Table 1 The primer pairs used in the PGASO construction
From: PGASO: A synthetic biology tool for engineering a cellulolytic yeast
Primer name | Sequence | |
|---|---|---|
Cassette construction | Â | |
Lac4-KanMx | Kl-PLac4-3’End-F | 5’-TAGGGCCTGTTTGGCCTCCCGCGGGGATC-3’ |
Kl-LAC4_46bpScPGap_Dra3_R | 5’-TAGCACTCAGTGATTATTTACGTATTCTTTGAAATGGCAGTATTGATAATGATAAACTTATACAACATCGAAGAAGAGTC-3’ | |
ScGapDH-EgIII | ScPGapDH-F-BglI | 5’-TAGGCCATGACGGCAGTTTATCATTATCAATACTGCC-3’ |
AFEgIII_ScPGapDH_R | 5’-GTAGAGAATTTCATTTTTTTGTTTGTTTATGTGTGTTTAT -3’ | |
ScPGapDH_AFEgIII_F | 5’-ATAAACACACATAAACAAACAAAAAAATGAAATTCTCTAC-3’ | |
ScTTGap_EgIII_R | 5’-AAGATTTAAAGTAAATTCACGCGGCCGCCTACTTTCTTGCGAGACACG -3’ | |
EgIII_ScTTGap_F | 5’-CGTGTCTCGCAAGAAAGTAGGCGGCCGCGTGAATTTACTTTAAATCTT-3’ | |
ScTTGap_Kl_PGapDH_R | 5’-CTTTTCCATTTGCCTTCGCGCTTGCCTGTACGGTCGTTACCATACTTGGCGGAAAAAATTCATTTG -3’ | |
KlGapDH-CBHI | Kl-PGapDH-F | 5’-AGTATGGTAACGACCGTACAGGCAA-3’ |
AFCBHI_KlPGapDH_R | 5’-GTAGAGAATTTCATTTTTTTTGTGTAATATTCTTTTTTTT-3’ | |
KlPGapDH_AFCBHI_F | 5’-AAAAAAAAGAATATTACACAAAAAAAATGAAATTCTCTAC -3’ | |
ScTTGap_CBHI_R | 5’-AAGATTTAAAGTAAATTCACGCGGCCGCTTACAGGCACTGAGAGTAGT -3’ | |
CBHI_ScTTGap_F | 5’-ACTACTCTCAGTGCCTGTAAGCGGCCGCGTGAATTTACTTTAAATCTT -3’ | |
ScTTGap_Kl_PADHI_R | 5’-TGGTAACGACCGTACAGGCAAGCGCGAAGGCAAATGGAAAAGCTGGTGGCGGAAAAAATTCATTTG-3’ | |
KlADHI-GFP | Kl-PADHI-F | 5’-CCAGCTTTTCCATTTGCCTTCGCGCTTGCC-3’ |
GFPKLADHI-R | 5’-TCCTCGCCCTTGCTCACCATTTTATCTTTTTTTAGTATAGAGT-3’ | |
KLADHIGFP-F | 5’-ACTCTATACTAAAAAAAGATAAAATGGTGAGCAAGGGCGAGGA-3’ | |
ScTTGap_46bpScPADHI_CGA-BglI_R | 5’-TAGGCCGTCGTGGCATGTATGGGTTTGGTTGCCAGAAAAGAGGAAGTCCATATTGTACAC-3’ | |
ScADHI-NpaBGS | ScPADHI_CGA-BglI_F | 5’-TAGGCCACGACGGCGTGTACAATATGGACTTCCTCTTTTC -3’ |
NpaBGS-BglII-F | 5’-ACGAGATCTAAAAAAATGAAATTCTCT-3’ | |
NpaBGS-SmaI-R | 5’-TATCCCGGGTTAGTAAAGTTTGTAAGC-3’ | |
Kl-PLac4 -5’End-R-SfiI | 5’-AGGGCCAAGAAGGCCAGCCGCGGAAATTTAGGAATTTTAAAC-3’ | |
Checking primer | Â | |
Kan | Kan-BglII-F | 5’-AAAAAGATCTGCCACCATGGGTAAGGAAAAGACTC-3’ |
