                         SEQUENCE LISTING

<110>  Dow AgroSciences LLC
       Ainley, William M.
       Armstrong, Janna
       Madduri, Krishna
       Merlo, Donald Joseph
       Smith, Kelley A.
       Thompson, Mark
       Webb, Steven R.
       Shen, Liu Yin
 
<120>  Poultry Vaccine, for Necrotic Enteritis, Comprising a Mutated 
       Clostridium perfringens Alpha Toxin Antigen, and Methods of 
       Producing the Vaccine

<130>  DAS-137X PCT

<140>  60/854,741
<141>  2006-10-27

<160>  12    

<170>  PatentIn version 3.3

<210>  1
<211>  1197
<212>  DNA
<213>  Clostridium perfringens


<220>
<221>  misc_feature
<222>  (1)..(1194)
<223>  Protein coding sequence

<220>
<221>  misc_feature
<222>  (1)..(84)
<223>  Encode native secretion signal peptide

<400>  1
atgaaaagaa agatttgtaa ggcacttatt tgtgctacgc tagcaactag cctatgggct     60

ggggcatcaa ctaaagtcta cgcttgggat ggaaagattg atggaacagg aactcatgct    120

atgattgtaa ctcaagggat ttcaatctta gaaaatgatc tgtccaaaaa tgaaccagaa    180

agtgtaagaa aaaacttaga gattttaaag cagaacatgc atgaacttca attaggttct    240

acttatccag attatgataa gaatgcttat gatctatatc aagatcattt ctgggatcct    300

gatacagata ataatttctc aaaggataat agttggtatt tagcttattc tatacctgat    360

acaggggaat cacaaataag aaaattttca gcattagcta gatatgaatg gcaaagagga    420

aactataaac aagctacatt ctatcttgga gaggctatgc actattttgg tgatatagat    480

actccatatc atcctgctaa tgttactgca gttgatagtg caggacatgt taagtttgag    540

acttttgcag aggaaagaaa agagcagtat aaaataaata cagcaggttg caaaactaat    600

gaggcttttt atgctgatat cttaaaaaac aaagatttta atgcatggtc aaaagaatat    660

gcaaggggtt ttgctaaaac aggaaaatca atatactaca gtcatgctag tatgagtcat    720

agttgggatg attgggatta tgcagcaaaa gtaactctag ctaactctca aaaaggaaca    780

gcaggatata tttatagatt cttacacgat gtatcagagg gtaatgatcc atcagttggc    840

aagaatgtaa aagaactagt agcttacata tcaactagtg gtgaaaaaga tgctggaaca    900

gatgactaca tgtattttgg aatcaaaaca aaggatggaa aaactcaaga atgggaaatg    960

gacaacccgg gaaatgactt tatgactgga agtaaagata cttatacttt caagttaaag   1020

gatgaaaatc taaaaattga tgacatacaa aatatgtgga ttagaaaaag aaaatataca   1080

gcattcccag atgcttataa gccagaaaat ataaaggtaa tagcaaatgg aaaagttgta   1140

gtggacaaag atataaatga gtggatttca ggaaattcaa cttataatat aaaataa      1197


<210>  2
<211>  398
<212>  PRT
<213>  Clostridium perfringens


<220>
<221>  MISC_FEATURE
<222>  (1)..(28)
<223>  Secretion signal peptide

<220>
<221>  MISC_FEATURE
<222>  (29)..(398)
<223>  Mature protein

<400>  2

Met Lys Arg Lys Ile Cys Lys Ala Leu Ile Cys Ala Thr Leu Ala Thr 
1               5                   10                  15      


Ser Leu Trp Ala Gly Ala Ser Thr Lys Val Tyr Ala Trp Asp Gly Lys 
            20                  25                  30          


Ile Asp Gly Thr Gly Thr His Ala Met Ile Val Thr Gln Gly Ile Ser 
        35                  40                  45              


Ile Leu Glu Asn Asp Leu Ser Lys Asn Glu Pro Glu Ser Val Arg Lys 
    50                  55                  60                  


Asn Leu Glu Ile Leu Lys Gln Asn Met His Glu Leu Gln Leu Gly Ser 
65                  70                  75                  80  


Thr Tyr Pro Asp Tyr Asp Lys Asn Ala Tyr Asp Leu Tyr Gln Asp His 
                85                  90                  95      


Phe Trp Asp Pro Asp Thr Asp Asn Asn Phe Ser Lys Asp Asn Ser Trp 
            100                 105                 110         


Tyr Leu Ala Tyr Ser Ile Pro Asp Thr Gly Glu Ser Gln Ile Arg Lys 
        115                 120                 125             


Phe Ser Ala Leu Ala Arg Tyr Glu Trp Gln Arg Gly Asn Tyr Lys Gln 
    130                 135                 140                 


