                         SEQUENCE LISTING

<110>  Oxford University Innovation Ltd
 
<120>  Mutant streptavidin with improved fluorescent brightness and uses
        thereof

<130>  20.130203/01

<150>  GB1616051.7
<151>  2016-09-21

<160>  26    

<170>  PatentIn version 3.5

<210>  1
<211>  183
<212>  PRT
<213>  Streptomyces avidinii

<400>  1

Met Arg Lys Ile Val Val Ala Ala Ile Ala Val Ser Leu Thr Thr Val 
1               5                   10                  15      


Ser Ile Thr Ala Ser Ala Ser Ala Asp Pro Ser Lys Asp Ser Lys Ala 
            20                  25                  30          


Gln Val Ser Ala Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln 
        35                  40                  45              


Leu Gly Ser Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr 
    50                  55                  60                  


Gly Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu 
65                  70                  75                  80  


Thr Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala 
                85                  90                  95      


Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser 
            100                 105                 110         


Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile 
        115                 120                 125             


Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp 
    130                 135                 140                 


Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser 
145                 150                 155                 160 


Ala Ala Ser Ile Asp Ala Ala Lys Lys Ala Gly Val Asn Asn Gly Asn 
                165                 170                 175     


Pro Leu Asp Ala Val Gln Gln 
            180             


<210>  2
<211>  159
<212>  PRT
<213>  Streptomyces avidinii

<400>  2

Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala Gly 
1               5                   10                  15      


Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val Thr 
            20                  25                  30          


Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val Gly 
        35                  40                  45              


Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro 
    50                  55                  60                  


Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys 
65                  70                  75                  80  


Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln Tyr 
                85                  90                  95      


Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser 
            100                 105                 110         


Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp 
        115                 120                 125             


Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala Lys 
    130                 135                 140                 


Lys Ala Gly Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln 
145                 150                 155                 


<210>  3
<211>  127
<212>  PRT
<213>  Streptomyces avidinii

<400>  3

Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr 
1               5                   10                  15      


Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu 
            20                  25                  30          


Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr 
        35                  40                  45              


Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr 
    50                  55                  60                  


Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp 
65                  70                  75                  80  


Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp 
                85                  90                  95      


Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu 
            100                 105                 110         


Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser 
        115                 120                 125         


<210>  4
<211>  127
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Flavidin

<400>  4

Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr 
1               5                   10                  15      


Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu 
            20                  25                  30          


Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr 
        35                  40                  45              


Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr 
    50                  55                  60                  


Val Ala Trp Arg Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp 
65                  70                  75                  80  


Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp 
                85                  90                  95      


Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Arg Ser Thr Leu 
            100                 105                 110         


Val Gly His Asp Thr Phe Thr Arg Val Arg Pro Ser Ala Ala Ser 
        115                 120                 125         


<210>  5
<211>  127
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Two amine version of core streptavidin

<400>  5

Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr 
1               5                   10                  15      


Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu 
            20                  25                  30          


Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr 
        35                  40                  45              


Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr 
    50                  55                  60                  


Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp 
65                  70                  75                  80  


Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp 
                85                  90                  95      


Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Arg Ser Thr Leu 
            100                 105                 110         


Val Gly His Asp Thr Phe Thr Arg Val Arg Pro Ser Ala Ala Ser 
        115                 120                 125         


<210>  6
<211>  127
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Three amine version of core streptavidin

<400>  6

Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr 
1               5                   10                  15      


Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu 
            20                  25                  30          


Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr 
        35                  40                  45              


Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr 
    50                  55                  60                  


Val Ala Trp Arg Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp 
65                  70                  75                  80  


Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp 
                85                  90                  95      


Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Arg Ser Thr Leu 
            100                 105                 110         


Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser 
        115                 120                 125         


<210>  7
<211>  127
<212>  PRT
<213>  Artificial sequence

<220>
<223>  Mutant flavidin

<400>  7

Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Ala Gln Leu Gly Asp Thr 
1               5                   10                  15      


Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu 
            20                  25                  30          


Ala Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr 
        35                  40                  45              


Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr 
    50                  55                  60                  


Val Ala Trp Arg Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp 
65                  70                  75                  80  


Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp 
                85                  90                  95      


Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Arg Ser Thr Leu 
            100                 105                 110         


Val Gly His Asp Thr Phe Thr Arg Val Arg Pro Ser Ala Ala Ser 
        115                 120                 125         


