Table 2 Oligonucleotides used in this study
From: Carbon dioxide fixation by Calvin-Cycle enzymes improves ethanol yield in yeast
Number | Name | Sequence (5′ to 3′) | Purpose |
|---|---|---|---|
Cloning | |||
1 | XbaI_prk_FW2 | TGACATCTAGATGTCACAACAACAAACAATTG | Cloning of PRK into pUDE046. |
2 | RV1 XhoI prk | TGACATCTAGATGTCACAACAACAAACAATTG | Cloning of PRK into pUDE046. |
Primers used for in vivo plasmid assembly | |||
3 | HR-cbbM-FW-65 | TTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACAGCTGGAGCTCAGTTTATCATTATC | Rubisco cbbM cassette for plasmids pUDC075, pUDC099, and pUDC100. |
4 | HR-cbbM-RV-65 | GGAATCTGTGTAGTATGCCTGGAATGTCTGCCGTGCCATAGCCATGTATGCTGATATGTCGGTACCGGCCGCAAATTAAAG | Rubisco cbbM cassette for plasmids pUDC075, pUDC099, and pUDC100 |
5 | linker-cbbO2-pRS416 | ATCACTCTTACCAGGCTAGGACGACCCTACTCATGTATTGAGATCGACGAGATTTCTAGGCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGC | Linker fragment for assembly of plasmid pUDC099. |
6 | linker-cbbM-GroEL | GACATATCAGCATACATGGCTATGGCACGGCAGACATTCCAGGCATACTACACAGATTCCATCACTCTTACCAGGCTAGGACGACCCTACTCATGTATTGAGATCGACGAGATTTCTAGG | Linker fragment for assembly of plasmid pUDC100. |
Primers used for in vivo integration assembly | |||
7 | FW pTDH3- HR-CAN1up | GTTGGATCCAGTTTTTAATCTGTCGTCAATCGAAAGTTTATTTCAGAGTTCTTCAGACTTCTTAACTCCTGTAAAAACAAAAAAAAAAAAAGGCATAGCAAGCTGGAGCTCAGTTTATC | 1st cloning expression cassette linker fragment between CAN1 upstream and PRK expression cassette (IMI229), and CAN1up-linker and KlLEU2 expression cassette (IMI232). |
8 | RV linker-iHR2B | AGATATACTGCAAAGTCCGGAGCAACAGTCGTATAACTCGAGCAGCCCTCTACTTTGTTGTTGCGCTAAGAGAATGGACC | 1st cloning fragment: linker fragment between CAN1up-linker and PRK expression cassette (IMI229). |
9 | RV linker-iHR6 | GCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGTTGCGCTAAGAGAATGGACC | 1st cloning fragment: linker fragment between CAN1up-linker and KlLEU2 expression cassette (IMI232). |
10 | FW pGAL1-prk HR2B | CAACAAAGTAGAGGGCTGCTCGAGTTATACGACTGTTGCTCCGGACTTTGCAGTATATCTGCTGGAGCTCTAGTACGGATT | 2nd cloning fragment: GAL1p-PRK-CYC1t expression cassette (IMI229) from pUDE046. |
11 | RV CYC1t-prk HR2 | GGAATCTGTGTAGTATGCCTGGAATGTCTGCCGTGCCATAGCCATGTATGCTGATATGTCGTACCGGCCGCAAATTAAAG | 2nd cloning fragment: GAL1 p-PRK-CYC1t expression cassette (IMI229) from pUDE046. |
12 | FW HR2-cbbQ2-HR3 | GACATATCAGCATACATGGCTATGG | 3rd cloning fragment: PGI1p-cbbQ2-TEF2 t cassette (IMI229). |
13 | RV HR2-cbbQ2-HR3 | GGACACGCTTGACAGAATGTCAAAGG | 3rd cloning fragment: PGI1p-cbbQ2-TEF2 t cassette (IMI229). |
14 | FW HR3-cbbO2-HR4 | CGTCCGATATGATCTGATTGG | 4th cloning fragment: PGK1p-cbbO2-ADH1t cassette (IMI229). |