Ala Thr Phe Tyr Leu Gly Glu Ala Met His Tyr Phe Gly Asp Ile Asp 
145                 150                 155                 160 


Thr Pro Tyr His Pro Ala Asn Val Thr Ala Val Asp Ser Ala Gly His 
                165                 170                 175     


Val Lys Phe Glu Thr Phe Ala Glu Glu Arg Lys Glu Gln Tyr Lys Ile 
            180                 185                 190         


Asn Thr Ala Gly Cys Lys Thr Asn Glu Ala Phe Tyr Ala Asp Ile Leu 
        195                 200                 205             


Lys Asn Lys Asp Phe Asn Ala Trp Ser Lys Glu Tyr Ala Arg Gly Phe 
    210                 215                 220                 


Ala Lys Thr Gly Lys Ser Ile Tyr Tyr Ser His Ala Ser Met Ser His 
225                 230                 235                 240 


Ser Trp Asp Asp Trp Asp Tyr Ala Ala Lys Val Thr Leu Ala Asn Ser 
                245                 250                 255     


Gln Lys Gly Thr Ala Gly Tyr Ile Tyr Arg Phe Leu His Asp Val Ser 
            260                 265                 270         


Glu Gly Asn Asp Pro Ser Val Gly Lys Asn Val Lys Glu Leu Val Ala 
        275                 280                 285             


Tyr Ile Ser Thr Ser Gly Glu Lys Asp Ala Gly Thr Asp Asp Tyr Met 
    290                 295                 300                 


Tyr Phe Gly Ile Lys Thr Lys Asp Gly Lys Thr Gln Glu Trp Glu Met 
305                 310                 315                 320 


Asp Asn Pro Gly Asn Asp Phe Met Thr Gly Ser Lys Asp Thr Tyr Thr 
                325                 330                 335     


Phe Lys Leu Lys Asp Glu Asn Leu Lys Ile Asp Asp Ile Gln Asn Met 
            340                 345                 350         


Trp Ile Arg Lys Arg Lys Tyr Thr Ala Phe Pro Asp Ala Tyr Lys Pro 
        355                 360                 365             


Glu Asn Ile Lys Val Ile Ala Asn Gly Lys Val Val Val Asp Lys Asp 
    370                 375                 380                 


Ile Asn Glu Trp Ile Ser Gly Asn Ser Thr Tyr Asn Ile Lys 
385                 390                 395             


<210>  3
<211>  1113
<212>  DNA
<213>  Clostridium perfringens


<220>
<221>  misc_feature
<222>  (1)..(1211)
<223>  Mutant Clostridium perfringens phospholipase C isolated from a 
       chicken with ER targeting and retention signals.  This DNA 
       sequence has reworked codon usage optimized for expression in 
       tobacco and rice

<220>
<221>  misc_feature
<222>  (1)..(63)
<223>  Encode 15 kDa Zein Endoplasmic Reticulum targeting signal

<220>
<221>  misc_feature
<222>  (229)..(231)
<223>  Encode Mutation D84L (Aspartic acid to Leucine at amino acid 84 
       of native protein)

<220>
<221>  misc_feature
<222>  (505)..(507)
<223>  Encode Mutation H176G (Histidine to Glycine at amino acid 176 of 
       native protein)

<220>
<221>  misc_feature
<222>  (517)..(519)
<223>  Encode Mutation E180G (Glutamic acid to Glycine at amino acid 180
       of native protein)

<220>
<221>  misc_feature
<222>  (1171)..(1182)
<223>  Encode KDEL Endoplasmic Reticulum retention signal

<220>
<221>  misc_feature
<222>  (1185)..(1211)
<223>  Six-frame translation stops

<400>  3
tgggatggaa agattgatgg cactggcact catgccatga ttgtgactca agggatttcc     60

