                         SEQUENCE LISTING

<110>  Omniome, Inc.
       Iyidogan, Pinar
       Wallen, Mark C.
       Liu, Ying L.
       Vijayan, Kandaswamy
 
<120>  Polymerases Engineered to Reduce Nucleotide-Independent DNA 
       Binding

<130>  OMNI-019

<150>  62/444,733
<151>  2017-01-10

<150>  62/329,489
<151>  2016-04-29

<150>  15/581,822
<151>  2017-04-28

<150>  62/534,871
<151>  2017-07-20

<160>  13    

<170>  PatentIn version 3.5

<210>  1
<211>  604
<212>  PRT
<213>  Bacillus stearothermophilus


<220>
<221>  MISC_FEATURE
<222>  (5)..(10)
<223>  Polyhistidine tag motif

<220>
<221>  MISC_FEATURE
<222>  (17)..(18)
<223>  Thrombin cleavage site

<220>
<221>  MISC_FEATURE
<222>  (23)..(23)
<223>  Cystine residue

<220>
<221>  MISC_FEATURE
<222>  (27)..(604)
<223>  Bst polymerase polypeptide sequence with replacement of naturally
       occurring cysteine residues

<400>  1

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


Arg Gly Ser His Met Ser Cys Gly Ala Ala Met Ala Phe Thr Leu Ala 
            20                  25                  30          


Asp Arg Val Thr Glu Glu Met Leu Ala Asp Lys Ala Ala Leu Val Val 
        35                  40                  45              


Glu Val Val Glu Glu Asn Tyr His Asp Ala Pro Ile Val Gly Ile Ala 
    50                  55                  60                  


Val Val Asn Glu His Gly Arg Phe Phe Leu Arg Pro Glu Thr Ala Leu 
65                  70                  75                  80  


Ala Asp Pro Gln Phe Val Ala Trp Leu Gly Asp Glu Thr Lys Lys Lys 
                85                  90                  95      


Ser Met Phe Asp Ser Lys Arg Ala Ala Val Ala Leu Lys Trp Lys Gly 
            100                 105                 110         


Ile Glu Leu Ala Gly Val Ser Phe Asp Leu Leu Leu Ala Ala Tyr Leu 
        115                 120                 125             


Leu Asp Pro Ala Gln Gly Val Asp Asp Val Ala Ala Ala Ala Lys Met 
    130                 135                 140                 


Lys Gln Tyr Glu Ala Val Arg Pro Asp Glu Ala Val Tyr Gly Lys Gly 
145                 150                 155                 160 


Ala Lys Arg Ala Val Pro Asp Glu Pro Val Leu Ala Glu His Leu Val 
                165                 170                 175     


Arg Lys Ala Ala Ala Ile Trp Glu Leu Glu Arg Pro Phe Leu Asp Glu 
            180                 185                 190         


Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu Val Glu Leu Glu Gln Pro 
        195                 200                 205             


Leu Ser Ser Ile Leu Ala Glu Met Glu Phe Ala Gly Val Lys Val Asp 
    210                 215                 220                 


Thr Lys Arg Leu Glu Gln Met Gly Lys Glu Leu Ala Glu Gln Leu Gly 
225                 230                 235                 240 


Thr Val Glu Gln Arg Ile Tyr Glu Leu Ala Gly Gln Glu Phe Asn Ile 
                245                 250                 255     


Asn Ser Pro Lys Gln Leu Gly Val Ile Leu Phe Glu Lys Leu Gln Leu 
            260                 265                 270         


Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr Ser Thr Ser Ala Asp Val 
        275                 280                 285             


Leu Glu Lys Leu Ala Pro Tyr His Glu Ile Val Glu Asn Ile Leu His 
    290                 295                 300                 


Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr Tyr Ile Glu Gly Leu Leu 
305                 310                 315                 320 


Lys Val Val Arg Pro Ala Thr Lys Lys Val His Thr Ile Phe Asn Gln 
                325                 330                 335     


Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Thr Glu Pro Asn Leu Gln 
            340                 345                 350         


Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Lys Ile Arg Gln Ala Phe 
        355                 360                 365             


Val Pro Ser Glu Ser Asp Trp Leu Ile Phe Ala Ala Asp Tyr Ser Gln 
    370                 375                 380                 


