Table 3.
Primer sequences.
| Primer name | Primer sequence (5′-3′) |
|---|---|
| Generation of trangenic cDNA lines | |
| attB1 IRP1A FP | CAAGTTTGTACAAAAAAGCAGGCTATGTCCGGCTCCGGCGCCAATC |
| attB2 IRP1A RP | TCCACTTTGTACAAGAAAGCTGGGTCTAATCCAGCATTTTGCGTATC |
| attB1 IRP1B FP | TCAAGTTTGTACAAAAAAGCAGGCTATGTCAGGCGCCAATCCCTTC |
| attB2 IRP1B RP | CCCACTTTGTACAAGAAAGCTGGGTTTAAGAGAGCATTTTGCGAATCATG |
| attB1 yeast Aco1 DelSp FP | CCAAGTTTGTACAAAAAAGCAGGCTGTCTCCAACTTGACTAGAGATTC |
| attB2 yeast Aco1 RP | CACTTTGTACAAGAAAGCTGGGTTTTCTTCTCATCGGCCTTAATTTTATTTAAG |
| attB1 yeast Aco1 WT FP | CCAAGTTTGTACAAAAAAGCAGGCTATGCTGTCTGCACGTTCTG |
| attB1 human IRP1 FP | CAAGTTTGTACAAAAAAGCAGGCTATGAGCAACCCATTCGCAC |
| attB2 human IRP1 RP | CCCACTTTGTACAAGAAAGCTGGGTCTACTTGGCCATCTTGCGGATC |
| attB1 human IRP2 FP | TCAAGTTTGTACAAAAAAGCAGGCTATGGACGCCCCAAAAGCAG |
| attB2 human IRP2 RP | TCCACTTTGTACAAGAAAGCTGGGTCTATGAGAATTTTCGTGCCAC |
| attB1 FG RP | AGCCTGCTTTTTTGTACAAACTTGATACCGGTGCTTGTCATCGTC |
| miniwhite FP | GAGTTCGATGTGTTTATTAAGGGTATCTAGCATTAC |
| miniwhite RP | GTAATGCTAGATACCCTTAATAAACACATCGAACTC |
| attB2 FG FP | ACCCAGCTTTCTTGTACAAAGTGGAGACGTAAGCTAGAGGATCTTTGTG |
| attB1 UASCG RP | AGCCTGCTTTTTTGTACAAACTTGAGATATCGAGCTCTCCCGGGAATTCGGATC |
| attB2 UASCG FP | ACCCAGCTTTCTTGTACAAAGTGGAGATATCGCATGCGGTACCTC |
| Yeast Aco1 WT RevMut FP | ATGCTGTCTGCACGTTCTGCCATCAAGAGACCCATTGTTCGTGGTCTTGCGACAGTCTCCAACTTGACTAGAGATTC |
| Mutagenesis of IRP1A variants | |
| IRP1A C450S Mut FP | ATCACCTCGAGCACGAACACTTC |
| IRP1A C450S Mut RP | GGCGGCAATCACAAAGATC |
| IRP1A R549Q Mut FP | CGGGCAATCAGAATTTCGAG |
| IRP1A R549Q Mut RP | ACAGGACGCCACAGCAAAC |
| IRP1A R554Q Mut FP | ATACTAGGGCCAATTATCTGGCCAG |
| IRP1A R554Q Mut RP | TGGGATGGATCTGACCCTC |
| IRP1A R712Q Mut FP | CTATTTGTCGGAACAGGGTCTAACGCCGCGCGAC |
| IRP1A R712Q Mut RP | CGCGCTGCCGGTGACTTTCGTG |
| IRP1A R793Q Mut FP | TGGCAGCTCACAGGATTGGGCCGCCAAG |
| IRP1A R793Q Mut RP | CTGCCGTAGTCCTTGCCTAC |
| Mutagenesis of IRP1B variants | |
| IRP1B C447S Mut FP | TCACATCCAGCACGAACACATC |
| IRP1B C447S Mut RP | TGGCCGCAATGACAACAGATC |
| IRP1B R546Q Mut FP | AGTTTTGTCCGGAAACCAGAACTTCGAG |
| IRP1B R546Q Mut RP | CCAGCACACACGAGGCCGTTCTTCTCGATG |
| IRP1B R551Q Mut FP | CAACTATCTGGCCAGTCCTCTG |
| IRP1B R551Q Mut RP | GCCCTGGTGTTGGGATGGATCTGACCCTCGAAG |
| IRP1B R709Q Mut FP | TCTTGTCCGAGCAGAACATCACACCCCGTG |
| IRP1B R709Q Mut RP | ATCGGGCAGCAGGCGAGGTTCTAG |
| IRP1B R790Q Mut FP | CGGAAGCTCTCAGGATTGGGCCGCCAAG |
| IRP1B R790Q Mut RP | CTGCCGTAGTCCTTGCCTAC |
