                         SEQUENCE LISTING

<110>  Neemo Inc
       Pyne, Michael
       Moo-Young, Murray
       Chung, Duane
       Chou, Chih-Hsiung
 
<120>  Expansion of the genetic toolkit for metabolic engineering of 
       Clostridium pasteurianum: Chromosomal gene disruption of the 
       endogenous CpaAI restriction enzyme

<130>  2015-01

<160>  38    

<170>  PatentIn version 3.5

<210>  1
<211>  60
<212>  DNA
<213>  Clostridium pasteurianum

<400>  1
ctagcgctat atgcgttgat gcaatttcta tgcactcgta gtagtctgag aaggcatatg       60


<210>  2
<211>  53
<212>  DNA
<213>  Clostridium pasteurianum

<400>  2
atgccttctc agactactac gagtgcatag aaattgcatc aacgcatata gcg              53


<210>  3
<211>  37
<212>  DNA
<213>  Clostridium pasteurianum

<400>  3
tttctactcg aggcgttgat gcaatttcta tgcactc                                37


<210>  4
<211>  24
<212>  DNA
<213>  Clostridium pasteurianum

<400>  4
ggcatcagag cagattgtac tgag                                              24


<210>  5
<211>  56
<212>  DNA
<213>  Clostridium pasteurianum

<400>  5
ggggttaatc atttaacata gataattaaa tagtaaaagg gagtgtcgag atatcc           56


<210>  6
<211>  60
<212>  DNA
<213>  Clostridium pasteurianum

<400>  6
tcgaggatat ctcgacactc ccttttacta tttaattatc tatgttaaat gattaacccc       60


<210>  7
<211>  60
<212>  DNA
<213>  Clostridium pasteurianum

<400>  7
aattgattta gtaatttcta taagcaggtt agctgtaaaa ctagcagtag cacgcatatg       60


<210>  8
<211>  53
<212>  DNA
<213>  Clostridium pasteurianum

<400>  8
atgcgtgcta ctgctagttt tacagctaac ctgcttatag aaattactaa atc              53


<210>  9
<211>  54
<212>  DNA
<213>  Clostridium pasteurianum

<400>  9
ctagcattta gtaatttcta taagcaggtt agctgtaaaa ctagcagtag cacc             54


<210>  10
<211>  54
<212>  DNA
<213>  Clostridium pasteurianum

<400>  10
aattggtgct actgctagtt ttacagctaa cctgcttata gaaattacta aatg             54


<210>  11
<211>  57
<212>  DNA
<213>  Clostridium pasteurianum

<400>  11
ctagcattta gtaatttcta taagcaggtt agctgtaaaa ctagcagtag cacccgc          57


<210>  12
<211>  51
<212>  DNA
<213>  Clostridium pasteurianum

<400>  12
gggtgctact gctagtttta cagctaacct gcttatagaa attactaaat g                51


<210>  13
<211>  30
<212>  DNA
<213>  Clostridium pasteurianum

<400>  13
gggatatgat atacgagtaa ggagatctgg                                        30


<210>  14
<211>  42
<212>  DNA
<213>  Clostridium pasteurianum

<400>  14
agtattaggc cctgacgtcc cacataattc acaacattta gc                          42


<210>  15
<211>  41
<212>  DNA
<213>  Clostridium pasteurianum

<400>  15
aacaggagat ctgctaaatg ttgtgaatta tgtgggacgt c                           41


<210>  16
<211>  34
<212>  DNA
<213>  Clostridium pasteurianum

<400>  16
tactctaggc cctggagacc ccacactacc atcg                                   34


<210>  17
<211>  33
<212>  DNA
<213>  Clostridium pasteurianum

<400>  17
tcgccaagat ctcgatggta gtgtggggtc tcc                                    33


<210>  18
<211>  24
<212>  DNA
<213>  Clostridium pasteurianum

<400>  18
gtgccacctg acgtctaaga aacc                                              24


<210>  19
<211>  25
<212>  DNA
<213>  Clostridium pasteurianum

<400>  19
tgggaaatgg caatgatagc gaaac                                             25


<210>  20
<211>  24
<212>  DNA
<213>  Clostridium pasteurianum

<400>  20
ataggcgtat cacgaggccc tttc                                              24