Ile Glu Leu Arg Val Leu Ala His Ile Ala Glu Asp Asp Asn Leu Met 
385                 390                 395                 400 


Glu Ala Phe Arg Arg Asp Leu Asp Ile His Thr Lys Thr Ala Met Asp 
                405                 410                 415     


Ile Phe Gln Val Ser Glu Asp Glu Val Thr Pro Asn Met Arg Arg Gln 
            420                 425                 430         


Ala Lys Ala Val Asn Tyr Gly Ile Val Tyr Gly Ile Ser Asp Tyr Gly 
        435                 440                 445             


Leu Ala Gln Asn Leu Asn Ile Ser Arg Lys Glu Ala Ala Glu Phe Ile 
    450                 455                 460                 


Glu Arg Tyr Phe Glu Ser Phe Pro Gly Val Lys Arg Tyr Met Glu Asn 
465                 470                 475                 480 


Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Val Thr Thr Leu Leu His 
                485                 490                 495     


Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Arg Asn Phe Asn Val Arg 
            500                 505                 510         


Ser Phe Ala Glu Arg Met Ala Met Asn Thr Pro Ile Gln Gly Ser Ala 
        515                 520                 525             


Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Leu Asn Ala Arg Leu Lys 
    530                 535                 540                 


Glu Glu Arg Leu Gln Ala His Leu Leu Leu Gln Val His Asp Glu Leu 
545                 550                 555                 560 


Ile Leu Glu Ala Pro Lys Glu Glu Met Glu Arg Leu Ala Arg Leu Val 
                565                 570                 575     


Pro Glu Val Met Glu Gln Ala Val Thr Leu Arg Val Pro Leu Lys Val 
            580                 585                 590         


Asp Tyr His Tyr Gly Ser Thr Trp Tyr Asp Ala Lys 
        595                 600                 


<210>  2
<211>  587
<212>  PRT
<213>  Bacillus stearothermophilus


<220>
<221>  MISC_FEATURE
<222>  (1)..(587)
<223>  Product resulting from thrombin cleavage of the protein sequence 
       identified as SEQ ID NO:1

<400>  2

Gly Ser His Met Ser Cys Gly Ala Ala Met Ala Phe Thr Leu Ala Asp 
1               5                   10                  15      


Arg Val Thr Glu Glu Met Leu Ala Asp Lys Ala Ala Leu Val Val Glu 
            20                  25                  30          


Val Val Glu Glu Asn Tyr His Asp Ala Pro Ile Val Gly Ile Ala Val 
        35                  40                  45              


Val Asn Glu His Gly Arg Phe Phe Leu Arg Pro Glu Thr Ala Leu Ala 
    50                  55                  60                  


Asp Pro Gln Phe Val Ala Trp Leu Gly Asp Glu Thr Lys Lys Lys Ser 
65                  70                  75                  80  


Met Phe Asp Ser Lys Arg Ala Ala Val Ala Leu Lys Trp Lys Gly Ile 
                85                  90                  95      


Glu Leu Ala Gly Val Ser Phe Asp Leu Leu Leu Ala Ala Tyr Leu Leu 
            100                 105                 110         


Asp Pro Ala Gln Gly Val Asp Asp Val Ala Ala Ala Ala Lys Met Lys 
        115                 120                 125             


Gln Tyr Glu Ala Val Arg Pro Asp Glu Ala Val Tyr Gly Lys Gly Ala 
    130                 135                 140                 


Lys Arg Ala Val Pro Asp Glu Pro Val Leu Ala Glu His Leu Val Arg 
145                 150                 155                 160 


Lys Ala Ala Ala Ile Trp Glu Leu Glu Arg Pro Phe Leu Asp Glu Leu 
                165                 170                 175     


Arg Arg Asn Glu Gln Asp Arg Leu Leu Val Glu Leu Glu Gln Pro Leu 
            180                 185                 190         


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


Lys Arg Leu Glu Gln Met Gly Lys Glu Leu Ala Glu Gln Leu Gly Thr 
    210                 215                 220                 


Val Glu Gln Arg Ile Tyr Glu Leu Ala Gly Gln Glu Phe Asn Ile Asn 
225                 230                 235                 240 