atcttggaaa atgacctctc caagaatgag ccagaaagtg tgaggaagaa cctggagatt    120

ttgaagcaga acatgcatga actccagctt ggttctacct atccgttgta tgacaagaat    180

gcttatgatc tgtaccaaga tcacttctgg gaccctgata cagataacaa tttcagcaag    240

gacaactcat ggtacctggc ttattcaatt ccagacaccg gggaatccca gataaggaag    300

ttctctgcac ttgcaagata tgaatggcaa cgtggcaact acaaacaagc aacattctat    360

cttggtgagg ccatgcacta ctttggtgat atagacacac cctaccaccc tgccaatgtc    420

actgctgttg acagtgctgg cggtgttaag tttgggacct ttgctgaaga gagaaaagag    480

cagtacaaga taaacacagc aggctgcaaa acaaatgagg ctttctatgc tgacatcttg    540

aagaacaaag atttcaatgc ctggtcaaaa gagtatgcca gaggttttgc caaaactgga    600

aaatccatct attacagcca tgccagcatg agccacagtt gggatgactg ggattatgct    660

gcaaaagtta ctttggcaaa ctctcagaaa ggaactgctg gttacatcta ccgattcctc    720

catgatgtca gtgagggcaa tgatccctct gttggcaaga atgtgaaaga gcttgttgct    780

tacatatcaa cttctggtga gaaagatgct ggaactgatg actacatgta tttcggaatc    840

aagacaaagg atggaaagac ccaagaatgg gaaatggaca accctggaaa tgactttatg    900

acaggttcaa aggacactta cactttcaag ctgaaggatg aaaatctcaa gattgatgac    960

attcaaaaca tgtggattcg taaaaggaaa tacacagcat ttccagatgc ctataagccg   1020

gaaaacatca aggtcatagc aaatggaaag gttgtcgtgg acaaggatat caatgagtgg   1080

atttctggca attcaaccta caatatcaaa tga                                1113


<210>  4
<211>  370
<212>  PRT
<213>  Clostridium perfringens


<220>
<221>  MISC_FEATURE
<222>  (56)..(56)
<223>  D56L mutated residue

<220>
<221>  MISC_FEATURE
<222>  (148)..(148)
<223>  H148G mutated residue

<220>
<221>  MISC_FEATURE
<222>  (152)..(152)
<223>  E152G mutated residue

<400>  4

Trp Asp Gly Lys Ile Asp Gly Thr Gly Thr His Ala Met Ile Val Thr 
1               5                   10                  15      


Gln Gly Ile Ser Ile Leu Glu Asn Asp Leu Ser Lys Asn Glu Pro Glu 
            20                  25                  30          


Ser Val Arg Lys Asn Leu Glu Ile Leu Lys Gln Asn Met His Glu Leu 
        35                  40                  45              


Gln Leu Gly Ser Thr Tyr Pro Leu Tyr Asp Lys Asn Ala Tyr Asp Leu 
    50                  55                  60                  


Tyr Gln Asp His Phe Trp Asp Pro Asp Thr Asp Asn Asn Phe Ser Lys 
65                  70                  75                  80  


Asp Asn Ser Trp Tyr Leu Ala Tyr Ser Ile Pro Asp Thr Gly Glu Ser 
                85                  90                  95      


Gln Ile Arg Lys Phe Ser Ala Leu Ala Arg Tyr Glu Trp Gln Arg Gly 
            100                 105                 110         


Asn Tyr Lys Gln Ala Thr Phe Tyr Leu Gly Glu Ala Met His Tyr Phe 
        115                 120                 125             


Gly Asp Ile Asp Thr Pro Tyr His Pro Ala Asn Val Thr Ala Val Asp 
    130                 135                 140                 


Ser Ala Gly Gly Val Lys Phe Gly Thr Phe Ala Glu Glu Arg Lys Glu 
145                 150                 155                 160 


Gln Tyr Lys Ile Asn Thr Ala Gly Cys Lys Thr Asn Glu Ala Phe Tyr 
                165                 170                 175     


Ala Asp Ile Leu Lys Asn Lys Asp Phe Asn Ala Trp Ser Lys Glu Tyr 
            180                 185                 190         


Ala Arg Gly Phe Ala Lys Thr Gly Lys Ser Ile Tyr Tyr Ser His Ala 
        195                 200                 205             


Ser Met Ser His Ser Trp Asp Asp Trp Asp Tyr Ala Ala Lys Val Thr 
    210                 215                 220                 


Leu Ala Asn Ser Gln Lys Gly Thr Ala Gly Tyr Ile Tyr Arg Phe Leu 
225                 230                 235                 240 


His Asp Val Ser Glu Gly Asn Asp Pro Ser Val Gly Lys Asn Val Lys 
                245                 250                 255     


Glu Leu Val Ala Tyr Ile Ser Thr Ser Gly Glu Lys Asp Ala Gly Thr 
            260                 265                 270         


Asp Asp Tyr Met Tyr Phe Gly Ile Lys Thr Lys Asp Gly Lys Thr Gln 
        275                 280                 285             


Glu Trp Glu Met Asp Asn Pro Gly Asn Asp Phe Met Thr Gly Ser Lys 
    290                 295                 300                 


Asp Thr Tyr Thr Phe Lys Leu Lys Asp Glu Asn Leu Lys Ile Asp Asp 
305                 310                 315                 320 


Ile Gln Asn Met Trp Ile Arg Lys Arg Lys Tyr Thr Ala Phe Pro Asp 
                325                 330                 335     


Ala Tyr Lys Pro Glu Asn Ile Lys Val Ile Ala Asn Gly Lys Val Val 
            340                 345                 350         


