DNA insert from MON89034 vector PV-ZMIR245:
Maize line MON89034 expresses two Bt-toxins encoded by Bacillus thuringiensis cry1A.105  and cry2Ab2.

Transcription of cry1A.105 begins at the Cauliflower Mosaic Virus (CaMV) Enhanced 35S promoter and finishes at the wheat (Triticum aestivum) wheat heat shock protein 17.3 terminator. The transcript initially includes (5' to 3'): wheat 5' untranslated leader from the chlorophyll a/b-binding protein, Oryza sativa (rice) actin 1 intron and Cry1A.105. The wheat 5' untranslated leader sequence and the rice intron enhance the expression of cry1A.105.

Transcription of cry2Ab2 commences from the Figwort Mosaic Virus (FMV) 35S promoter and terminates at the Agrobacterium tumefaciens nopaline synthase (nos) terminator. The transcript initially includes (5' to 3'): maize heat shock protein 70 (hsp70) intron, maize transit peptide and first intron from the small subunit of Ribulose-1,5-bisphosphate carboxylase/oxygenase and cry2Ab32. The hsp70 regulates and enhances gene expression, while the transit peptide targets Cry2Ab2 to the chloroplast.

Note:
- The viral promoters are expected to be constitutively active and promote high levels of transcription.
- The coding sequence of cry2Ab2 was codon-optimized for expression within plant systems.
- A second T-DNA insertion (containing CaMV 35S promoter, Escherichia coli neomycin phosphotransferase and A. tumefaciens nos  terminator) was initially inserted into the genome for kanamycin selection during transformation. However, once transformants were regenerated, the selectable marker was bred out of the parental line using convention breeding techniques.
- Southern blot analyses indicated a single copy of the cry1A.105 and the cry2Ab2 cassettes. No backbone plasmid DNA or nptII sequences were detected. PCR and DNA sequence analyses provided the complete DNA sequence of the insert and confirmed the organization of the elements within the insert. Furthermore, sequence analysis indicated that MON 89034 no longer has the duplicated enhancer elements compared to the original e35S promoter in PV-ZMIR245, possibly due to a recombination event that resulted in its deletion.

DNA insert from GA21 vector pDPG434
Transcription of Zea mays modified 5-enolpyruvylshikimate-3-phosphate synthase (mepsps) commences from the Oryza sativa (rice) actin 1 promoter and terminates at the Agrobacterium tumefaciens nopaline synthase terminator. The transcribed elements (from 5’ to 3’) are expected to be as follows: first intron of rice actin 1, a synthetic transit peptide and mepsps. Transcription of mepsps is expected to occur constitutively due to the rice actin promoter. Gene expression is additionally enhanced by the rice actin intron. Post-translation, the optimized transit peptide targets mEPSPS to the chloroplasts.

Note:
- The coding sequence of mepsps was obtained through site-directed mutagenesis to create a modified version of the native enzyme to confer glyphosate tolerance with similar enzymatic function.
- The Rice Actin 1 promoter contains a portion of the first intron of the Actin 1 and thus corresponds to the 5’ end of the gene.
- The optimized transit peptide was derived from maize and sunflower (Helianthus sp.) ribulose 1,5 –bisphosphate carboxylase oxygenase sequences.
- Southern blot analysis indicated that an insert containing three complete tandem copies of the insert and one incomplete copy were inserted into the parental genome. The incomplete copy contains rice actin promoter, the optimized transit peptide and a truncated mepsps sequence without the nos 3’ untranslated region (as uncovered by sequence analysis).
- Sequencing analysis indicated a truncated rice actin promoter in the 5’ end of the insertion event, only containing 148 bp of the promoter region.
- The modified maize expresses only the full-length mEPSPS protein.

For more information, kindly refer to the parental records