Genetic elements from PV-ZMAP1043
Transcription of  Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase (cp4 epsps) commences from the Cauliflower mosaic virus (CaMV) enhanced 35S promoter and terminates at the A. tumefaciens nopaline synthase (nos) terminator. The transcript contains a Zea mays heat shock protein 70 (hsp70) intron for enhanced gene expression, an Arabidopsis thaliana N-terminal chloroplast transit peptide sequence for chloroplast targeting of the protein and cp4 epsps.  The CaMV enhanced 35S promoter-hsp70 combination promotes gene expression in female and vegetative tissues, but not in male reproductive tissues (pollen microspores and tapetum).

Note:
- Southern blot analyses indicate that a single copy of the T-DNA was inserted at a single site in the parental maize genome and no plasmid vector backbone sequences were detected to have been integrated. DNA sequencing analyses further indicated that the expected T-DNA sequences were integrated.
-The cp4 epsps coding sequence is the codon optimized coding sequence of the aroA gene from Agrobacterium sp. strain CP4 encoding CP4 EPSPS.


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.