Ser Pro Lys Gln Leu Gly Val Ile Leu Phe Glu Lys Leu Gln Leu Pro 
                245                 250                 255     


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


Glu Lys Leu Ala Pro Tyr His Glu Ile Val Glu Asn Ile Leu His Tyr 
        275                 280                 285             


Arg Gln Leu Gly Lys Leu Gln Ser Thr Tyr Ile Glu Gly Leu Leu Lys 
    290                 295                 300                 


Val Val Arg Pro Ala Thr Lys Lys Val His Thr Ile Phe Asn Gln Ala 
305                 310                 315                 320 


Leu Thr Gln Thr Gly Arg Leu Ser Ser Thr Glu Pro Asn Leu Gln Asn 
                325                 330                 335     


Ile Pro Ile Arg Leu Glu Glu Gly Arg Lys Ile Arg Gln Ala Phe Val 
            340                 345                 350         


Pro Ser Glu Ser Asp Trp Leu Ile Phe Ala Ala Asp Tyr Ser Gln Ile 
        355                 360                 365             


Glu Leu Arg Val Leu Ala His Ile Ala Glu Asp Asp Asn Leu Met Glu 
    370                 375                 380                 


Ala Phe Arg Arg Asp Leu Asp Ile His Thr Lys Thr Ala Met Asp Ile 
385                 390                 395                 400 


Phe Gln Val Ser Glu Asp Glu Val Thr Pro Asn Met Arg Arg Gln Ala 
                405                 410                 415     


Lys Ala Val Asn Tyr Gly Ile Val Tyr Gly Ile Ser Asp Tyr Gly Leu 
            420                 425                 430         


Ala Gln Asn Leu Asn Ile Ser Arg Lys Glu Ala Ala Glu Phe Ile Glu 
        435                 440                 445             


Arg Tyr Phe Glu Ser Phe Pro Gly Val Lys Arg Tyr Met Glu Asn Ile 
    450                 455                 460                 


Val Gln Glu Ala Lys Gln Lys Gly Tyr Val Thr Thr Leu Leu His Arg 
465                 470                 475                 480 


Arg Arg Tyr Leu Pro Asp Ile Thr Ser Arg Asn Phe Asn Val Arg Ser 
                485                 490                 495     


Phe Ala Glu Arg Met Ala Met Asn Thr Pro Ile Gln Gly Ser Ala Ala 
            500                 505                 510         


Asp Ile Ile Lys Lys Ala Met Ile Asp Leu Asn Ala Arg Leu Lys Glu 
        515                 520                 525             


Glu Arg Leu Gln Ala His Leu Leu Leu Gln Val His Asp Glu Leu Ile 
    530                 535                 540                 


Leu Glu Ala Pro Lys Glu Glu Met Glu Arg Leu Ala Arg Leu Val Pro 
545                 550                 555                 560 


Glu Val Met Glu Gln Ala Val Thr Leu Arg Val Pro Leu Lys Val Asp 
                565                 570                 575     


Tyr His Tyr Gly Ser Thr Trp Tyr Asp Ala Lys 
            580                 585         


<210>  3
<211>  578
<212>  PRT
<213>  Bacillus stearothermophilus


<220>
<221>  MISC_FEATURE
<222>  (1)..(578)
<223>  Protein expression product of the cysteine-substituted Bst-f 
       polymerase

<400>  3

Met Ala Phe Thr Leu Ala Asp Arg Val Thr Glu Glu Met Leu Ala Asp 
1               5                   10                  15      


Lys Ala Ala Leu Val Val Glu Val Val Glu Glu Asn Tyr His Asp Ala 
            20                  25                  30          


Pro Ile Val Gly Ile Ala Val Val Asn Glu His Gly Arg Phe Phe Leu 
        35                  40                  45              


Arg Pro Glu Thr Ala Leu Ala Asp Pro Gln Phe Val Ala Trp Leu Gly 
    50                  55                  60                  


Asp Glu Thr Lys Lys Lys Ser Met Phe Asp Ser Lys Arg Ala Ala Val 
65                  70                  75                  80  


Ala Leu Lys Trp Lys Gly Ile Glu Leu Ala Gly Val Ser Phe Asp Leu 
                85                  90                  95      


