Patent ID: 8771984

Claim:
A method for utilizing a multicistronic transcription unit to produce a peptide of interest from a host cell, the method comprising: introducing into a plurality of host cells of a eukaryotic cell line a DNA molecule comprising, in 5′-3′ direction: a promoter, a bicistronic transcription unit comprising: a first upstream polynucleotide with the translation start codon selected from the group consisting of GTG and TTG, wherein the first upstream polynucleotide encodes a selectable marker polypeptide with no methionine amino acids in its sequence that provides resistance against the lethal or growth-inhibitory effects of a selection marker, and a second downstream polynucleotide encoding the peptide of interest, wherein the second downstream polynucleotide has the translation initiation sequence ATG, wherein the bicistronic transcription unit has no ATG sequence between the start codon of the first upstream polynucleotide and the start codon of the second downstream polynucleotide, and a transcription termination sequence, wherein the DNA molecule further comprises at least two anti-repressor (STAR) sequences flanking the bicistronic transcription unit, wherein the at least two anti-repressor (STAR) sequences flanking the bicistronic transcription unit are a STAR7 sequence (SEQ ID NO:7) and a STAR67 sequence (SEQ ID NO:66) located upstream of the promoter, and a STAR7 sequence (SEQ ID NO:7) located downstream of the transcription termination sequence; culturing the plurality of host cells to select for expression of the selectable marker polypeptide; selecting at least one host cell producing the peptide of interest; and producing the peptide of interest from the host cell.