Val Asp Lys Asp Ile Asn Glu Trp Ile Ser Gly Asn Ser Thr Tyr Asn 
        355                 360                 365             


Ile Lys 
    370 


<210>  5
<211>  63
<212>  DNA
<213>  Artificial sequence

<220>
<223>  Chemically synthesized plant-optimized DNA sequence for modified 
       15 kDa zein Endoplasmic Reticulum targeting peptide

<400>  5
atggctaaga tggtcattgt gcttgttgtg tgcttggctc tctctgctgc ctcagcttct     60

gcc                                                                   63


<210>  6
<211>  21
<212>  PRT
<213>  Artificial sequence

<220>
<223>  Chemically synthesized amino acid sequence of modified 15 kDa 
       zein Endoplasmic Reticulum targeting peptide encoded by SEQ ID 
       NO:5

<400>  6

Met Ala Lys Met Val Ile Val Leu Val Val Cys Leu Ala Leu Ser Ala 
1               5                   10                  15      


Ala Ser Ala Ser Ala 
            20      


<210>  7
<211>  1185
<212>  DNA
<213>  Artificial sequence

<220>
<223>  Chemically synthesized plant-optimized DNA sequence encoding 
       mature mutated C. perfringens alpha toxin protein with 15 kDA 
       zein endoplasmic reticulum targeting peptide, and KDEL ER 
       retention peptide.


<220>
<221>  misc_feature
<222>  (1)..(63)
<223>  15 kDa zein ER targeting peptide

<220>
<221>  misc_feature
<222>  (64)..(1173)
<223>  mutated mature C. perfringens alpha toxin protein

<220>
<221>  misc_feature
<222>  (1171)..(1182)
<223>  KDEL ER retention peptide

<400>  7
atggctaaga tggtcattgt gcttgttgtg tgcttggctc tctctgctgc ctcagcttct     60

gcctgggatg gaaagattga tggcactggc actcatgcca tgattgtgac tcaagggatt    120

tccatcttgg aaaatgacct ctccaagaat gagccagaaa gtgtgaggaa gaacctggag    180

attttgaagc agaacatgca cgaactccag cttggttcta cctatccgtt gtatgacaag    240

aatgcttatg atctgtacca agatcacttc tgggaccctg atacagataa caatttcagc    300

aaggacaact catggtacct ggcttattca attccagaca ccggggaatc ccagataagg    360

aagttctctg cacttgcaag atatgaatgg caacgtggca actacaaaca agcaacattc    420

tatcttggtg aggccatgca ctactttggt gatatagaca caccctacca ccctgccaat    480

gtcactgctg ttgacagtgc tggcggtgtt aagtttggga cctttgctga agagagaaaa    540

gagcagtaca agataaacac agccggctgc aaaacaaatg aggctttcta tgctgacatc    600

ttgaagaaca aagatttcaa tgcctggtca aaagagtatg ccagaggttt tgccaaaact    660

ggaaaatcca tctattacag ccatgccagc atgagccaca gttgggatga ctgggattat    720

gctgcaaaag ttactttggc aaactctcag aaaggaactg ctggttacat ctaccgattc    780

ctccatgatg tcagtgaggg caatgatccc tctgttggca agaatgtgaa agagcttgtt    840

gcttacatat caacttctgg tgagaaagat gctggaactg atgactacat gtatttcgga    900

atcaagacaa aggatggaaa gacccaagaa tgggaaatgg acaaccctgg aaatgacttt    960

atgaccggtt caaaggacac ttacactttc aagctgaagg atgaaaatct caagattgat   1020

gacattcaaa acatgtggat tcgtaaaagg aaatacacag catttccaga tgcctataag   1080

ccggaaaaca tcaaggtcat agcaaatgga aaggttgtcg tggacaagga tatcaatgag   1140

tggatttctg gcaattcaac ctacaatatc aaagatgagc tttga                   1185


<210>  8
<211>  394
<212>  PRT
<213>  Artificial sequence

<220>
<223>  Chemically synthesized fusion protein of mature mutated C. 
       perfringens alpha toxin protein with 15 kDA zein endoplasmic 
       reticulum targeting peptide, and KDEL ER retention peptide


<220>
<221>  misc_feature
<222>  (1)..(21)
<223>  15 kDa zein ER targeting peptide

<220>
<221>  misc_feature
<222>  (22)..(391)
<223>  mutated mature C. perfringens alpha toxin protein

<220>
<221>  misc_feature
<222>  (391)..(394)
<223>  KDEL ER retention peptide

<400>  8

Met Ala Lys Met Val Ile Val Leu Val Val Cys Leu Ala Leu Ser Ala 
1               5                   10                  15      


Ala Ser Ala Ser Ala Trp Asp Gly Lys Ile Asp Gly Thr Gly Thr His 
            20                  25                  30          