Leu Leu Ala Ala Tyr Leu Leu Asp Pro Ala Gln Gly Val Asp Asp Val 
            100                 105                 110         


Ala Ala Ala Ala Lys Met Lys Gln Tyr Glu Ala Val Arg Pro Asp Glu 
        115                 120                 125             


Ala Val Tyr Gly Lys Gly Ala Lys Arg Ala Val Pro Asp Glu Pro Val 
    130                 135                 140                 


Leu Ala Glu His Leu Val Arg Lys Ala Ala Ala Ile Trp Glu Leu Glu 
145                 150                 155                 160 


Arg Pro Phe Leu Asp Glu Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu 
                165                 170                 175     


Val Glu Leu Glu Gln Pro Leu Ser Ser Ile Leu Ala Glu Met Glu Phe 
            180                 185                 190         


Ala Gly Val Lys Val Asp Thr Lys Arg Leu Glu Gln Met Gly Lys Glu 
        195                 200                 205             


Leu Ala Glu Gln Leu Gly Thr Val Glu Gln Arg Ile Tyr Glu Leu Ala 
    210                 215                 220                 


Gly Gln Glu Phe Asn Ile Asn Ser Pro Lys Gln Leu Gly Val Ile Leu 
225                 230                 235                 240 


Phe Glu Lys Leu Gln Leu Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr 
                245                 250                 255     


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


Val Glu Asn Ile Leu His Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr 
        275                 280                 285             


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


His Thr Ile Phe Asn Gln Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser 
305                 310                 315                 320 


Thr Glu Pro Asn Leu Gln Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg 
                325                 330                 335     


Lys Ile Arg Gln Ala Phe Val Pro Ser Glu Ser Asp Trp Leu Ile Phe 
            340                 345                 350         


Ala Ala Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Ile Ala 
        355                 360                 365             


Glu Asp Asp Asn Leu Met Glu Ala Phe Arg Arg Asp Leu Asp Ile His 
    370                 375                 380                 


Thr Lys Thr Ala Met Asp Ile Phe Gln Val Ser Glu Asp Glu Val Thr 
385                 390                 395                 400 


Pro Asn Met Arg Arg Gln Ala Lys Ala Val Asn Tyr Gly Ile Val Tyr 
                405                 410                 415     


Gly Ile Ser Asp Tyr Gly Leu Ala Gln Asn Leu Asn Ile Ser Arg Lys 
            420                 425                 430         


Glu Ala Ala Glu Phe Ile Glu Arg Tyr Phe Glu Ser Phe Pro Gly Val 
        435                 440                 445             


Lys Arg Tyr Met Glu Asn Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr 
    450                 455                 460                 


Val Thr Thr Leu Leu His Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser 
465                 470                 475                 480 


Arg Asn Phe Asn Val Arg Ser Phe Ala Glu Arg Met Ala Met Asn Thr 
                485                 490                 495     


Pro Ile Gln Gly Ser Ala Ala Asp Ile Ile Lys Lys Ala Met Ile Asp 
            500                 505                 510         


Leu Asn Ala Arg Leu Lys Glu Glu Arg Leu Gln Ala His Leu Leu Leu 
        515                 520                 525             


Gln Val His Asp Glu Leu Ile Leu Glu Ala Pro Lys Glu Glu Met Glu 
    530                 535                 540                 


Arg Leu Ala Arg Leu Val Pro Glu Val Met Glu Gln Ala Val Thr Leu 
545                 550                 555                 560 


Arg Val Pro Leu Lys Val Asp Tyr His Tyr Gly Ser Thr Trp Tyr Asp 
                565                 570                 575     


Ala Lys 
        


<210>  4
<211>  578
<212>  PRT
<213>  Bacillus stearothermophilus


<220>
<221>  MISC_FEATURE
<222>  (1)..(578)
<223>  Bacillus DNA polymerase C-terminal fragment lacking 5'-3' 
       exonuclease activity

<400>  4

Met Ala Phe Thr Leu Ala Asp Arg Val Thr Glu Glu Met Leu Ala Asp 
1               5                   10                  15      


Lys Ala Ala Leu Val Val Glu Val Val Glu Glu Asn Tyr His Asp Ala 
            20                  25                  30          