<210>  21
<211>  29
<212>  DNA
<213>  Clostridium pasteurianum

<400>  21
cgagtaagga gatctggaac gataaaacg                                         29


<210>  22
<211>  51
<212>  DNA
<213>  Clostridium pasteurianum

<400>  22
ctagagtcga cgtcacgcgt ccaaggagat ctccaggcct gcagacatgc a                51


<210>  23
<211>  51
<212>  DNA
<213>  Clostridium pasteurianum

<400>  23
agcttgcatg tctgcaggcc tggagatctc cttggacgcg tgacgtcgac t                51


<210>  24
<211>  30
<212>  DNA
<213>  Clostridium pasteurianum

<400>  24
ggaggtcaat ctatgaaaat gcgattaagc                                        30


<210>  25
<211>  27
<212>  DNA
<213>  Clostridium pasteurianum

<400>  25
ctttcgtttc gttcccatag gttctcc                                           27


<210>  26
<211>  45
<212>  DNA
<213>  Clostridium pasteurianum

<400>  26
gtatttaaag cttataatta tccttaaatt tcttaaaagt gcgcc                       45


<210>  27
<211>  26
<212>  DNA
<213>  Clostridium pasteurianum

<400>  27
ctacttgagg tctaggactt ctatct                                            26


<210>  28
<211>  25
<212>  DNA
<213>  Clostridium pasteurianum

<400>  28
acagataggc cattaaaggt attca                                             25


<210>  29
<211>  24
<212>  DNA
<213>  Clostridium pasteurianum

<400>  29
ccaacgcgtc gccacgtaat aaat                                              24


<210>  30
<211>  24
<212>  DNA
<213>  Clostridium pasteurianum

<400>  30
atgggaacga aacgaaagcg atgc                                              24


<210>  31
<211>  134
<212>  DNA
<213>  Clostridium pasteurianum

<400>  31
tcatgtttga cagcttatca tcgataagct ttaatgcggt agtttatcac agttaaattg       60

ctaacgcagt cagaggagaa aaatagtcta tgttacacac agaaattaag agtaaaattg      120

ataaactatg gaac                                                        134


<210>  32
<211>  107
<212>  DNA
<213>  Clostridium pasteurianum

<400>  32
taggctgcaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca       60

tcgtaaagaa caaattacgc ctacttcacc attcccttta actcagc                    107


<210>  33
<211>  119
<212>  DNA
<213>  Clostridium pasteurianum

<400>  33
tcatgtttga cagcttatca tcgataagct ttaatgcggt agtttatcac agttaaattg       60

ctaacgcagt cagaggagaa aaatagtcta tggaaaatgt taagttacct gaaggatgg       119


<210>  34
<211>  117
<212>  DNA
<213>  Clostridium pasteurianum

<400>  34
taggctgcaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca       60

tcgtaaagaa caaattacgc ttagctatac aattctccat taaaagcttt ttgcatc         117


<210>  35
<211>  1551
<212>  DNA
<213>  Clostridium pasteurianum

<400>  35
atgttacaca cagaaattaa gagtaaaatt gataaactat ggaacaattt ttggtcaggg       60