Ala Met Ile Val Thr Gln Gly Ile Ser Ile Leu Glu Asn Asp Leu Ser 
        35                  40                  45              


Lys Asn Glu Pro Glu Ser Val Arg Lys Asn Leu Glu Ile Leu Lys Gln 
    50                  55                  60                  


Asn Met His Glu Leu Gln Leu Gly Ser Thr Tyr Pro Leu Tyr Asp Lys 
65                  70                  75                  80  


Asn Ala Tyr Asp Leu Tyr Gln Asp His Phe Trp Asp Pro Asp Thr Asp 
                85                  90                  95      


Asn Asn Phe Ser Lys Asp Asn Ser Trp Tyr Leu Ala Tyr Ser Ile Pro 
            100                 105                 110         


Asp Thr Gly Glu Ser Gln Ile Arg Lys Phe Ser Ala Leu Ala Arg Tyr 
        115                 120                 125             


Glu Trp Gln Arg Gly Asn Tyr Lys Gln Ala Thr Phe Tyr Leu Gly Glu 
    130                 135                 140                 


Ala Met His Tyr Phe Gly Asp Ile Asp Thr Pro Tyr His Pro Ala Asn 
145                 150                 155                 160 


Val Thr Ala Val Asp Ser Ala Gly Gly Val Lys Phe Gly Thr Phe Ala 
                165                 170                 175     


Glu Glu Arg Lys Glu Gln Tyr Lys Ile Asn Thr Ala Gly Cys Lys Thr 
            180                 185                 190         


Asn Glu Ala Phe Tyr Ala Asp Ile Leu Lys Asn Lys Asp Phe Asn Ala 
        195                 200                 205             


Trp Ser Lys Glu Tyr Ala Arg Gly Phe Ala Lys Thr Gly Lys Ser Ile 
    210                 215                 220                 


Tyr Tyr Ser His Ala Ser Met Ser His Ser Trp Asp Asp Trp Asp Tyr 
225                 230                 235                 240 


Ala Ala Lys Val Thr Leu Ala Asn Ser Gln Lys Gly Thr Ala Gly Tyr 
                245                 250                 255     


Ile Tyr Arg Phe Leu His Asp Val Ser Glu Gly Asn Asp Pro Ser Val 
            260                 265                 270         


Gly Lys Asn Val Lys Glu Leu Val Ala Tyr Ile Ser Thr Ser Gly Glu 
        275                 280                 285             


Lys Asp Ala Gly Thr Asp Asp Tyr Met Tyr Phe Gly Ile Lys Thr Lys 
    290                 295                 300                 


Asp Gly Lys Thr Gln Glu Trp Glu Met Asp Asn Pro Gly Asn Asp Phe 
305                 310                 315                 320 


Met Thr Gly Ser Lys Asp Thr Tyr Thr Phe Lys Leu Lys Asp Glu Asn 
                325                 330                 335     


Leu Lys Ile Asp Asp Ile Gln Asn Met Trp Ile Arg Lys Arg Lys Tyr 
            340                 345                 350         


Thr Ala Phe Pro Asp Ala Tyr Lys Pro Glu Asn Ile Lys Val Ile Ala 
        355                 360                 365             


Asn Gly Lys Val Val Val Asp Lys Asp Ile Asn Glu Trp Ile Ser Gly 
    370                 375                 380                 


Asn Ser Thr Tyr Asn Ile Lys Asp Glu Leu 
385                 390                 


<210>  9
<211>  1269
<212>  DNA
<213>  Artificial sequence

<220>
<223>  Encodes mutant phospholipase C with his-tag, thrombin recognition
       site, S-tag and enterokinase recognition site


<220>
<221>  misc_feature
<222>  (1)..(1269)
<223>  Protein coding sequence

<220>
<221>  misc_feature
<222>  (1)..(159)
<223>  Sequence coding for his-tag, thrombin recognition site, S-tag and
       enterokinase recognition site

<220>
<221>  misc_feature
<222>  (325)..(327)
<223>  Encode Mutation D84L (Aspartic acid to Leucine at amino acid 84 
       of native protein)

<220>
<221>  misc_feature
<222>  (601)..(603)
<223>  Encode Mutation H176G (Histidine to Glycine at amino acid 176 of 
       native protein)

<220>
<221>  misc_feature
<222>  (613)..(615)
<223>  Encode Mutation E180G (Glutamic acid to Glycine at amino acid 180
       of native protein)

