Genetic elements from PV-ZMIR245:
Two insecticidal protein expression cassettes were inserted into the genome. Bacillus thuringiensis Cry1A.105 expression is under the control of the Cauliflower Mosaic Virus (CaMV) 35S enhanced promoter, which first transcribes wheat (Triticum aestivum) 5' untranslated region of the chlorophyll a/b-binding protein (cab) and a rice actin 1 intron before transcribing cry1A.105. Transcription terminates at the wheat heat shock protein 17.3 terminator. Expression of the B. thuringiensis Cry2Ab2 gene starts at the Figwort Mosaic Virus (FMV) promoter, which transcribes the Zea mays heat shock protein 70 (Hsp70), then the Z. mays transit peptide and the Cry2Ab2 coding sequence, before terminating at the nos terminator.

Note:
- The Cry2Ab2 coding sequence was modified for optimal expression in plants.
- South blot analysis confirmed that single insertions of both cry2Ab2 and cry1A.105, as well as no vector backbone were present and in the parent.
- A deletion removed the duplicated enhancer elements compared to the original CaMV e35S promoter in PV-ZMIR245.
- The selectable marker, nphII, cassette was bred out of the parental line and became not associated with this transformation event.

Genetic elements from PHI8999A:
DNA fragment PHI8999A contains two adjacent plant gene expression cassettes for the cry1F and pat genes. Transcription of the cry1F gene was directed by the promoter and first exon and intron of the maize ubiquitin gene. The 3' terminator sequence used was the 3' polyadenylation signal from ORF25 (A. tumefaciens). Transcriptional regulation of the pat gene from Streptomyces viridochromogenes was via promoter and terminator sequences derived from the 35S transcript of CaMV. The coding sequence of both genes has been optimized to achieve a high level of expression in maize.

Note: The sequences of the complete cry1F and pat genes are identical to those in the original plasmid. The proteins produced in the modified plants are the ones intended, including a leucine residue (replacing a phenylalanine) at position 604 (of 605 amino acids in total) of Cry1F. This modification was introduced to create a specific restriction site for cloning purposes.

Genetic elements from PV-ZMGT32:
The plant expression plasmid vector, PV-ZMGT32 contains two adjacent plant gene expression cassettes each containing a single copy of the cp4 epsps. In the first (5' end) expression cassette, the cp4 epsps gene is under the regulation of the rice actin promoter (P-Ract1) and the rice actin intron (I-Ract1). The second cassette, which is fused to the 3' end of the first, consists of the cp4epsps gene regulated by the enhanced CaMV 35S promoter (e35S)  and intron from the corn heat shock protein 70 (HSP70). Both expression cassettes incorporate the 3'untranslated region of the nopaline synthase gene (nos) for signal polyadenylation.

Note:
- P-e35S is a 0.61Kb long sequence containing the promoter and leader for the cauliflower mosaic virus (CaMV) 35S RNA containing the duplicated enhancer region. This modification was made to enhance the activity of this promoter in plants.
- The parental NK603 contains one insertion site containing a single copy of the linear DNA of PV-ZMGT32 used for transformation. Both cp4 epsps gene cassettes within the single insert which are intact.

Genetic elements from pNOV1300:
In the parental MIR162 maize, a variant of the native B. thuringiensis vegetative insecticidal protein 3Aa (Vip3Aa), named vip3Aa19, which has codon changes that result in a single  M129I amino acid substitution was inserted into the transformation cassette. During the transformation process an additional DNA mutation resulted in a K284Q amino acid substitution. This final form was designated the name Vip3Aa20. Transcription of Vip3Aa20 commences at the Z. mays ubiquitin gene promoter and then transcribes Vip3Aa20 followed by intron 9 of Z. mays phosphoenolpyruvate carboxylase, before terminating at the CaMV 35S terminator.
A second expression cassette, containing the E. coli phosphomannose isomerase gene, was also inserted into the parental genome. The gene is under the control of another ubiquitin promoter and transcription terminates at the Agrobacterium tumefaciens nopaline synthase gene terminator.

Note: Southern blot analyses demonstrated that the T-DNA insert contains: i) single copies of a vip3Aa20 gene and a pmi gene; ii) two copies of the ZmUbiInt promoter; iii) one copy of the NOS terminator; and iv) no backbone sequences from transformation plasmid pNOV1300.

Genetic elements from pDAS1740:
The LMO was generated using the Whiskers mediated transformation method. The aryloxyalkanoate dioxygenase-1 (aad-1)  is under the control of the Z. mays ubiquitin gene promoter and transcription terminates at Z. mays root preferential cationic peroxidase terminator. The aad-1 coding sequence was modified for plant-optimized expression.

Note:
- Southern blot analysis indicated that a single complete copy of the transformation cassette was stably integrated into the host genome at a single locus
- No integration of segments from the vector backbone occured.