15 | RV HR3-cbbO2-HR4 | CCTAGAAATCTCGTCGATCTC | 4th cloning fragment: PGK1p-cbbO2-ADH1t cassette (IMI229). |
16 | FW HR4-GroEL-HR5 | ATCACTCTTACCAGGCTAGG | 5th cloning fragment: TEF1p-groEL-ACT1t cassette (IMI229). |
17 | RV HR4-GroEL-HR5 | CTGGACCTTAATCGTGTGCGCATCCTC | 5th cloning fragment: TEF1p-groEL-ACT1t cassette (IMI229). |
18 | FW HR5-GroES-HR6 | CCGTATAGCTTAATAGCCAGCTTTATC | 6th cloning fragment: TPI1p-groES-PGI1t cassette (IMI229). |
19 | RV HR5-GroES-HR6 | GCTATGACCATGATTACGCCAAGC | 6th cloning fragment: TPI1p-groES-PGI1t cassette (IMI229). |
20 | FW HR6-LEU2-CAN1dwn | CCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCCTGTGAAGATCCCAGCAAAG | 7th (IMI229) or 2nd (IMI232) cloning fragment: KlLEU2 cassette from pUG73. |
21 | RV LEU2 HR-CAN1 | AGCTCATTGATCCCTTAAACTTTCTTTTCGGTGTATGACTTATGAGGGTGAGAATGCGAAATGGCGTGGAAATGTGATCAAAGGTAATAAAACGTCATATATCCGCAGGCTAACCGGAAC | 7th (IMI229) or 2nd (IMI232) cloning fragment: KlLEU2 cassette from pUG73. |
Primers used for verification of the in vivo assembled constructs | |||
22 | m-PCR-HR1-FW | GGCGATTAAGTTGGGTAACG | Diagnostic for assembly of plasmids pUDC075, pUDC099, and pUDC100, and integration in strain IMI229. |
23 | m-PCR-HR1-RV | AACTGAGCTCCAGCTGTACC | Diagnostic for assembly of plasmids pUDC075, pUDC099, pUDC100, and integration in strain IMI229. |
24 | m-PCR-HR2-FW | ACGCGTGTACGCATGTAAC | Diagnostic for assembly of pUDC075, pUDC099, pUDC100, and integration in strain IMI229 |
25 | m-PCR-HR2-RV | GCGCGTGGCTTCCTATAATC | Diagnostic for assembly of pUDC075, pUDC099, pUDC100, and integration in strain IMI229 |
26 | m-PCR-HR3-FW | GTGAATGCTGGTCGCTATAC | Diagnostic for assembly of pUDC075, pUDC099, pUDC100, and integration in strain IMI229. |
27 | m-PCR-HR3-RV | GTAAGCAGCAACACCTTCAG | Diagnostic for assembly of pUDC075, pUDC099, pUDC100, and integration in strain IMI229. |
28 | m-PCR-HR4-FW | ACCTGACCTACAGGAAAGAG | Diagnostic for assembly of pUDC075, pUDC099, pUDC100, and integration in strain IMI229. |
29 | m-PCR-HR4-RV | TGAAGTGGTACGGCGATGC | Diagnostic for assembly of pUDC075, pUDC099, pUDC100, and integration in strain IMI229. |
30 | m-PCR-HR5-FW | ATAGCCACCCAAGGCATTTC | Diagnostic for assembly of pUDC075, pUDC099, pUDC100, and integration in strain IMI229. |
31 | m-PCR-HR5-RV | CCGCACTTTCTCCATGAGG | Diagnostic for assembly of pUDC075, pUDC099, pUDC100, and integration in strain IMI229. |
32 | m-PCR-HR6-FW | CGACGGTTACGGTGTTAAG | Diagnostic for assembly of pUDC075, pUDC099, pUDC100, and integration in strain IMI229. |
33 | m-PCR-HR6-RV | CTTCCGGCTCCTATGTTGTG | Diagnostic for assembly of pUDC075, pUDC099, pUDC100, and integration in strain IMI229. |