<400>  9
atgcatcacc atcaccatca ctccgcgggt ctggtgccac gcggtagtac tgcaattggt     60

atgaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagccc agatctgggt    120

accggtggtg gctccggtga tgacgacgac aagagtccat gggatggaaa gattgatggc    180

actggcactc atgccatgat tgtgactcaa gggatttcca tcttggaaaa tgacctctcc    240

aagaatgagc cagaaagtgt gaggaagaac ctggagattt tgaagcagaa catgcacgaa    300

ctccagcttg gttctaccta tccgttgtat gacaagaatg cttatgatct gtaccaagat    360

cacttctggg accctgatac agataacaat ttcagcaagg acaactcatg gtacctggct    420

tattcaattc cagacaccgg ggaatcccag ataaggaagt tctctgcact tgcaagatat    480

gaatggcaac gtggcaacta caaacaagca acattctatc ttggtgaggc catgcactac    540

tttggtgata tagacacacc ctaccaccct gccaatgtca ctgctgttga cagtgctggc    600

ggtgttaagt ttgggacctt tgctgaagag agaaaagagc agtacaagat aaacacagcc    660

ggctgcaaaa caaatgaggc tttctatgct gacatcttga agaacaaaga tttcaatgcc    720

tggtcaaaag agtatgccag aggttttgcc aaaactggaa aatccatcta ttacagccat    780

gccagcatga gccacagttg ggatgactgg gattatgctg caaaagttac tttggcaaac    840

tctcagaaag gaactgctgg ttacatctac cgattcctcc atgatgtcag tgagggcaat    900

gatccctctg ttggcaagaa tgtgaaagag cttgttgctt acatatcaac ttctggtgag    960

aaagatgctg gaactgatga ctacatgtat ttcggaatca agacaaagga tggaaagacc   1020

caagaatggg aaatggacaa ccctggaaat gactttatga ccggttcaaa ggacacttac   1080

actttcaagc tgaaggatga aaatctcaag attgatgaca ttcaaaacat gtggattcgt   1140

aaaaggaaat acacagcatt tccagatgcc tataagccgg aaaacatcaa ggtcatagca   1200

aatggaaagg ttgtcgtgga caaggatatc aatgagtgga tttctggcaa ttcaacctac   1260

aatatcaaa                                                           1269


<210>  10
<211>  423
<212>  PRT
<213>  Artificial sequence

<220>
<223>  Chemically synthesized protein encoded by SEQ ID NO:9


<220>
<221>  misc_feature
<222>  (1)..(423)
<223>  mutant phospholipase C with his-tag, thrombin recognition site, 
       S-tag and enterokinase recognition site

<220>
<221>  misc_feature
<222>  (1)..(53)
<223>  his-tag, thrombin recognition site, S-tag and enterokinase 
       recognition site

<220>
<221>  misc_feature
<222>  (109)..(109)
<223>  Mutation D84L (Aspartic acid to Leucine at amino acid 84 of 
       native protein)

<220>
<221>  misc_feature
<222>  (201)..(201)
<223>  Mutation H176G (Histidine to Glycine at amino acid 176 of native 
       protein)

<220>
<221>  misc_feature
<222>  (205)..(205)
<223>  Mutation E180G (Glutamic acid to Glycine at amino acid 180 of 
       native protein)

<400>  10

Met His His His His His His Ser Ala Gly Leu Val Pro Arg Gly Ser 
1               5                   10                  15      


Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln 
            20                  25                  30          


His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly Asp Asp 
        35                  40                  45              


Asp Asp Lys Ser Pro Trp Asp Gly Lys Ile Asp Gly Thr Gly Thr His 
    50                  55                  60                  


Ala Met Ile Val Thr Gln Gly Ile Ser Ile Leu Glu Asn Asp Leu Ser 
65                  70                  75                  80  


Lys Asn Glu Pro Glu Ser Val Arg Lys Asn Leu Glu Ile Leu Lys Gln 
                85                  90                  95      


Asn Met His Glu Leu Gln Leu Gly Ser Thr Tyr Pro Leu Tyr Asp Lys 
            100                 105                 110         


Asn Ala Tyr Asp Leu Tyr Gln Asp His Phe Trp Asp Pro Asp Thr Asp 
        115                 120                 125             


Asn Asn Phe Ser Lys Asp Asn Ser Trp Tyr Leu Ala Tyr Ser Ile Pro 
    130                 135                 140                 


Asp Thr Gly Glu Ser Gln Ile Arg Lys Phe Ser Ala Leu Ala Arg Tyr 
145                 150                 155                 160 


Glu Trp Gln Arg Gly Asn Tyr Lys Gln Ala Thr Phe Tyr Leu Gly Glu 
                165                 170                 175     


Ala Met His Tyr Phe Gly Asp Ile Asp Thr Pro Tyr His Pro Ala Asn 
            180                 185                 190         


Val Thr Ala Val Asp Ser Ala Gly Gly Val Lys Phe Gly Thr Phe Ala 
        195                 200                 205             