Pro Ile Val Gly Ile Ala Val Val Asn Glu His Gly Arg Phe Phe Leu 
        35                  40                  45              


Arg Pro Glu Thr Ala Leu Ala Asp Pro Gln Phe Val Ala Trp Leu Gly 
    50                  55                  60                  


Asp Glu Thr Lys Lys Lys Ser Met Phe Asp Ser Lys Arg Ala Ala Val 
65                  70                  75                  80  


Ala Leu Lys Trp Lys Gly Ile Glu Leu Cys Gly Val Ser Phe Asp Leu 
                85                  90                  95      


Leu Leu Ala Ala Tyr Leu Leu Asp Pro Ala Gln Gly Val Asp Asp Val 
            100                 105                 110         


Ala Ala Ala Ala Lys Met Lys Gln Tyr Glu Ala Val Arg Pro Asp Glu 
        115                 120                 125             


Ala Val Tyr Gly Lys Gly Ala Lys Arg Ala Val Pro Asp Glu Pro Val 
    130                 135                 140                 


Leu Ala Glu His Leu Val Arg Lys Ala Ala Ala Ile Trp Glu Leu Glu 
145                 150                 155                 160 


Arg Pro Phe Leu Asp Glu Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu 
                165                 170                 175     


Val Glu Leu Glu Gln Pro Leu Ser Ser Ile Leu Ala Glu Met Glu Phe 
            180                 185                 190         


Ala Gly Val Lys Val Asp Thr Lys Arg Leu Glu Gln Met Gly Lys Glu 
        195                 200                 205             


Leu Ala Glu Gln Leu Gly Thr Val Glu Gln Arg Ile Tyr Glu Leu Ala 
    210                 215                 220                 


Gly Gln Glu Phe Asn Ile Asn Ser Pro Lys Gln Leu Gly Val Ile Leu 
225                 230                 235                 240 


Phe Glu Lys Leu Gln Leu Pro Val Leu Lys Lys Thr Lys Thr Gly Tyr 
                245                 250                 255     


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


Val Glu Asn Ile Leu His Tyr Arg Gln Leu Gly Lys Leu Gln Ser Thr 
        275                 280                 285             


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


His Thr Ile Phe Asn Gln Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser 
305                 310                 315                 320 


Thr Glu Pro Asn Leu Gln Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg 
                325                 330                 335     


Lys Ile Arg Gln Ala Phe Val Pro Ser Glu Ser Asp Trp Leu Ile Phe 
            340                 345                 350         


Ala Ala Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Ile Ala 
        355                 360                 365             


Glu Asp Asp Asn Leu Met Glu Ala Phe Arg Arg Asp Leu Asp Ile His 
    370                 375                 380                 


Thr Lys Thr Ala Met Asp Ile Phe Gln Val Ser Glu Asp Glu Val Thr 
385                 390                 395                 400 


Pro Asn Met Arg Arg Gln Ala Lys Ala Val Asn Tyr Gly Ile Val Tyr 
                405                 410                 415     


Gly Ile Ser Asp Tyr Gly Leu Ala Gln Asn Leu Asn Ile Ser Arg Lys 
            420                 425                 430         


Glu Ala Ala Glu Phe Ile Glu Arg Tyr Phe Glu Ser Phe Pro Gly Val 
        435                 440                 445             


Lys Arg Tyr Met Glu Asn Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr 
    450                 455                 460                 


Val Thr Thr Leu Leu His Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser 
465                 470                 475                 480 


Arg Asn Phe Asn Val Arg Ser Phe Ala Glu Arg Met Ala Met Asn Thr 
                485                 490                 495     


Pro Ile Gln Gly Ser Ala Ala Asp Ile Ile Lys Lys Ala Met Ile Asp 
            500                 505                 510         


Leu Asn Ala Arg Leu Lys Glu Glu Arg Leu Gln Ala His Leu Leu Leu 
        515                 520                 525             


Gln Val His Asp Glu Leu Ile Leu Glu Ala Pro Lys Glu Glu Met Glu 
    530                 535                 540                 


Arg Leu Cys Arg Leu Val Pro Glu Val Met Glu Gln Ala Val Thr Leu 
545                 550                 555                 560 


Arg Val Pro Leu Lys Val Asp Tyr His Tyr Gly Ser Thr Trp Tyr Asp 
                565                 570                 575     


