Table 1.
Oligonucleotides for QRT-PCR and genetic manipulations.
| Primers for QRT-PCR | |||
|---|---|---|---|
| Name | Purpose | Location | Sequence (5' to 3') |
| MA0326 | spacer #2 | CCTGGTTGAGGTTAGCGTTGA | |
| PCR of spacer #1 |
|||
| MA0327 | outside CRISPR | AATTCGGTGGCCAGTTGTTC | |
| MA0328 | sense strand | CAGGAAGCCACCAAGGAT | |
| PCR of hisS Pcar_1041 |
|||
| MA0329 | antisense strand | TGGGAGCCGAGTTGATTG | |
| MA0441 | sense strand | CAAACTGATTGCCGTTCCTT | |
| PCR of hisZ GSU3307 |
|||
| MA0442 | antisense strand | AGGCCGATGAGTTCTACGC | |
| Primers for construction of hisS transgenic strain MA159 | |||
| Name | Purpose | Sequence (5' to 3') | |
| MA0330 | TGACATCTCGCTGGACCGGG | ||
| PCR on 5' side of hisS GSU1659 |
|||
| MA0331 | CTATGCTAGCACTAGTTTGTAATCATGAACGTACCTACTC CTTTAATTG |
||
| MA0332 | GTACGTTCATGATTACAAACTAGTGCTAGCATAGCAATAC CTGCATTG |
||
| PCR on 3' side of hisS GSU1659 |
|||
| MA0333 | AGTCCATTCCTCCTGTGG | ||
| MA0334 | AAGGGATCTATCATGAGCATATCAGGCATTAAGGG | ||
| PCR of hisS Pcar_1041 |
|||
| MA0335 | GCGCGGCGCGACTAGTTTCCTCGTGTCTTTTCC | ||
| MA0052 | TGCATATGGCTCTAGAATAACTTCGTATAGC | ||
| gentamicin marker |
|||
| MA0053 | TCGATAAGCTTCTAGAATAACTTCGTATAATG | ||
| Oligonucleotides for construction of chimeric CRISPR expression plasmid pMA35 | |||
| Name | Purpose | Sequence (5' to 3') | |
| MA0269 | ACATGTCACTGCCCGCTTTCCAGTC | ||
| PCR of lacI- taclacUV5 promoter |
|||
| MA0270 | GCATGCGTGTGAAATTGTTATCCGC | ||
| MA0429 | AATTCGGTTCATCCCCGCGCATGCGGGGAACACATACAT GAGGGCAAACGCCTTTTGGCCGGCGGCGGTTCATCCCCG CGCATGCGGGGAACACG |
||
| synthetic CRISPR of spacer #1 |
|||
| MA0430 | GATCCGTGTTCCCCGCATGCGCGGGGATGAACCGCCGCC GGCCAAAAGGCGTTTGCCCTCATGTATGTGTTCCCCGCAT GCGCGGGGATGAACCG |
||
| MA36R | sequencing | CGACATCATAACGGTTC | |
Note: Within the sequence of the chimeric CRISPR, a single base pair (underlined) has been duplicated in plasmid pMA35-!, at the exact centre of spacer #1.