Glu Glu Arg Lys Glu Gln Tyr Lys Ile Asn Thr Ala Gly Cys Lys Thr 
    210                 215                 220                 


Asn Glu Ala Phe Tyr Ala Asp Ile Leu Lys Asn Lys Asp Phe Asn Ala 
225                 230                 235                 240 


Trp Ser Lys Glu Tyr Ala Arg Gly Phe Ala Lys Thr Gly Lys Ser Ile 
                245                 250                 255     


Tyr Tyr Ser His Ala Ser Met Ser His Ser Trp Asp Asp Trp Asp Tyr 
            260                 265                 270         


Ala Ala Lys Val Thr Leu Ala Asn Ser Gln Lys Gly Thr Ala Gly Tyr 
        275                 280                 285             


Ile Tyr Arg Phe Leu His Asp Val Ser Glu Gly Asn Asp Pro Ser Val 
    290                 295                 300                 


Gly Lys Asn Val Lys Glu Leu Val Ala Tyr Ile Ser Thr Ser Gly Glu 
305                 310                 315                 320 


Lys Asp Ala Gly Thr Asp Asp Tyr Met Tyr Phe Gly Ile Lys Thr Lys 
                325                 330                 335     


Asp Gly Lys Thr Gln Glu Trp Glu Met Asp Asn Pro Gly Asn Asp Phe 
            340                 345                 350         


Met Thr Gly Ser Lys Asp Thr Tyr Thr Phe Lys Leu Lys Asp Glu Asn 
        355                 360                 365             


Leu Lys Ile Asp Asp Ile Gln Asn Met Trp Ile Arg Lys Arg Lys Tyr 
    370                 375                 380                 


Thr Ala Phe Pro Asp Ala Tyr Lys Pro Glu Asn Ile Lys Val Ile Ala 
385                 390                 395                 400 


Asn Gly Lys Val Val Val Asp Lys Asp Ile Asn Glu Trp Ile Ser Gly 
                405                 410                 415     


Asn Ser Thr Tyr Asn Ile Lys 
            420             


<210>  11
<211>  1116
<212>  DNA
<213>  Artificial sequence

<220>
<223>  Encodes mutant phospholipase C with two extra amino acids


<220>
<221>  misc_feature
<222>  (1)..(1116)
<223>  Protein coding sequence

<220>
<221>  misc_feature
<222>  (1)..(6)
<223>  Sequence coding for two amino acids introduced during PCR 
       amplification

<220>
<221>  misc_feature
<222>  (172)..(174)
<223>  Encode Mutation D84L (Aspartic acid to Leucine at amino acid 84 
       of native protein)

<220>
<221>  misc_feature
<222>  (448)..(450)
<223>  Encode Mutation H176G (Histidine to Glycine at amino acid 176 of 
       native protein)

<220>
<221>  misc_feature
<222>  (460)..(462)
<223>  Encode Mutation E180G (Glutamic acid to Glycine at amino acid 180
       of native protein)

<400>  11
atgagttggg atggaaagat tgatggcact ggcactcatg ccatgattgt gactcaaggg     60

atttccatct tggaaaatga cctctccaag aatgagccag aaagtgtgag gaagaacctg    120

gagattttga agcagaacat gcacgaactc cagcttggtt ctacctatcc gttgtatgac    180

aagaatgctt atgatctgta ccaagatcac ttctgggacc ctgatacaga taacaatttc    240

agcaaggaca actcatggta cctggcttat tcaattccag acaccgggga atcccagata    300

aggaagttct ctgcacttgc aagatatgaa tggcaacgtg gcaactacaa acaagcaaca    360

ttctatcttg gtgaggccat gcactacttt ggtgatatag acacacccta ccaccctgcc    420

aatgtcactg ctgttgacag tgctggcggt gttaagtttg ggacctttgc tgaagagaga    480

aaagagcagt acaagataaa cacagccggc tgcaaaacaa atgaggcttt ctatgctgac    540

atcttgaaga acaaagattt caatgcctgg tcaaaagagt atgccagagg ttttgccaaa    600

actggaaaat ccatctatta cagccatgcc agcatgagcc acagttggga tgactgggat    660

tatgctgcaa aagttacttt ggcaaactct cagaaaggaa ctgctggtta catctaccga    720

ttcctccatg atgtcagtga gggcaatgat ccctctgttg gcaagaatgt gaaagagctt    780

gttgcttaca tatcaacttc tggtgagaaa gatgctggaa ctgatgacta catgtatttc    840

ggaatcaaga caaaggatgg aaagacccaa gaatgggaaa tggacaaccc tggaaatgac    900

tttatgaccg gttcaaagga cacttacact ttcaagctga aggatgaaaa tctcaagatt    960

gatgacattc aaaacatgtg gattcgtaaa aggaaataca cagcatttcc agatgcctat   1020

aagccggaaa acatcaaggt catagcaaat ggaaaggttg tcgtggacaa ggatatcaat   1080

gagtggattt ctggcaattc aacctacaat atcaaa                             1116


<210>  12
<211>  372
<212>  PRT
<213>  Artificial sequence

<220>
<223>  Chemically synthesized protein encoded by SEQ ID NO:11


<220>
<221>  MISC_FEATURE
<222>  (1)..(423)
<223>  mutant phospholipase C with two extra amino acids