ggaatatcta atcctttgac agtaatagag cagatatctt atttattgtt tataaagaga      120

ttagatgata tagatactac taatgaaaaa atggccaata ggatgggtaa gccatttaaa      180

tccttattta tggaattaag tgaaaagtta aagaaagaga ataaaataaa ggatgattct      240

aattgggaat tattaaagtg gagtcaattt aagcatttag aagtagaaaa gatgtttgaa      300

gtagtatctc aaaaggtatt tccatttata aaatctatgg gaggagaaga atcgagcttt      360

acagctgaaa tgaaggatgc tgtatttatg atacctaagc catcattact tcaagaatct      420

gtaagactta tagatggtat aaatatggat gatgcagata caaaagggga tttatatgaa      480

tatctgttat caaaacttgc cacatcaggg gtaaatggtc agtttagaac accaagacac      540

ataataagga tgatggttga gcttatggat ccatcagctg aagataagat atgtgaccct      600

gcctgtggta cagcaggttt tttaattaat tcattagaat atatacttga aaaatataca      660

agatcagaaa gtgtatttac agatgaggaa gggaatatac ataacagaat tggagatatg      720

atgagtccaa aagactggga tcattttaag aaggatatgt tctacggttt tgactttgat      780

gcatcaatgg tcagaatagc ttcaatgaac ttaatgcttc atagcattga tgatccaaat      840

gttagacaaa tggattcgtt atcaaaaaga tatgaagaag aaaataaata tactttggtt      900

ttagctaatc ctccttttaa aggcagtata gataaggaag atattaatcc ttctttatta      960

aaaggtgcca aaactactaa gacggaatta ttatttataa aactcataaa cagaatactt     1020

gacttaggtg gaagatgcgc agttatagta ccagatggag tactatttgg gtcaactaag     1080

gcacataagg atataagaaa aaatttagta gaagaaaatc aattagaagg tgtaatatca     1140

atgccttcag gagtatttaa gccctatgca ggagtatcaa cagcagtgct tttatttact     1200

aaaggtggag aaacaaataa agtatggttt tatgatatgg aaagcgatgg atactctcta     1260

gatgataaga gaaataaact tgatactgat ggagatattc cagatataat tgaaaaatgg     1320

agagatataa agaaagataa tactttggaa cctagtaaag aagatagatg gttctgggta     1380

gaaaaagaag aaatagcagg taatgattat gatttatcaa taaataagta taaagagata     1440

gaatatgaag aagttgtgta tgaaaagcca gaggttatat tggagaggat tgaaattttg     1500

gagaaatcta ttttagagaa tatagctgag ttaaagggaa tggtgaagta g              1551


<210>  36
<211>  516
<212>  PRT
<213>  Clostridium pasteurianum

<400>  36

Met Leu His Thr Glu Ile Lys Ser Lys Ile Asp Lys Leu Trp Asn Asn 
1               5                   10                  15      


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


Ser Tyr Leu Leu Phe Ile Lys Arg Leu Asp Asp Ile Asp Thr Thr Asn 
        35                  40                  45              


Glu Lys Met Ala Asn Arg Met Gly Lys Pro Phe Lys Ser Leu Phe Met 
    50                  55                  60                  


Glu Leu Ser Glu Lys Leu Lys Lys Glu Asn Lys Ile Lys Asp Asp Ser 
65                  70                  75                  80  


Asn Trp Glu Leu Leu Lys Trp Ser Gln Phe Lys His Leu Glu Val Glu 
                85                  90                  95      


Lys Met Phe Glu Val Val Ser Gln Lys Val Phe Pro Phe Ile Lys Ser 
            100                 105                 110         


Met Gly Gly Glu Glu Ser Ser Phe Thr Ala Glu Met Lys Asp Ala Val 
        115                 120                 125             


Phe Met Ile Pro Lys Pro Ser Leu Leu Gln Glu Ser Val Arg Leu Ile 
    130                 135                 140                 


Asp Gly Ile Asn Met Asp Asp Ala Asp Thr Lys Gly Asp Leu Tyr Glu 
145                 150                 155                 160 


Tyr Leu Leu Ser Lys Leu Ala Thr Ser Gly Val Asn Gly Gln Phe Arg 
                165                 170                 175     


Thr Pro Arg His Ile Ile Arg Met Met Val Glu Leu Met Asp Pro Ser 
            180                 185                 190         


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


Ile Asn Ser Leu Glu Tyr Ile Leu Glu Lys Tyr Thr Arg Ser Glu Ser 
    210                 215                 220                 


Val Phe Thr Asp Glu Glu Gly Asn Ile His Asn Arg Ile Gly Asp Met 
225                 230                 235                 240 


Met Ser Pro Lys Asp Trp Asp His Phe Lys Lys Asp Met Phe Tyr Gly 
                245                 250                 255     


Phe Asp Phe Asp Ala Ser Met Val Arg Ile Ala Ser Met Asn Leu Met 
            260                 265                 270         


Leu His Ser Ile Asp Asp Pro Asn Val Arg Gln Met Asp Ser Leu Ser 
        275                 280                 285             


Lys Arg Tyr Glu Glu Glu Asn Lys Tyr Thr Leu Val Leu Ala Asn Pro 
    290                 295                 300                 


