The promoter of the GLDT gene from F. trinervia was inserted in the pAUL1 vector (Lyska et al., 2013 (link)). The coding sequence of NAC052 was isolated from cDNA from the Columbia-0 accession of A. thaliana using the primers listed in Supplementary Table S1 at JXB online.
To introduce the (truncated) NAC052 coding sequence (CDS) into the Gateway entry vector pDONR221, the BP Clonase reaction (Gateway ‘BP Clonase II’ enzyme mix, ThermoFisher Scientific) was carried out as described by the manufacturer. The resulting pENTRY221-(truncated)NAC052 was subsequently used for the LR Clonase reaction (Gateway ‘LR Clonase II’ enzyme mix, ThermoFisher Scientific) to transfer the (truncated )NAC052 CDS into pAUL1-GLDTFt (pAUL1-GLDTFt::NAC052 and pAUL1-GLDTFt::5'truncatedNAC052).
To introduce the (truncated) NAC052 coding sequence (CDS) into the Gateway entry vector pDONR221, the BP Clonase reaction (Gateway ‘BP Clonase II’ enzyme mix, ThermoFisher Scientific) was carried out as described by the manufacturer. The resulting pENTRY221-(truncated)NAC052 was subsequently used for the LR Clonase reaction (Gateway ‘LR Clonase II’ enzyme mix, ThermoFisher Scientific) to transfer the (truncated )NAC052 CDS into pAUL1-GLDTFt (pAUL1-GLDTFt::NAC052 and pAUL1-GLDTFt::5'truncatedNAC052).
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