Table 1.
Vectors, selective markers, and primers used in this study.
Vector | Selective marker | Primers |
---|---|---|
pDONR 221-AnargB | argB A. nidulans | ArgB1-ggggacaagtttgtacaaaaaagcaggctaagctttatttcgcggtttt ArgB2-ggggaccactttgtacaagaaagctgggtgtcgacctacagccattggg |
pDONR 221-AnpyrG | pyrG A. nidulans | attB1-AnpyrG5F-ggggacaagtttgtacaaaaaagcaggctgcctatgccagcattgagcaatg attB2-AnpyrG3R-ggggaccactttgtacaagaaagctgggtcagtggagcgaaccaattctctc |
pDONR 221-AfpyroA | pyroA A. fumigatus | attBpyroA1-ggggacaagtttgtacaaaaaagcaggctaagttgatcgatgaatgacattc attBpyroA2-ggggaccactttgtacaagaaagctgggtctaaggcacccagcatgagtgc |
pDONR 221-AfpyrG | pyrG A. fumigatus | attB1-AfpyrGF-ggggacaagtttgtacaaaaaagcaggctgcctcaaacaatgctcttcac attB2-AfpyrGR-ggggaccactttgtacaagaaagctgggtattctgtctgagaggaggcac |
pDONR 221-HPH | HPH Escherichia coli | attB1-HPH
F-ggggacaagtttgtacaaaaaagcaggctgaattcgtcgacgttaactgg attB2-HPH R-ggggaccactttgtacaagaaagctgggtgaattcgtcgacgttaactgc |
pDONR 221-BAR | BAR Streptomyces hygroscopicus | attB BAR
1-ggggacaagtttgtacaaaaaagcaggctgcaagcccagatgagaaccgac attB BAR 2-ggggaccactttgtacaagaaagctgggtcggagagagttgaacctggacg |
pDONR A | pyroA A. fumigatus | pyroA
F-GCGCGCACTAGTgcatcccaaggggaaggtagc pyroA FR-ctagcgttaacgcgagagtagggaactgcctaaggcacccagcatgagtgc pDONR221 FFA-cagttgacggcactcatgctgggtgccttaggcagttccctactctcgcgt pDONR221 R1-GCGCGCACTAGTaattgcgttgcgctcactgcc |
pDONR G | pyrG A. fumigatus | pyrG
F-GCGCGCACTAGTatgctcttcaccctcttcgcg pyrG FR-ctagcgttaacgcgagagtagggaactgccctgagaggaggcactgatgcg pDONR221 FFG-ttggcatcacgcatcagtgcctcctctcagggcagttccctactctcgcgt pDONR221 R1-GCGCGCACTAGTaattgcgttgcgctcactgcc |
pDONR NG | pyrG A. nidulans | AnpyrG
F-GCGCGCACTAGTctatgccagcattgagcaatg AnpyrG FR-gagtagggaactgccagtggagcgaaccaattctct pDONR221 FFG-AN-ttggttcgctccactggcagttccctactctcgcgt pDONR221 R1-GCGCGCACTAGTaattgcgttgcgctcactgcc |
pDONR B | argB A. fumigatus | AfargB
F-GCGCGCACTAGTaacgctgacgatgaggctgat AfargB FR-gagtagggaactgccaaacggttcttggctggaagg pDONR221 FF-AfargB-agccaagaaccgtttggcagttccctactctcgcgt pDONR221 R1-GCGCGCACTAGTaattgcgttgcgctcactgcc |
pDONR HPH A pDONR HPH B | HPH Escherichia coli | HPH
F-ACTAGTgaattcgtcgacgttaactgc HPH FR-ctagcgttaacgcgagagtagggaactgcccgtcgacgttaactggttccc |
pDONR BAR | BAR (Bernhard Straubinger 1992) Streptomyces hygroscopicus | BAR
F-GCGCGCACTAGTaagcccagatgagaaccgac BAR FR-gagtagggaactgccggagagagttgaacctggacg pDONR221 FF-BAR-ggttcaactctctccggcagttccctactctcgcgt pDONR221 R1-GCGCGCACTAGTaattgcgttgcgctcactgcc |
pDONR AMA | pyr4 Neurospora crassa | ccd
F-GGGGCATGCcccaaataatgattttatttt ccd R-GGGGGTACCaaataatgattttattttgactga |
pDONR AMA/HPH | HPH Escherichia coli | pDONR HindIII
F-GCGCGCAAGCTTaagcggaagagcgcccaatac pDONR 221 R3-GCGCGCAAGCTTggcggtaatacggttatccacagaatcag |
pDONR P4-P1R-mkkA 5' FR | mpkkA
1-ggggacaactttgtatagaaaagttgaaGCGGCCGCtcaatatatcgtcaactcaga mpkkA 2- ggggactgcttttttgtacaaacttgcggcgagactgttttgcctctc |
|
pDONR P2R-P3-mkkA 3' FR | mpkkA 3-
ggggacagctttcttgtacaaagtggaactctttggtctttgacgctta mpkkA 4- ggggacaactttgtataataaagttgcacttcccggggtctacttcgt |
|
pDONR A-mkkA CR | mkkA C1 -
ggggacaagtttgtacaaaaaagcaggctaagcggccgcgagcccagaccctagatatcg mkkA C2- ggggaccactttgtacaagaaagctgggtctacagcctgcaagcttttcag |
|
pDONR AMA-gliZ | gliZ attB 1-
ggggacaagtttgtacaaaaaagcaggctgcgaccgcagctgattggag gliZ attB 2- ggggaccactttgtacaagaaagctgggtcgattccctttgtgccgcc |
attB sites are underlined. All primers are listed 5' to 3'. Bolded sequences are extension regions for f-PCR. Uppercase regions are SpeI site in all primers, except ccd F (SphI), ccd R (KpnI), pDONR HindIII F (HindIII), pDONR R3 (HindIII), and mpkkA 1 (NotI). All nutritional markers are flanked by their respective promoter and terminator regions. HPH is preceded by the A. nidulans trpC promoter region (http://www.fgsc.net/fgn41/carroll.html), and BAR is flanked by the amdS promoter region from A. nidulans (with increased expression from I9 and I66 mutations) and the glucoamylase terminator from A. niger (Bernhard Straubinger 1992).