Ala Lys 
        


<210>  5
<211>  9
<212>  PRT
<213>  Bacillus stearothermophilus


<220>
<221>  MISC_FEATURE
<222>  (1)..(9)
<223>  N-terminal sequence containing Cys residue

<400>  5

Gly Ser His Met Ser Cys Gly Ala Ala 
1               5                   


<210>  6
<211>  26
<212>  PRT
<213>  Bacillus stearothermophilus


<220>
<221>  MISC_FEATURE
<222>  (1)..(26)
<223>  N-terminal sequence containing polyhistidine tag, thrombin 
       cleavage site, and Cys residue

<400>  6

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


Arg Gly Ser His Met Ser Cys Gly Ala Ala 
            20                  25      


<210>  7
<211>  10
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  DNA sequence to be determined using synthetic template

<400>  7
tagcatcaga                                                              10


<210>  8
<211>  10
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  DNA sequence to be determined using synthetic template

<400>  8
ccctgtcatg                                                              10


<210>  9
<211>  10
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  DNA sequence to be determined using synthetic template

<400>  9
cccatttatg                                                              10


<210>  10
<211>  10
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  DNA sequence to be determined using synthetic template

<400>  10
ccgattcgtc                                                              10


<210>  11
<211>  10
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  DNA sequence to be determined using synthetic template

<400>  11
ccatgtttca                                                              10


<210>  12
<211>  585
<212>  PRT
<213>  Bacillus subtilis

<400>  12

Met Asp Gln Ser Leu Glu Asp Ile Asn Val Lys Thr Val Thr Asp Val 
1               5                   10                  15      


Thr Ser Asp Ile Leu Val Ser Pro Ser Ala Phe Val Val Glu Gln Ile 
            20                  25                  30          


Gly Asp Asn Tyr His Glu Glu Pro Ile Leu Gly Phe Ser Ile Val Asn 
        35                  40                  45              


Glu Thr Gly Ala Tyr Phe Ile Pro Lys Asp Ile Ala Val Glu Ser Glu 
    50                  55                  60                  


Val Phe Lys Glu Trp Val Glu Asn Asp Glu Gln Lys Lys Trp Val Phe 
65                  70                  75                  80  


Asp Ser Lys Arg Ala Val Val Ala Leu Arg Trp Gln Gly Ile Glu Leu 
                85                  90                  95      


Lys Gly Ala Glu Phe Asp Thr Leu Leu Ala Ala Tyr Ile Ile Asn Pro 
            100                 105                 110         


Gly Asn Ser Tyr Asp Asp Val Ala Ser Val Ala Lys Asp Tyr Gly Leu 
        115                 120                 125             


His Ile Val Ser Ser Asp Glu Ser Val Tyr Gly Lys Gly Ala Lys Arg 
    130                 135                 140                 


Ala Val Pro Ser Glu Asp Val Leu Ser Glu His Leu Gly Arg Lys Ala 
145                 150                 155                 160 


Leu Ala Ile Gln Ser Leu Arg Glu Lys Leu Val Gln Glu Leu Glu Asn 
                165                 170                 175     


Asn Asp Gln Leu Glu Leu Phe Glu Glu Leu Glu Met Pro Leu Ala Leu 
            180                 185                 190         


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


Leu Lys Arg Met Gly Glu Glu Leu Gly Ala Lys Leu Lys Glu Tyr Glu 
    210                 215                 220                 


Glu Lys Ile His Glu Ile Ala Gly Glu Pro Phe Asn Ile Asn Ser Pro 
225                 230                 235                 240 


Lys Gln Leu Gly Val Ile Leu Phe Glu Lys Ile Gly Leu Pro Val Val 
                245                 250                 255     


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


Leu Ala Asp Lys His Asp Ile Val Asp Tyr Ile Leu Gln Tyr Arg Gln 
        275                 280                 285             


Ile Gly Lys Leu Gln Ser Thr Tyr Ile Glu Gly Leu Leu Lys Val Thr 
    290                 295                 300                 


Arg Pro Asp Ser His Lys Val His Thr Arg Phe Asn Gln Ala Leu Thr 
305                 310                 315                 320 