Pro Phe Lys Gly Ser Ile Asp Lys Glu Asp Ile Asn Pro Ser Leu Leu 
305                 310                 315                 320 


Lys Gly Ala Lys Thr Thr Lys Thr Glu Leu Leu Phe Ile Lys Leu Ile 
                325                 330                 335     


Asn Arg Ile Leu Asp Leu Gly Gly Arg Cys Ala Val Ile Val Pro Asp 
            340                 345                 350         


Gly Val Leu Phe Gly Ser Thr Lys Ala His Lys Asp Ile Arg Lys Asn 
        355                 360                 365             


Leu Val Glu Glu Asn Gln Leu Glu Gly Val Ile Ser Met Pro Ser Gly 
    370                 375                 380                 


Val Phe Lys Pro Tyr Ala Gly Val Ser Thr Ala Val Leu Leu Phe Thr 
385                 390                 395                 400 


Lys Gly Gly Glu Thr Asn Lys Val Trp Phe Tyr Asp Met Glu Ser Asp 
                405                 410                 415     


Gly Tyr Ser Leu Asp Asp Lys Arg Asn Lys Leu Asp Thr Asp Gly Asp 
            420                 425                 430         


Ile Pro Asp Ile Ile Glu Lys Trp Arg Asp Ile Lys Lys Asp Asn Thr 
        435                 440                 445             


Leu Glu Pro Ser Lys Glu Asp Arg Trp Phe Trp Val Glu Lys Glu Glu 
    450                 455                 460                 


Ile Ala Gly Asn Asp Tyr Asp Leu Ser Ile Asn Lys Tyr Lys Glu Ile 
465                 470                 475                 480 


Glu Tyr Glu Glu Val Val Tyr Glu Lys Pro Glu Val Ile Leu Glu Arg 
                485                 490                 495     


Ile Glu Ile Leu Glu Lys Ser Ile Leu Glu Asn Ile Ala Glu Leu Lys 
            500                 505                 510         


Gly Met Val Lys 
        515     


<210>  37
<211>  1239
<212>  DNA
<213>  Clostridium pasteurianum

<400>  37
atggaaaatg ttaagttacc tgaaggatgg agattagaaa aactaaaaga tttattagaa       60

gataaatatg caggggaatg gggtgatgaa gatactgata acagtggaat ttgtgtaata      120

agaactacta attttactaa tagtggaagg ctagatttta gcaatgttgt aactagaaaa      180

atagataaaa aaataattga aaagaaagag ttaaagtatg gagatattat tttagaaaaa      240

tcaggcggta gtgaaaatca accggtaggt agagtagtat actttgataa atatgaagat      300

aatcatattt gcaataattt tacacatgta ttaagaatat atgataaaaa agctgtttca      360

aaatatgtta tgtacttttt actagacata tataaaaaag gaataacaga atattttcaa      420

aataaaacaa caggaataag aaatcttcaa atgaagagat atttagagtt agatatagta      480

ttaccaccat tagaaactca aaagaaaata gttgatgttt tagaaaaggc tgaaaagact      540

ttagaaaaga gaaaagaagc cattaagttg ttagatgaat tagtcaaatc gagatttatc      600

gagatgtttg gtgatccagc tactaattct aagggatgga tagttgaaga gctttcaaag      660

tgtttaatta atattgaaaa tggaaaaagc tttgtatgtg aaaactatag tagaaaaggt      720

gaatatccag ctatattaaa attaagtgct gtaacatatg gaatatataa ttcttctgaa      780

aataaagcat tgacagatga aaatttattt gtagaaagtg ttgaggtgaa aaatggagat      840

ttgttattta caagaaaaaa cactccagaa ttagtaggaa tgagtgcata tgtttatgaa      900

acttctccta atttaatgat gcctgattta atatttaggt ttaatactaa tgaaaaatgt      960

aacaaagttt atttgtggaa attaattaat cacgatttgt ttagagaaaa tattaaggct     1020

ttatctaatg gttctgcgaa gtctatgtct aatatatcaa aacaaagatt aatgagtctg     1080

aatattccaa taccaccact agaactccaa aaccaatttg cagatttcgt caagcaagtc     1140

gacaaattga aatttgaaat gcaaaagagt ttaaaagaac tagaggacaa ctttaattct     1200

ttgatgcaaa aagcttttaa tggagaattg tatagctaa                            1239


<210>  38
<211>  412
<212>  PRT
<213>  Clostridium pasteurianum

<400>  38

Met Glu Asn Val Lys Leu Pro Glu Gly Trp Arg Leu Glu Lys Leu Lys 
1               5                   10                  15      