| Generation of AGBE CRISPR lines | |
| AGBE LA FP | CGCTGAAGCAGGTGGAATTCTATGGCAACAGTCGGTGGCTTCTG |
| AGBE FRT LA RP | GAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAAACAGCTCTGCTCCACTG |
| AGBE FRT Middle FP | GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGAGTGGCGACCTAATCTGTG |
| AGBE 3xFlag RP | AATATCATGATCCTTGTAGTCTCCGTCGTGGTCCTTATAGTCCATCTAGTCACTGACGCGGGCATAAAC |
| AGBE 3xFlag_3UTR FP | ACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGTAGCTAGTCAGACGCAATTAAC |
| AGBE 3UTR RP | ACTACGATCGCAGGTGTGCAAAAGCAAGCCCAAATCCCTAAAATTC |
| AGBE RA FRT FP | ACTCATCAATGTATCTTAGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC CATTGGCCAATAACAAAG |
| AGBE RA RP | TGCATGGAGATCTTTACTAGCATTTAGTTCTGCTCTCTTTGTTG |
| AGBE 3xMyc RP | TGCTCGAGGTCCTCCTCGGAGATGAGCTTTTGCTCAAGATCCTCTTCAGAAATAAGTTTTTGTTCTCTAGTCACTGACGCGGGCATAAAC |
| AGBE 3xMyc_3UTR FP | TCCGAGGAGGACCTCGAGCAGAAGTTGATCAGCGAGGAAGACTTGTAGCTAGTCAGACGCAATTAAC |
| pCFD3 AGBE Left gRNA FP | GTCGGAGCAGAGCTGTTTCCGAG |
| pCFD3 AGBE Left gRNA RP | AAACTCGGAAACAGCTCTGCTCC |
| pCFD3 AGBE Right gRNA FP | GTCGGATTTGGGCTTGCTTTCAT |
| pCFD3 AGBE Right gRNA RP | AAACATGAAAGCAAGCCCAAATC |
| Generation of IRP1AKO CRISPR line | |
| IRP1A LA FP | TGTCGCCCTTCGCTGAAGCAGGTGGGTACGAGTGGGCGGGACAGAAG |
| IRP1A KO LA RP | GCACTACGATCGCAGGTGTGCATATAGTGGAATAATTTATCATTTTTGTGATTC |
| IRP1A KO RA FP | TATACGAAGTTATAGAAGAGCGCCAAACCAGTCCTGCTAAAAATGCCTAAC |
| IRP1A RA RP | GATTGACGGAAGAGCCTCGAGCGCTGGTGGTGTTGGTGATGTTGCTG |
| pCFD5 IRP1A Left gRNA FP | CGGCCCGGGTTCGATTCCCGGCCGATGCAGAAACATTTGTAAATTATAG GTTTTAGAGCTAGAAATAGCAAG |
| pCFD5 IRP1A Right gRNA RP | ATTTTAACTTGCTATTTCTAGCTCTAAAACATGGCCAAACCAGTCCTGCTTGCACCAGCCGGGAATCGAACCC |
| Generation of IRP1A3F CRISPR line | |
| IRP1A 3 F LA FP | CCCTTCGCTGAAGCAGGTGGTGACCTCGGTTTCGGGGCCCAAG |
| IRP1A 3 F LA RP | GATCCGGCTGGCGAGATGTGGTCGGTGGTCACTGAATCACCGAG |
| IRP1A 3 F Middle FP | CTCGGTGATTCAGTGACCACCGACCACATCTCGCCAGCCGGATC |
| IRP1A 3 F 3xFlag RP | CTTGTAATCGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCATCCAGCATTTTGCGTATCATATAG |
| IRP1A 3 F 3xFlag FP | AGATCATGACATCGATTACAAGGATGACGATGACAAGTAGTTAGTGCGTTCGTTGACTTTTATATTC |
| IRP1A 3 F Middle RP | CGATCGCAGGTGTGCATAGTTAGGCATTTTTAGCAG |
| pCFD3 IRP1A 3 F Left gRNA FP | GTCGGCTGGCGAGATGTGGTCGG |
| pCFD3 IRP1A 3 F Left gRNA RP | AAACCGACCACATCTCGCCAGCC |
| pCFD3 IRP1A 3 F Right gRNA FP | GTCGAGCAGGACTGGTTTGGCCAT |
| pCFD3 IRP1A 3 F Right gRNA RP | AAACATGGCCAAACCAGTCCTGCT |
| Generation of IRP1AC450S.3F and IRP1A3R3Q.3F CRISPR lines | |