Gln Thr Gly Arg Leu Ser Ser Thr Asp Pro Asn Leu Gln Asn Ile Pro 
                325                 330                 335     


Ile Arg Leu Glu Glu Gly Arg Lys Ile Arg Gln Ala Phe Val Pro Ser 
            340                 345                 350         


Glu Lys Asp Trp Leu Ile Phe Ala Ala Asp Tyr Ser Gln Ile Glu Leu 
        355                 360                 365             


Arg Val Leu Ala His Ile Ser Lys Asp Glu Asn Leu Ile Glu Ala Phe 
    370                 375                 380                 


Thr Asn Asp Met Asp Ile His Thr Lys Thr Ala Met Asp Val Phe His 
385                 390                 395                 400 


Val Ala Lys Asp Glu Val Thr Ser Ala Met Arg Arg Gln Ala Lys Ala 
                405                 410                 415     


Val Asn Phe Gly Ile Val Tyr Gly Ile Ser Asp Tyr Gly Leu Ser Gln 
            420                 425                 430         


Asn Leu Gly Ile Thr Arg Lys Glu Ala Gly Ala Phe Ile Asp Arg Tyr 
        435                 440                 445             


Leu Glu Ser Phe Gln Gly Val Lys Ala Tyr Met Glu Asp Ser Val Gln 
    450                 455                 460                 


Glu Ala Lys Gln Lys Gly Tyr Val Thr Thr Leu Met His Arg Arg Arg 
465                 470                 475                 480 


Tyr Ile Pro Glu Leu Thr Ser Arg Asn Phe Asn Ile Arg Ser Phe Ala 
                485                 490                 495     


Glu Arg Thr Ala Met Asn Thr Pro Ile Gln Gly Ser Ala Ala Asp Ile 
            500                 505                 510         


Ile Lys Lys Ala Met Ile Asp Met Ala Ala Lys Leu Lys Glu Lys Gln 
        515                 520                 525             


Leu Lys Ala Arg Leu Leu Leu Gln Val His Asp Glu Leu Ile Phe Glu 
    530                 535                 540                 


Ala Pro Lys Glu Glu Ile Glu Ile Leu Glu Lys Leu Val Pro Glu Val 
545                 550                 555                 560 


Met Glu His Ala Leu Ala Leu Asp Val Pro Leu Lys Val Asp Phe Ala 
                565                 570                 575     


Ser Gly Pro Ser Trp Tyr Asp Ala Lys 
            580                 585 


<210>  13
<211>  611
<212>  PRT
<213>  Bacillus subtilis


<220>
<221>  MISC_FEATURE
<222>  (1)..(611)
<223>  Cys-Bsu (CBU) polypeptide sequence

<220>
<221>  MISC_FEATURE
<222>  (5)..(10)
<223>  Polyhistidine tag motif

<220>
<221>  MISC_FEATURE
<222>  (17)..(18)
<223>  Thrombin cleavage site

<220>
<221>  MISC_FEATURE
<222>  (27)..(611)
<223>  Bsu DNA polymerase polypeptide sequence

<400>  13

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


Arg Gly Ser His Met Ser Cys Gly Ala Ala Gly Asp Gln Ser Leu Glu 
            20                  25                  30          


Asp Ile Asn Val Lys Thr Val Thr Asp Val Thr Ser Asp Ile Leu Val 
        35                  40                  45              


Ser Pro Ser Ala Phe Val Val Glu Gln Ile Gly Asp Asn Tyr His Glu 
    50                  55                  60                  


Glu Pro Ile Leu Gly Phe Ser Ile Val Asn Glu Thr Gly Ala Tyr Phe 
65                  70                  75                  80  


Ile Pro Lys Asp Ile Ala Val Glu Ser Glu Val Phe Lys Glu Trp Val 
                85                  90                  95      


Glu Asn Asp Glu Gln Lys Lys Trp Val Phe Asp Ser Lys Arg Ala Val 
            100                 105                 110         


Val Ala Leu Arg Trp Gln Gly Ile Glu Leu Lys Gly Ala Glu Phe Asp 
        115                 120                 125             


Thr Leu Leu Ala Ala Tyr Ile Ile Asn Pro Gly Asn Ser Tyr Asp Asp 
    130                 135                 140                 


