Table 2.
Oligonucleotides used in this study
| 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: GAL1p-PRK-CYC1t expression cassette (IMI229) from pUDE046. |
| 12 |
FW HR2-cbbQ2-HR3 |
GACATATCAGCATACATGGCTATGG |
3rd cloning fragment: PGI1p-cbbQ2-TEF2t cassette (IMI229). |
| 13 |
RV HR2-cbbQ2-HR3 |
GGACACGCTTGACAGAATGTCAAAGG |
3rd cloning fragment: PGI1p-cbbQ2-TEF2t 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. |