| IRP1A CRISPR Mut LA FP | TGGAATTCTTGCATGCTAGCGTGACAACTTTCATGTGCTG |
| IRP1A CRISPR Mut LA RP | TGGATATCAAGTAATAAATTTAGATAATTTTTAAG |
| IRP1A CRISPR Mut Middle FP | TGGATGTGGATCATTAGATCGCTCCGAGAAGAAAATCGATATTATCCGGAAG |
| IRP1A CRISPR Mut Middle RP | GCACTACGATCGCAGGTGTGCATAGTTAGGCATTTTTAGCAGGACTG |
| IRP1A CRISPR Mut RA RP | TGTATGCTATACGAAGTTATAGAAGAGCGCCTGCTGGTGAATCATCGACAAG |
| IRP1A CRISPR Mut RA FP | GATTGACGGAAGAGCCTCGAGCTGCACCTGGTGCTGGTGGTG |
| IRP1A pCFD5 FP | GCGGCCCGGGTTCGATTCCCGGCCGATGCAAATTTATTACTTGATATCCAGTTTTAGAGCTAGAAATAGCAAG |
| IRP1A pCFD5 RP | ATTTTAACTTGCTATTTCTAGCTCTAAAACATGATTCACCAGCAGGCGTTTGCACCAGCCGGGAATCGAAC |
| Generation of IRP1AFCF CRISPR line | |
| IRP1A FCF FRT LA RP | GAAGTTCCTATACTTTCTAGAGAATAGGAACTTCTAGTGGAATAATTTATCATTTTTG |
| IRP1A FCF FRT Middle FP | GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCTAATTTACAAATGTTTCATTTTAAG |
| IRP1A FCF FRT Middle RP | GCACTACGATCGCAGGTGTGCATAGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCCATCGGCATTTCTGCTATC |
| Generation of IRP1AgRNA CRISPR line | |
| IRP1A gRNA FP | GCGGCCCGGGTTCGATTCCCGGCCGATGCCTTTATCCGGATAGCGTTGT GTTTTAGAGCTAGAAATAGCAAG |
| IRP1A gRNA FP | ATTTTAACTTGCTATTTCTAGCTCTAAAACCCCAGCTCGCGGACAGCATCTGCACCAGCCGGGAATCGAACCC |
| Generation of IRP1BKO CRISPR line | |
| IRP1B KO LA FP | TCGCCCTTCGCTGAAGCAGGTCACAGCAGACAGTTAATAC |
| IRP1B KO LA RP | TACGATCGCAGGTGTGCATAAGTAATCGACAGAGCTCGTGCAATC |
| IRP1B KO RA FP | ACGAAGTTATAGAAGAGCAGGGTGGCTTCCGCAAACGAATTG |
| IRP1B KO RA RP | CTTATGCATGGAGATCTTTACTAGCGTAGAGCATCTGCACCAGATTTCG |
| pCFD5 IRP1B Left gRNA FP | CGGCCCGGGTTCGATTCCCGGCCGATGCAGAGCTCTGTCGATTACTGATGTTTTAGAGCTAGAAATAGCAAG |
| pCFD5 IRP1B Right gRNA RP | ATTTTAACTTGCTATTTCTAGCTCTAAAACCCCGTGGCCCCACCGCAACCTGCACCAGCCGGGAATCGAACCC |
| Generation of IRP1B3F CRISPR line | |
| IRP1B 3 F LA FP | TGGAATTCTTGCATGCTAGCACTTCCCCATCGATGAGAATACTC |
| IRP1B 3 F LA RP | GATGTCCTGCAAGAACACATTCTTGCCATTG |
| IRP1B 3 F Middle FP | CAATGGCAAGAATGTGTTCTTGCAGGACATC |
| IRP1B 3 F 3xFlag RP | TCCTTGTAATCGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCAGAGAGCATTTTGCGAATCATGTAGTTG |
| IRP1B 3 F 3xFlag FP | TATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGTAAGCAACTCATCTTATTTTG |
| IRP1B 3 F Middle RP | ACGATCGCAGGTGTGCATAGTGGCCCCACCGCAACCCCTTAAG |
| pCFD3 IRP1B 3 F Left gRNA FP | GTCGATGTGTTCTTGCAGGACATC |
| pCFD3 IRP1B 3 F Left gRNA RP | AAACGATGTCCTGCAAGAACACAT |
| pCFD3 IRP1B 3F Right gRNA FP | GTCGGTTGCGGTGGGGCCACGGG |
| pCFD3 IRP1B 3F Right gRNA RP | AAACCCGTGGCCCCACCGCAACC |