<220>
<221>  MISC_FEATURE
<222>  (1)..(2)
<223>  two amino acids introduced during PCR amplification

<220>
<221>  MISC_FEATURE
<222>  (58)..(58)
<223>  Mutation D84L (Aspartic acid to Leucine at amino acid 84 of 
       native protein)

<220>
<221>  MISC_FEATURE
<222>  (150)..(150)
<223>  Mutation H176G (Histidine to Glycine at amino acid 176 of native 
       protein)

<220>
<221>  MISC_FEATURE
<222>  (154)..(154)
<223>  Mutation E180G (Glutamic acid to Glycine at amino acid 180 of 
       native protein)

<400>  12

Met Ser Trp Asp Gly Lys Ile Asp Gly Thr Gly Thr His Ala Met Ile 
1               5                   10                  15      


Val Thr Gln Gly Ile Ser Ile Leu Glu Asn Asp Leu Ser Lys Asn Glu 
            20                  25                  30          


Pro Glu Ser Val Arg Lys Asn Leu Glu Ile Leu Lys Gln Asn Met His 
        35                  40                  45              


Glu Leu Gln Leu Gly Ser Thr Tyr Pro Leu Tyr Asp Lys Asn Ala Tyr 
    50                  55                  60                  


Asp Leu Tyr Gln Asp His Phe Trp Asp Pro Asp Thr Asp Asn Asn Phe 
65                  70                  75                  80  


Ser Lys Asp Asn Ser Trp Tyr Leu Ala Tyr Ser Ile Pro Asp Thr Gly 
                85                  90                  95      


Glu Ser Gln Ile Arg Lys Phe Ser Ala Leu Ala Arg Tyr Glu Trp Gln 
            100                 105                 110         


Arg Gly Asn Tyr Lys Gln Ala Thr Phe Tyr Leu Gly Glu Ala Met His 
        115                 120                 125             


Tyr Phe Gly Asp Ile Asp Thr Pro Tyr His Pro Ala Asn Val Thr Ala 
    130                 135                 140                 


Val Asp Ser Ala Gly Gly Val Lys Phe Gly Thr Phe Ala Glu Glu Arg 
145                 150                 155                 160 


Lys Glu Gln Tyr Lys Ile Asn Thr Ala Gly Cys Lys Thr Asn Glu Ala 
                165                 170                 175     


Phe Tyr Ala Asp Ile Leu Lys Asn Lys Asp Phe Asn Ala Trp Ser Lys 
            180                 185                 190         


Glu Tyr Ala Arg Gly Phe Ala Lys Thr Gly Lys Ser Ile Tyr Tyr Ser 
        195                 200                 205             


His Ala Ser Met Ser His Ser Trp Asp Asp Trp Asp Tyr Ala Ala Lys 
    210                 215                 220                 


Val Thr Leu Ala Asn Ser Gln Lys Gly Thr Ala Gly Tyr Ile Tyr Arg 
225                 230                 235                 240 


Phe Leu His Asp Val Ser Glu Gly Asn Asp Pro Ser Val Gly Lys Asn 
                245                 250                 255     


Val Lys Glu Leu Val Ala Tyr Ile Ser Thr Ser Gly Glu Lys Asp Ala 
            260                 265                 270         


Gly Thr Asp Asp Tyr Met Tyr Phe Gly Ile Lys Thr Lys Asp Gly Lys 
        275                 280                 285             


Thr Gln Glu Trp Glu Met Asp Asn Pro Gly Asn Asp Phe Met Thr Gly 
    290                 295                 300                 


Ser Lys Asp Thr Tyr Thr Phe Lys Leu Lys Asp Glu Asn Leu Lys Ile 
305                 310                 315                 320 


Asp Asp Ile Gln Asn Met Trp Ile Arg Lys Arg Lys Tyr Thr Ala Phe 
                325                 330                 335     


Pro Asp Ala Tyr Lys Pro Glu Asn Ile Lys Val Ile Ala Asn Gly Lys 
            340                 345                 350         


Val Val Val Asp Lys Asp Ile Asn Glu Trp Ile Ser Gly Asn Ser Thr 
        355                 360                 365             


Tyr Asn Ile Lys 
    370         