Asp Leu Leu Glu Asp Lys Tyr Ala Gly Glu Trp Gly Asp Glu Asp Thr 
            20                  25                  30          


Asp Asn Ser Gly Ile Cys Val Ile Arg Thr Thr Asn Phe Thr Asn Ser 
        35                  40                  45              


Gly Arg Leu Asp Phe Ser Asn Val Val Thr Arg Lys Ile Asp Lys Lys 
    50                  55                  60                  


Ile Ile Glu Lys Lys Glu Leu Lys Tyr Gly Asp Ile Ile Leu Glu Lys 
65                  70                  75                  80  


Ser Gly Gly Ser Glu Asn Gln Pro Val Gly Arg Val Val Tyr Phe Asp 
                85                  90                  95      


Lys Tyr Glu Asp Asn His Ile Cys Asn Asn Phe Thr His Val Leu Arg 
            100                 105                 110         


Ile Tyr Asp Lys Lys Ala Val Ser Lys Tyr Val Met Tyr Phe Leu Leu 
        115                 120                 125             


Asp Ile Tyr Lys Lys Gly Ile Thr Glu Tyr Phe Gln Asn Lys Thr Thr 
    130                 135                 140                 


Gly Ile Arg Asn Leu Gln Met Lys Arg Tyr Leu Glu Leu Asp Ile Val 
145                 150                 155                 160 


Leu Pro Pro Leu Glu Thr Gln Lys Lys Ile Val Asp Val Leu Glu Lys 
                165                 170                 175     


Ala Glu Lys Thr Leu Glu Lys Arg Lys Glu Ala Ile Lys Leu Leu Asp 
            180                 185                 190         


Glu Leu Val Lys Ser Arg Phe Ile Glu Met Phe Gly Asp Pro Ala Thr 
        195                 200                 205             


Asn Ser Lys Gly Trp Ile Val Glu Glu Leu Ser Lys Cys Leu Ile Asn 
    210                 215                 220                 


Ile Glu Asn Gly Lys Ser Phe Val Cys Glu Asn Tyr Ser Arg Lys Gly 
225                 230                 235                 240 


Glu Tyr Pro Ala Ile Leu Lys Leu Ser Ala Val Thr Tyr Gly Ile Tyr 
                245                 250                 255     


Asn Ser Ser Glu Asn Lys Ala Leu Thr Asp Glu Asn Leu Phe Val Glu 
            260                 265                 270         


Ser Val Glu Val Lys Asn Gly Asp Leu Leu Phe Thr Arg Lys Asn Thr 
        275                 280                 285             


Pro Glu Leu Val Gly Met Ser Ala Tyr Val Tyr Glu Thr Ser Pro Asn 
    290                 295                 300                 


Leu Met Met Pro Asp Leu Ile Phe Arg Phe Asn Thr Asn Glu Lys Cys 
305                 310                 315                 320 


Asn Lys Val Tyr Leu Trp Lys Leu Ile Asn His Asp Leu Phe Arg Glu 
                325                 330                 335     


Asn Ile Lys Ala Leu Ser Asn Gly Ser Ala Lys Ser Met Ser Asn Ile 
            340                 345                 350         


Ser Lys Gln Arg Leu Met Ser Leu Asn Ile Pro Ile Pro Pro Leu Glu 
        355                 360                 365             


Leu Gln Asn Gln Phe Ala Asp Phe Val Lys Gln Val Asp Lys Leu Lys 
    370                 375                 380                 


Phe Glu Met Gln Lys Ser Leu Lys Glu Leu Glu Asp Asn Phe Asn Ser 
385                 390                 395                 400 


Leu Met Gln Lys Ala Phe Asn Gly Glu Leu Tyr Ser 
                405                 410         


