A polycistronic expression vector, pKGA-NTF1, was constructed for the cyanobacterium. Within this vector, the spectinomycin/streptomycin resistance gene (aadA) facilitated the selection of transformants when co-transcribed with favorite genes. A natural glnA gene was selected as the platform to introduce the plasmid into a neutral site of the Synechococcus sp. PCC 7002 chromosome. Function of the vector was demonstrated by the insertion of a modified human Trefoil factor 3 gene (NTF1 ) to upstream of the aadA gene and by the analyses of the transformed strains. Antibiotics resistance assays showed that the dicistronic expression cassette conferred high spectinomycin resistance to both the E. coli cells and the Synechococcus cells. PCR analysis and Western-blot analysis were carried out to confirm the integration and expression of the NTF1 gene, respectively. Through simple molecular manipulations, the artificial polycistronic structure described here can be conveniently used to express other favorable genes or operons in cyanobacteria, and to study the cyanobacterial gene expression as well.