Maize with increased production of the amino acid lycine through introduction of the cordapA gene from Corynebacterium glutamicum and regulated by a promoter from globulin 1 (Glb1) gene from Zea mays, rice actin gene intron (rAct1) and Z. mays chloroplast transit peptide sequence for DHDPS and a Glb1 gene 3' terminator from a non-translated region from Z. mays. The DNA used in the microprojectile bombardment also comprised a nptII cassette as a selectable marker which was flanked by loxP recombination sites. The progeny were screened for plants that did not contain the nptII gene cassette, but contained the cordapA gene cassette. The Cre recombinase protein gene, which was also integrated into the maize genome, was segregated away from the cordapA gene through subsequent breeding.

Information on the inserted DNA sequences:
The linear DNA fragment used in the transformation included two expression cassettes, each with a single copy of a gene: cordapA, and  nptII. The 3′ nontranslated region of the globulin 1 gene contains the polyadenylation signal.The nptII gene cassette confers the paromomycin resistance that permits the selection of cells containing the expression cassette. 

Vector information
The plant expression plasmid vector, PV-ZMPQ76, contains three expression cassettes, the cordapA sequence encoding a lysine-insensitive cDHDPS protein, nptII, a selectable marker flanked by loxP recombination sites and amp, a selectable marker encoding ampicillin resistance which, along with nptII was used for selection in E.coli. The 5.9 Kb fragment of this plasmid used for transformation contained only the cordapA and nptII cassettes and in the final product only the cordapA cassette was present. This was accomplished by crossing plants positive for cordapA with a maize line engineered to express the Cre recombinase protein.  The Cre recombinase in the resulting hybrid initiated the excision of the DNA fragment containing the nptII cassette at the loxP sites.

The nptII genetic element was constructed as follows: loxp >> CaMV 35S promoter >> nptII coding sequence >> ble >> NOS terminator >> loxp