Kan-XbaI-R | 5’-AAAAATCTAGATTAGAAAAACTCATCGAGCAT-3’ | |
EgIII | EgIII-1084 F | 5’-GACATGTGCCAGCAAATCCAATATC-3’ |
ScTTGap_Kl_PGapDH_R | 5’CTTTTCCATTTGCCTTCGCGCTTGCCTGTACGGTCGTTACCATACTTGGCGGAAAAAATTCATTTG-3’ | |
CBHI | Kl-PGapDH-F | 5’-AGTATGGTAACGACCGTACAGGCAA-3’ |
CBHI-218R | 5’-AAGTGTTGCCATCGTAGCAGTTCGT-3’ | |
GFP | GFP-BglII-F | 5’-ACGAGATCTATGGTGAGCAAGGGCGA-3’ |
GFP-SmaI-R | 5’-TATCCCGGGTTACTTGTACAGCTCGTCCA-3’ | |
NpaBGS | NpaBGS-1422-F | 5’-TCCAGGTCCAGTTAATGTTCCATTC-3’ |
NpaBGS-SmaI-R | 5’-TATCCCGGGTTAGTAAAGTTTGTAAGC-3’ | |
Internal primer | Â | |
amplicon L-K | Lac4-Primer1 | 5’-ACACACGTAAACGCGCTCGGT-3’ |
Kan-126R | 5’-TACAATCGATAGATTGTCGCACCTG-3’ | |
Kan-673 F | 5’-CAGGATCTTGCCATCCTATGGAACT-3’ | |
amplicon K-E | EgIII-528R | 5’-TACTTGGAAATGCTCGTGGAATCAA-3’ |
amplicon E-C | EgIII-1084 F | 5’-GACATGTGCCAGCAAATCCAATATC-3’ |
CBHI-218R | 5’-AAGTGTTGCCATCGTAGCAGTTCGT-3’ | |
amplicon C-G | CBHI-585 F | 5’-CGATCTGAAGTTCATCAATGGCCAG-3’ |
GFP-150R | 5’-GTGCAGATGAACTTCAGGGTCAGCT-3’ | |
amplicon G-N | GFP-492 F | 5’-GAACTTCAAGATCCGCCACAACATC-3’ |
NpaBGS-403R | 5’-CACATTCACCAACATAGAATGGATC-3’ | |
amplicon N-L | NpaBGS −1422 F | 5’-TCCAGGTCCAGTTAATGTTCCATTC-3’ |
Lac4-3'-436-R | 5’-ACTCTACATGCGACTTGGAAGGC-3’ | |
UPL system QPCR primer | Â | |
Kan | Kan-UPL#144 F | 5’- AGACTAAACTGGCTGACGGAAT-3’ |
Kan-UPL#144R | 5’- CATCAGGAGTACGGATAAAATGC -3’ | |
EgIII | EgIII-UPL#77 F | 5’- TGGCTCCGACAGAACAATC -3’ |
EgIII-UPL#77R | 5’- GTCTTGTATGCAGGACTGAACG -3’ | |
CBHI | CBHI-UPL#77 F | 5’- ACATCAAGTTCGGACCCATT-3’ |
CBHI-UPL#77R | 5’- GGTAGGTCCGGGAGAGCTT-3’ | |
GFP | GFP-UPL#148 F | 5’- TCTATATCATGGCCGACAAGC-3’ |
GFP-UPL#148 F | 5’- GTTGTGGCGGATCTTGAAGT-3’ | |
NpaBGS | NpaBGS-UPL#150 F | 5’- GAAGCTGTAATGGAAGAAGATGG-3’ |
NpaBGS-UPL#150R | 5’- CTGGGAATGAAAGGAAAATCAT-3’ | |
Alg9 | ALG9-UPL#151 F | 5’- GTGGGTCTATACCACGTCTCATC-3’ |
Actin | ALG9-UPL#151R | 5’- TCCAAATATAACGAATTTAAGCAACTT-3’ |
ACTIN-UPL #9 F | 5’- GCGTAGATTGGAACAACGTG-3’ | |
ACTIN-UPL #9R | 5’- AGAACTACCGGTATTGTGTTGGA-3’ | |