Selection of host cells expressing protein at high levels

A DNA molecule comprising a multicistronic transcription unit, wherein the multicistronic transcription unit comprises:

• a polynucleotide encoding a polypeptide of interest;
• a polynucleotide encoding a first selectable marker polypeptide;
• a polynucleotide encoding a second selectable marker polypeptide;
• an anti-repressor (STAR) element that is STAR67 (SEQ ID NO:66);
• and
• a transcription terminator,
• wherein the polynucleotide encoding the polypeptide of interest has a translation initiation sequence separate from that of the polynucleotide encoding the first selectable marker polypeptide,
• wherein the polynucleotide encoding the polypeptide of interest is upstream from the polynucleotide coding for the first selectable marker polypeptide in the multicistronic transcription unit,
• wherein the polynucleotide encoding the first selectable marker polypeptide is under translational control of an internal ribosome entry site (IRES) that is present downstream of the polynucleotide coding for the polypeptide of interest and upstream of the polynucleotide coding for the first selectable marker polypeptide,
• wherein the polynucleotide encoding the first selectable marker polypeptide comprises a GTG start codon, a TTG start codon, a CTG start codon, an ATT start codon or an ACG start codon, and
• wherein the polynucleotide encoding the second selectable marker polypeptide:
• comprises a translation initiation sequence separate from that of the polypeptide of interest,
• is positioned upstream of the polynucleotide encoding the polypeptide of interest,
• does not comprise an ATG codon in the coding strand between the start codon of the second selectable marker polypeptide and the start codon of the polypeptide of interest, and
• comprises a GTG start codon or a TTG start codon,
• wherein the multicistronic transcription unit further comprises an anti-repressor (STAR) element that is STAR7 (SEQ ID NO:7).