TABLE 2.
PCR primers for hASBT mutant constructs with FLAG insertions
| FLAG constructa |
Insertion sites b |
Position (7TM/9TM)c |
Forward Primer d | Reverse Primer e |
|---|---|---|---|---|
| I | S15-G16 | NT/NT | 5' Xggtgcatcctgtgtgg3' | 5' Zagagcaaactgttgcattg3' |
| II | F56-L57 | IL1/IL1 | 5' Xggccacataaagcgg3' | 5' Ztagaaatttcttgatctccac3' |
| III | D91-I92 | TMD2/EL1 | 5' Xatcctcccgctccaggcc3' | 5' Zgtcaaaggccaccgacagg3' |
| IV | L115-A116 | EL1/IL2 | 5' Xgcctattgggtcgatggc3' | 5' Zcaagatattggaggcagttcc3' |
| V | D120-G121 | EL1/IL2 | 5' Xgatggcgacatggacctg3' | 5' Zgacccaataggccaag3' |
| VI | D155-S156 | IL2/EL2 | 5' Xtctgggagcatcgtaattc3' | 5' Zgtcgacccacattttggtatag3' |
| VII | K185-W186 | EL2/IL3 | 5' Xtggccccaaaaagcaaagatc3' | 5' Ztttgtgattaacaaacattcc3' |
| VIII | W220-IL221 | IL2/EL3 | 5' Xatcattgctcccaaactgtg3' | 5' Zccaggcgctttggtacaatatg3' |
| IX | L250-P251 | TMD6/IL4 | 5' Xccctggtacaggtgccgaac3' | 5' Ztagaccagcaattctagccag3' |
| X | L269-C270 | EL3/TMD8 | 5' Xtgttccaccatcgttcagc3' | 5' Ztagctgcgtgttctgcatccc3' |
| XI | L283-N284 | EL3/EL4 | 5' Xaatgtcgtattcaccttcccg3' | 5' Zgagctcctcaggagtgaaggag3' |
| XII | K319-A320 | CT/CT | 5' Xaaggcagaaattccag3' | 5' Zgttttttccatgacatttc3' |
a
hASBT-FLAG constructs I-XII as illustrated in Fig.1
b
Location of HA epitope insertion in hASBT primary sequence; NT, N-terminus; CT, C-terminus.
c
Localization of the construct relative to the putative 7TM or 9TM topology
d
Forward primer where ‘X’ is the second half of FLAG sequence-gat gac gac aag
e
Reverse primer where ‘Z’ is the first half of the FLAG epitope sequence – gtc ctt gta gtc