Val Ala Ser Val Ala Lys Asp Tyr Gly Leu His Ile Val Ser Ser Asp 
145                 150                 155                 160 


Glu Ser Val Tyr Gly Lys Gly Ala Lys Arg Ala Val Pro Ser Glu Asp 
                165                 170                 175     


Val Leu Ser Glu His Leu Gly Arg Lys Ala Leu Ala Ile Gln Ser Leu 
            180                 185                 190         


Arg Glu Lys Leu Val Gln Glu Leu Glu Asn Asn Asp Gln Leu Glu Leu 
        195                 200                 205             


Phe Glu Glu Leu Glu Met Pro Leu Ala Leu Ile Leu Gly Glu Met Glu 
    210                 215                 220                 


Ser Thr Gly Val Lys Val Asp Val Asp Arg Leu Lys Arg Met Gly Glu 
225                 230                 235                 240 


Glu Leu Gly Ala Lys Leu Lys Glu Tyr Glu Glu Lys Ile His Glu Ile 
                245                 250                 255     


Ala Gly Glu Pro Phe Asn Ile Asn Ser Pro Lys Gln Leu Gly Val Ile 
            260                 265                 270         


Leu Phe Glu Lys Ile Gly Leu Pro Val Val Lys Lys Thr Lys Thr Gly 
        275                 280                 285             


Tyr Ser Thr Ser Ala Asp Val Leu Glu Lys Leu Ala Asp Lys His Asp 
    290                 295                 300                 


Ile Val Asp Tyr Ile Leu Gln Tyr Arg Gln Ile Gly Lys Leu Gln Ser 
305                 310                 315                 320 


Thr Tyr Ile Glu Gly Leu Leu Lys Val Thr Arg Pro Asp Ser His Lys 
                325                 330                 335     


Val His Thr Arg Phe Asn Gln Ala Leu Thr Gln Thr Gly Arg Leu Ser 
            340                 345                 350         


Ser Thr Asp Pro Asn Leu Gln Asn Ile Pro Ile Arg Leu Glu Glu Gly 
        355                 360                 365             


Arg Lys Ile Arg Gln Ala Phe Val Pro Ser Glu Lys Asp Trp Leu Ile 
    370                 375                 380                 


Phe Ala Ala Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Ile 
385                 390                 395                 400 


Ser Lys Asp Glu Asn Leu Ile Glu Ala Phe Thr Asn Asp Met Asp Ile 
                405                 410                 415     


His Thr Lys Thr Ala Met Asp Val Phe His Val Ala Lys Asp Glu Val 
            420                 425                 430         


Thr Ser Ala Met Arg Arg Gln Ala Lys Ala Val Asn Phe Gly Ile Val 
        435                 440                 445             


Tyr Gly Ile Ser Asp Tyr Gly Leu Ser Gln Asn Leu Gly Ile Thr Arg 
    450                 455                 460                 


Lys Glu Ala Gly Ala Phe Ile Asp Arg Tyr Leu Glu Ser Phe Gln Gly 
465                 470                 475                 480 


Val Lys Ala Tyr Met Glu Asp Ser Val Gln Glu Ala Lys Gln Lys Gly 
                485                 490                 495     


Tyr Val Thr Thr Leu Met His Arg Arg Arg Tyr Ile Pro Glu Leu Thr 
            500                 505                 510         


Ser Arg Asn Phe Asn Ile Arg Ser Phe Ala Glu Arg Thr Ala Met Asn 
        515                 520                 525             


Thr Pro Ile Gln Gly Ser Ala Ala Asp Ile Ile Lys Lys Ala Met Ile 
    530                 535                 540                 


Asp Met Ala Ala Lys Leu Lys Glu Lys Gln Leu Lys Ala Arg Leu Leu 
545                 550                 555                 560 


Leu Gln Val His Asp Glu Leu Ile Phe Glu Ala Pro Lys Glu Glu Ile 
                565                 570                 575     


Glu Ile Leu Glu Lys Leu Val Pro Glu Val Met Glu His Ala Leu Ala 
            580                 585                 590         


Leu Asp Val Pro Leu Lys Val Asp Phe Ala Ser Gly Pro Ser Trp Tyr 
        595                 600                 605             


Asp Ala Lys 
    610     