<210>  8
<211>  127
<212>  PRT
<213>  Artificial sequence

<220>
<223>  Mutant flavidin

<400>  8

Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr 
1               5                   10                  15      


Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu 
            20                  25                  30          


Ser Ala Val Gly Asn Ala Glu Gly Asp Tyr Val Leu Thr Gly Arg Tyr 
        35                  40                  45              


Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr 
    50                  55                  60                  


Val Ala Trp Arg Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp 
65                  70                  75                  80  


Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp 
                85                  90                  95      


Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Arg Ser Thr Leu 
            100                 105                 110         


Val Gly His Asp Thr Phe Thr Arg Val Arg Pro Ser Ala Ala Ser 
        115                 120                 125         


<210>  9
<211>  6
<212>  PRT
<213>  Artificial sequence

<220>
<223>  Peptide linker

<400>  9

Gly Ser Gly Ser Gly Gly 
1               5       


<210>  10
<211>  12
<212>  PRT
<213>  Artificial sequence

<220>
<223>  SpyTag

<400>  10

Ala His Ile Val Met Val Asp Ala Tyr Arg Pro Thr 
1               5                   10          


<210>  11
<211>  145
<212>  PRT
<213>  Artificial sequence

<220>
<223>  Mutant Flavidin + SpyTag

<400>  11

Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Ala Gln Leu Gly Asp Thr 
1               5                   10                  15      


Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu 
            20                  25                  30          


Ala Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr 
        35                  40                  45              


Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr 
    50                  55                  60                  


Val Ala Trp Arg Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp 
65                  70                  75                  80  


Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp 
                85                  90                  95      


Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Arg Ser Thr Leu 
            100                 105                 110         


Val Gly His Asp Thr Phe Thr Arg Val Arg Pro Ser Ala Ala Ser Gly 
        115                 120                 125             


Ser Gly Ser Gly Gly Ala His Ile Val Met Val Asp Ala Tyr Arg Pro 
    130                 135                 140                 


Thr 
145 


<210>  12
<211>  145
<212>  PRT
<213>  Artificial sequence

<220>
<223>  Flavidin + SpyTag

<400>  12

Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr 
1               5                   10                  15      


Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu 
            20                  25                  30          


Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr 
        35                  40                  45              


Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr 
    50                  55                  60                  


Val Ala Trp Arg Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp 
65                  70                  75                  80  


Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp 
                85                  90                  95      


Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Arg Ser Thr Leu 
            100                 105                 110         


Val Gly His Asp Thr Phe Thr Arg Val Arg Pro Ser Ala Ala Ser Gly 
        115                 120                 125             


Ser Gly Ser Gly Gly Ala His Ile Val Met Val Asp Ala Tyr Arg Pro 
    130                 135                 140                 


Thr 
145 


<210>  13
<211>  29
<212>  DNA
<213>  Artificial sequence

<220>
<223>  Primer

<400>  13
gctaacgcgt ggcgatccac cctggttgg                                         29


<210>  14
<211>  29
<212>  DNA
<213>  Artificial sequence

<220>
<223>  Primer

<400>  14
gctaacgcgt ggcaatccac cctggttgg                                         29


<210>  15
<211>  29
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  15
gctaacgcgt gggaatccac cctggttgg                                         29


<210>  16
<211>  29
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  16
gctaacgcgt gggcatccac cctggttgg                                         29


<210>  17
<211>  47
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  17
ccgttgcttg gaaaaacaaa taccgtaacg ctcactccgc taccacc                     47


<210>  18
<211>  25
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  18
gatcgttgtc agaagtaagt tggcc                                             25


<210>  19
<211>  24
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  19
gtttttccaa gcaacggtcc aacc                                              24


<210>  20
<211>  25
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  20
ggccaactta cttctgacaa cgatc                                             25


<210>  21
<211>  51
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  21
cagtacgttg gtggtgctga agctaaaatc aacacccagt ggttgttgac c                51


<210>  22
<211>  23
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  22
accaccaacg tactggccag acc                                               23


<210>  23
<211>  46
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  23
tctgggttgg accgttgctt ggcgcaacaa ctaccgtaac gctcac                      46


<210>  24
<211>  23
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  24
ccaagcaacg gtccaaccca gag                                               23


<210>  25
<211>  43
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  25
ggtcacgaca ccttcacccg tgttcgtccg tccgctgctt ccg                         43


<210>  26
<211>  22
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Primer

<400>  26
ggtgaaggtg tcgtgaccaa cc                                                22


