Primers to introduce mutations into BDD-F8 by splicing overlap extension
| Location . | Mutation* . | Primer name . | Sequence . |
|---|---|---|---|
| External | F8-4162f-P1† | GCTGGGATGAGCACACTTTT | |
| External | F8-5302r-P4† | CCACCAAAGAAATGCAGGAC | |
| Internal | 2196A | F8-4709f-2196A-P2‡ | ATTACTGCTTCATCCTACGCTACCAATATGTTTGCCACC |
| Internal | 2196A | F8-4748r-2196A-P3‡ | GGTGGCAAACATATTGGTAGCGTAGGATGAAGCAGTAAT |
| Internal | 2196L | F8-4709f-2196L-P2 | ATTACTGCTTCATCCTACCTTACCAATATGTTTGCCACC |
| Internal | 2196L | F8-4748r-2196L-P3 | GGTGGCAAACATATTGGTAAGGTAGGATGAAGCAGTAAT |
| Internal | 2196K | F8-4709f-2196K-P2 | ATTACTGCTTCATCCTACAAAACCAATATGTTTGCCACC |
| Internal | 2196K | F8-4748r-2196K-P3 | GGTGGCAAACATATTGGTTTTGTAGGATGAAGCAGTAAT |
| Internal | 2199A | F8-4718f-2199A-P2 | TCATCCTACTTTACCAATGCTTTTGCCACCTGGTCTCCT |
| Internal | 2199A | F8-4757r-2199A-P3 | AGGAGACCAGGTGGCAAAAGCATTGGTAAAGTAGGATGA |
| Internal | 2199W | F8-4718f-2199W-P2 | TCATCCTACTTTACCAATTGGTTTGCCACCTGGTCTCCT |
| Internal | 2199W | F8-4757r-2199W-P3 | AGGAGACCAGGTGGCAAACCAATTGGTAAAGTAGGATGA |
| Internal | 2199R | F8-4718f-2199R-P2 | TCATCCTACTTTACCAATCGTTTTGCCACCTGGTCTCCT |
| Internal | 2199R | F8-4757r-2199R-P3 | AGGAGACCAGGTGGCAAAACGATTGGTAAAGTAGGATGA |
| Location . | Mutation* . | Primer name . | Sequence . |
|---|---|---|---|
| External | F8-4162f-P1† | GCTGGGATGAGCACACTTTT | |
| External | F8-5302r-P4† | CCACCAAAGAAATGCAGGAC | |
| Internal | 2196A | F8-4709f-2196A-P2‡ | ATTACTGCTTCATCCTACGCTACCAATATGTTTGCCACC |
| Internal | 2196A | F8-4748r-2196A-P3‡ | GGTGGCAAACATATTGGTAGCGTAGGATGAAGCAGTAAT |
| Internal | 2196L | F8-4709f-2196L-P2 | ATTACTGCTTCATCCTACCTTACCAATATGTTTGCCACC |
| Internal | 2196L | F8-4748r-2196L-P3 | GGTGGCAAACATATTGGTAAGGTAGGATGAAGCAGTAAT |
| Internal | 2196K | F8-4709f-2196K-P2 | ATTACTGCTTCATCCTACAAAACCAATATGTTTGCCACC |
| Internal | 2196K | F8-4748r-2196K-P3 | GGTGGCAAACATATTGGTTTTGTAGGATGAAGCAGTAAT |
| Internal | 2199A | F8-4718f-2199A-P2 | TCATCCTACTTTACCAATGCTTTTGCCACCTGGTCTCCT |
| Internal | 2199A | F8-4757r-2199A-P3 | AGGAGACCAGGTGGCAAAAGCATTGGTAAAGTAGGATGA |
| Internal | 2199W | F8-4718f-2199W-P2 | TCATCCTACTTTACCAATTGGTTTGCCACCTGGTCTCCT |
| Internal | 2199W | F8-4757r-2199W-P3 | AGGAGACCAGGTGGCAAACCAATTGGTAAAGTAGGATGA |
| Internal | 2199R | F8-4718f-2199R-P2 | TCATCCTACTTTACCAATCGTTTTGCCACCTGGTCTCCT |
| Internal | 2199R | F8-4757r-2199R-P3 | AGGAGACCAGGTGGCAAAACGATTGGTAAAGTAGGATGA |
Production of BDD-FVIII muteins F2196A, F2196K, and M2199A was reported previously.53
F8-4162f-P1 and F8-5302r-P4 primers hybridize at the ends of the target sequence and amplify an ApaI-NotI fragment.
The P2 and P3 primers are internal primers that contain the mutation, which are underlined within the primer sequences.