Table II. List of oligonucleotides used for amplification and mutation of HYDA1 promoter fragments for generation of reporter gene constructs as well as oligonucleotides used in EMSAs.
ApaI and XhoI restriction sites are underlined. Inserted mutations are indicated in boldface type.
| Name | Template | Oligonucleotide |
|---|---|---|
| Constructs created by PCR | ||
| HYDA1_rev: GCCTCGAGCTTGTCGCGTCTACGATATTAG | ||
| CL33 | gDNA | CL33_for: GGGGCCCGATGATATGGATCGTCGTCTGGTG |
| CL39 | CL33 | CL39_for: GGGGGCCCGCCGAGTGTCCGCTGCATTC |
| CL40 | CL33 | CL40_for: ATGGGCCCGGTCGCGCATAGGCAAGCTC |
| MP01 | CL33 | MP01_for: TGGGGCCCCATAGGCGTACTCGCAAATGCTGT |
| CL42 | CL33 | CL42_for: CTGGGCCCTGCTGTCAGCTTATCTTACAT |
| Constructs created by site-directed mutagenesis | ||
| 1: SDM_UP: GGGGCCCGATGATATGGATCGTCGTCTGGTG | ||
| 2: SDM_RP: GCCTCGAGCTTGTCGCGTCTACGATATTAG | ||
| CL34 | CL33 | A: SDM_pCL34_A: ACAGCATTTGCGATGAGGCCTAT |
| B: SDM_pCL34_B: CATAGGCCTCATCGCAAATGCTGT | ||
| CL35 | CL33 | A: SDM_pCL35_A: TTGAGGCTAGTGCCTTGTCGAGTGTT |
| B: SDM_pCL35_B: AACACTCGACAAGGCACTAGCCTCAA | ||
| BB01 | CL33 | A: SDM_pBB01_A: CTCCTATACTAGGCCGCATTGTTC |
| B: SDM_pBB01_B: GGAACAATGCGGCCTAGTATAGGA | ||
| Constructs created by QC-PCR | ||
| CL44 | CL33 | QC_for: GTTGACCGCCGAGTGTCCGCTGCATTC |
| QC_rev: CACTCGGCGGTCAACCCCGTCAGCAC | ||
| CL46 | BB01 | QC_for: GTTGACCGCCGAGTGTCCGCTGCATTC |
| QC_rev: CACTCGGCGGTCAACCCCGTCAGCAC | ||
| Fragments used in EMSAs | ||
| I | I-pHYDA1for: GACGGGGTGTACCGCCGAGTGTC | |
| I-pHYDA1rev: GACACTCGGCGGTACACCCCGTC | ||
| II | II-pHYDA1for: GACGGGGTTAGACGCCGAGTGTC | |
| II-pHYDA1rev: GACACTCGGCGTCTAACCCCGTC | ||
| III | III-pHYDA1for: GGTCGCGCATAGGCAAGCTCGCAAATGCTGTCAGCT | |
| III-pHYDA1rev: AGCTGACAGCATTTGCGAGCTTGCCTATGCGCGACC | ||
| IV | IV-pHYDA1for:TGAACACACAAACACTCTCGCAGGCACTAGCCTCAA | |
| IV-pHYDA1rev: TTGAGGCTAGTGCCTGCGAGAGTGTTTGTGTGTTCA | ||