0.4 kb <i>oriV</i> fragment from the RK2 plasmid fused to the 3.0 kb segment of pBR322 allowing maintenance in <i>E. coli</i> and in <i>A. tumefaciens</i>. This was fused to the 90 bp DNA fragment from pTiT37 plasmid, which contained the 25 bp nopaline-type T-DNA right border.</p>

<p style="text-align: justify;">The nucleotide sequence of the CP4 EPSPS encoding gene was modified by site-directed mutagenesis to contain plant-preferred codons in order to maximize protein expression in plant cells. These modifications did not alter the expected amino acid sequence of the CP4 EPSPS. Expression of the CP4-EPSPS gene in plant cells was under the control of the CMoVb promoter from a modified figwort mosaic virus (FMV) and the 3&rsquo; non-translated region of the rubisco (rbcS, small subunit ribulose bisphosphate carboxylase oxygenase) E9 gene (E9 3&rsquo;) terminator sequence from pea (<i>Pisum sativum</i>).</p>

<p style="text-align: justify;">Additionally, the synthetic CP4 EPSPS gene was fused at the 5&rsquo; end to the region that codes for the chloroplast transit peptide (CPT2) from <i>Arabidopsis thaliana</i>. The CTP2 peptide sequence targets CP4 EPSPS to the chloroplasts where the aromatic amino acid biosynthetic pathway and endogenous EPSPS activity are located.</p>

<p style="text-align: justify;">The aminoglycoside adenyltransferase (<i>aad</i>) gene was derived from <i>E. coli</i> bacterial transposon Tn7 and encodes aminoglycoside adenyltransferase (AAD), which confers resistance to the antibiotics spectinomycin and streptomycin. The <i>aad</i> gene was under the control of its own bacterial promoter and terminator and was not expressed in plant tissues.</p>

<p style="text-align: justify;">The NPTII encoding <i>neo</i> gene was located downstream of the <i>aad</i> gene and was under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the non-translated region of the 3&rsquo; region of the nopaline synthase gene (<i>nos</i> 3&rsquo;) from the pTiT37 plasmid of <i>A. tumefaciens</i> strain T37.</p>

<p style="text-align: justify;">The GOX encoding <i>gox</i> gene from the bacterium <i>Ochromobactrum anthropii</i> strain LBAA (formerly Achromobacter sp.) was fused at the 5&rsquo; end to the sequence coding for the chloroplast transit peptide from <i>A. thaliana</i> EPSPS and the CMoVb promoter. The 3&rsquo; region was derived from the 3&rsquo; non-translated region of the nopaline synthase gene (<i>nos</i> 3&rsquo;). The <i>gox</i> gene was not integrated into the transgenic cotton lines.</p>