| Generation of S2 cells transfection constructs | |
| attB1 eGFP FP | CAAGTTTGTACAAAAAAGCAGGCTATGGTGAGCAAGGGCGAGGAGCTGTTC |
| attB2 eGFP no stop codon RP | CACTTTGTACAAGAAAGCTGGGTCTTGTACAGCTCGTCCATGCCGAG |
| attB1 AGBE FP | CCAAGTTTGTACAAAAAAGCAGGCTATGGCCGAGGCTAAGGACATC |
| attB2 AGBE no stop codon RP | CACTTTGTACAAGAAAGCTGGGTGTCACTGACGCGGGCATAAAC |
| AGBE Y314S FP | CTGCGTTTCTTGCTATCCAACCTGCGTTG |
| AGBE Y314S RP | CACCTCGTACTCCACTGAGTTGAAGAGACGACTGTC |
| attB1 hGBE1 FP | TCAAGTTTGTACAAAAAAGCAGGCTATGGCGGCTCCGATGACTC |
| attB2 hGBE1 RP | CCCACTTTGTACAAGAAAGCTGGGTTCAATTCGGCAGATCCACATTC |
| attB1 hIRP1 FP | CAAGTTTGTACAAAAAAGCAGGCTATGAGCAACCCATTCGCAC |
| attB2 hIRP1 RP | CCCACTTTGTACAAGAAAGCTGGGTCTACTTGGCCATCTTGCGGATC |
| attB1 Cisd2 FP | ACAAGTTTGTACAAAAAAGCAGGCTATGGAGCCCATATCACATCTG |
| attB2 Cisd2 RP | ACCACTTTGTACAAGAAAGCTGGGTCTTCTTGATGACAATTGGTC |
| pAFW attB1 RP | AGCCTGCTTTTTTGTACAAACTTGATACCGGTGCTTGTCATCGTCATC |
| pAFW attB2 FP | ACCCAGCTTTCTTGTACAAAGTGGGACGTAAGCTAGCAGGATCTTTG |
| pAMW attB1 RP | AGCCTGCTTTTTTGTACAAACTTGATACCGGTGATTCAAGTCCTCTTC |
| pAMW attB2 FP | ACCCAGCTTTCTTGTACAAAGTGGACGTAAGCTAGCAGGATCTTTGTG |
| pAHW attB1 RP | AGCCTGCTTTTTTGTACAAACTTGATACCGGTGTCCGCCATGAGCAG |
| pAHW attB2 FP | ACCCAGCTTTCTTGTACAAAGTGGGACGTAAGCTAGCAGGATCTTTG |
| pAc5 STABLE2 RP | CATGGTGGCGAATTCCACCAC |
| pAc5 STABLE2 FP | GAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT |
| pAc5 3xFlag FP | TGGTGGAATTCGCCACCATGGACTACAAAGACCATGACGGTG |
| pAc5 3xFlag T2A RP | ACCAGGGCCAGGGTTCTCTTCGACATCTCCGCAAGTCAGTAGGCTGCCGCGTCCTTCGCGGCCCCACTTTGTACAAGAAAG |
| pAc5 3xFlag T2A 6xMyc FP | TACTGACTTGCGGAGATGTCGAAGAGAACCCTGGCCCTGGTTCCGATATCTCTAGAGCCACCGAGCAAAAGCTCATTTCTGAAG |
| pAc5 6xMyc attB2 RP | CGGGATTCTCCTCCACGTCACCGCATGTTAGAAGACTTCCTCTGCCCTCAAGCCACTTTGTACAAGAAAG |
| pAc5 6xMyc T2A RP | CGGGATTCTCCTCCACGTCACCGCATGTTAGAAGACTTCCTCTGCCCTCAAGCCACTTTGTACAAGAAAG |
| pAc5 3xHA FP | AGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTGCTAGCTACCCATACGATGTTCCTGAC |
| pAc5 3xHA attB2 RP | TCATGTCTGGATCCCTCGAGCCCACTTTGTACAAGAAAGCTG |
| Real-time PCR | |
| SdhB FP | ACGAGCAGTACCGCAACAT |
| SdhB RP | GGCCTTGCCCTCTTCTC |
| AGBE qPCR FP | GGCCGTTTGAGCATGAGA |
| AGBE qPCR RP | CGCTTTGGTTTATCTTATTCAGC |
| rp49 qPCR FP | TTCCTTGACGTGCCAAAACT |
| rp49 qPCR RP | AATGATCTATAACAAAATCCCCTGA |
A list of primers and their sequences that were used for generating S2 cell constructs, transgenic and CRISPR constructs, PCR-based mutagenesis, as well as for quantitative real-time PCR.