TABLE 2.
Oligonucleotide primers
| Primer | Sequence (5′-3′)a | Positionb | Used |
|---|---|---|---|
| P1 | TTTGGATCCGCACGTACCGCACATC (BamHI) | 6310-6335 | A, B, C |
| P2 | AAAAAGCTTCCACTTTTTCATTCTATGTCTCC (HindIII) | 7266-7298c | A |
| P3 | TTTAAGCTTATGTTCTCTGTCGTCTTC (HindIII) | 8150-8179c | A |
| P4 | TACAAGCTTCTCATGATTTCCAACGCCG (HindIII) | 8729-8757c | B |
| P5 | GACTTTTAAAAAAGGATTGATTCTAATGAAG | −90 to −59 | B |
| P6 | GATCTGGAGCCCTAATATAAAAACCTTCTTC | +718 to +749c | B |
| P7 | CAGGAATTCCCGCACATCTGGTATGAAGAG (EcoRI) | 6330-6360 | B |
| P8 | TCCGGATCCTTGGATTAGTTTTTGAATAATTCGGTAC (BamHI) | 6458-6495c | B |
| P9 | CCAGGATCCAGAAGGAGACATAGAATG (BamHI) | 7241-7268 | B |
| P10 | GCCTAAGCGCTGCGTCATCAACGAATGCTG (Cys19 to Ala) | 7308-7338 | E |
| P11 | CAGCATTCGTTGATGACGCAGCGCTTAGGC (Cys19 to Ala) | 7308-7338c | E |
| P12 | CTAAGCGCTTGTGATTCAACGAATGCTGATG (Ser20 to Asp) | 7310-7341 | E |
| P13 | CATCAGCATTCGTTGAATCACAAGCGCTTAG (Ser20 to Asp) | 7310-7341c | E |
| P14 | CTAAGCGCTTGTGATGATACGAATGCTGATG (Ser20, Ser21 to Asp) | 7310-7341 | E |
| P15 | CATCAGCATTCGTATCATCACAAGCGCTTAG (Ser20, Ser21 to Asp) | 7310-7341c | E |
| P16 | AAAAAGTGGATGCTATTTCTGTTC | 7268-7292 | F |
| P17 | TAAGCTCGAGTTCTCTGTCGTCTTC (XhoI) | 8139-8164c | F |
| P18 | AGAAGGAACATATGGAGATCATGGGCGAG (NdeI) | 7455-7484 | D |
| P19 | TCCACTCGAGACGTAGTTCATGCTTTTTTG (XhoI) | 7733-7763c | D |
| P20 | ATTAAGCTTGGATCCTTACCAAACGGTCACGTTGGA (BamHI) | 7120-7156c | C |
a
Restriction sites or mutations are given in parentheses after sequences. Restriction sites or mutated nucleotides in sequences are underlined.
b
Nucleotide numbers correspond to the published sequence (accession no. AB274730).
c
Complementary strands.
d
A, cloning of xyn1 and xyn1-lppX; B, construction of lppX knockout mutant; C, xyn1 probe used in Northern blotting; D, construction of pEN2; E, site-directed mutagenesis of lppX; F, construction of pC19A2.