Genetic elements from PV-ZMIR245
Two insecticidal protein expression cassettes were inserted into the genome. Bacillus thuringiensis cry1A.105 is transcribed from the Cauliflower mosaic virus 35S enhanced promoter an terminates at the Triticum aestivum (wheat) heat shock protein 17.3 terminator. The transcript is expected to contain a wheat  5' untranslated region of the chlorophyll a/b-binding protein (cab) and an Oryza sativa actin 1 intron before (5') the Cry1A.105 coding sequence. Expression of the B. thuringiensis cry2Ab2 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. Transcription of both gene cassettes is expected to occur at high levels.

Note:
- The cry2Ab2 coding sequence was modified for optimal expression in plants.
- Southern 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 pNOV1300
In the parental MIR162 maize, a variant of the native B. thuringiensis vegetative insecticidal protein 3Aa (vip3Aa), termed vip3Aa20, was inserted into the transformation cassette. 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. The intron enhances expression of the transgene.

A second expression cassette, containing E. coli phosphomannose isomerase (pmi), 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 (nos) terminator.

Note:
- Southern blot analyses demonstrated that the T-DNA insert contains:
(i) single copies of vip3Aa20 and pmi gene;
(ii) two copies of the maize ubiquitin promoter;
(iii) one copy of the nos terminator; and
(iv) no backbone sequences from transformation plasmid pNOV1300.
- Vip3Aa20 is a variant of the native Vip3Aa, which has codon changes that result in M129I (methionine to isoleucine at position 129) and K284Q (lysine to glutamine at position 284) amino acid substitutions.


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 transcriptional regulation of an Oryza sativa actin promoter and a nos terminator. An O. sativa actin intron is also present in the transcript for enhanced expression of the coding sequence. The second cassette consists of another cp4 epsps gene regulated by an CaMV enhanced 35S promoter (containing a duplicated enhancer region) and a nos terminator. Similarly, an intron from the maize heat shock protein 70 (hsp70) was included for enhancing expression of the coding sequence. Both promoters of the gene cassettes are expected to promoter high levels of transcription.

Note:
- The parental NK603 line contained a single, intact insertion containing both cp4 epsps gene cassettes.
- Due to restriction digest prior to particle bombardment, the vector backbone, containing E. coli neomycin phosphotransferase II and origin of replication, were not incorporated into the parental genome.

For more information, kindly refer to the parental line records.