Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

Table 2 Oligonucleotide-primers used for A, amplification of DAS1/FLD1/TKL1 from chromosomal DNA; B, evaluation of DAS1/DAS2 transcription induction; C, the analysis of DAS1/FLD1/TKL1 overexpression; D, copy number determination of the reporter genes (via verification of the Zeo^R copy number)

A.	*target*	*orientation*	*sequence 5'-3'*
	DAS1	forward	AAAAGGCGCGCCGAAACGATGGCTAGAATTCCCAAAGCAG
		reverse	TTTGCGGCCGCTTACAACTTGTCATGCTTTGGTTTTC
	FLD1	forward	AACACTAGTATGTCTACCGAAGGTCAA
		reverse	AACGCGGCCGCTTAGTGCATAGTAATCAC
	TKL1	forward	AGAGAATTCGAAACGATGTCTGATCTCTTAGCTATCAACAC
		reverse	AGAGCGGCCGCCTACGCATGAACAGACTCAAAAG
B .	*target*	*orientation*	*sequence 5'-3'*
	ARG4	forward	TGCTGGCTACAGATCTTGCCGACT
		reverse	CTCGGCTTGTCTGACACATTCACCAG
	AOX2	forward	ATACTCATCCGAGGCCAGAGCTTACG
		reverse	ACCGTGAGCAAGACCAGCAGTCAA
	DAS1	forward	CTGAGAAACCAGCTAAAGGTGACGAGT
		reverse	TCTTGTCCCTCACGAGGGTACTCT
	DAS2	forward	CTGAAAAACCAGCCGAGGGTGATC
		reverse	TTCCTCACCTTCTTGAGGATAGTTCTTAACG
C .	*target*	*orientation*	*sequence 5'-3'*
	ARG4	forward	TGCTGGCTACAGATCTTGCCGACT
		reverse	CTCGGCTTGTCTGACACATTCACCAG
	AOX2	forward	ATACTCATCCGAGGCCAGAGCTTAC
		reverse	ACCGTGAGCAAGACCAGCAGTCAA
	DAS1	forward	CTGAGAAACCAGCTAAAGGTGACGAGT
		reverse	TCTTGTCCCTCACGAGGGTACTCT
	FLD1	forward	TGGATTATCTGTCATCCAAGGTGCAGTTTC
		reverse	GTCCGCCCATGCCTTCTTTGAATC
	TKL1	forward	GTCGCTACACATGACTCGATTGGTC
		reverse	CATGAGGTTTGGAAGAGCTCTCAAGTG
D .	*target*	*orientation*	*sequence 5'-3'*
	Zeo^R	forward	GACTCGGTTTCTCCCGTGACT
		reverse	CTGCGGAGATGAACAGGGTAA
	ARG4	forward	TCCTCCGGTGGCAGTTCTT
		reverse	TCCATTGACTCCCGTTTTGAG

ISSN: 1475-2859