Summary of the invention
The present invention aim to provide a kind of can in vitro, under normal temperature isothermal condition, realize lengthy motion picture fast to break the proteolytic enzyme of nucleic acid amplification and new amplification method.
First object of the present invention is to provide the proteolytic enzyme for isothermal nucleic acid large fragment amplification in vitros such as normal temperature.
For the proteolytic enzyme of the isothermal nucleic acid large fragment amplification in vitros such as normal temperature, it is characterized in that, described proteolytic enzyme comprises: (1) recombinase; (2) single strand binding protein; (3) archaeal dna polymerase; (4) accessory protein; The aminoacid sequence of described recombinase is as shown in SEQ IDNo.35; The aminoacid sequence of single strand binding protein is as shown in SEQ ID No.36; The aminoacid sequence of archaeal dna polymerase is as shown in SEQ ID No.37; The aminoacid sequence of accessory protein is as shown in SEQ ID No.38.
Further, in amplification system, recombinase concentration range is 0.5 ~ 0.8mg/ml; Single strand binding protein concentration range is 0.9 ~ 1.2mg/ml; Archaeal dna polymerase concentration range is 0.3 ~ 0.6mg/ml; Accessory protein concentration range is 0.1 ~ 0.3mg/ml.
Described proteolytic enzyme also comprises: (5) topoisomerase or helicase; The aminoacid sequence of topoisomerase is as SEQ ID No.39; The aminoacid sequence of helicase is as shown in SEQ ID No.40.
In amplification system, topoisomerase concentration range is 0.1 ~ 0.5U/ul; Helicase concentration range is 0.1 ~ 0.5U/ul.
Normal temperature isothermal of the present invention, refer to and to hatch under a certain steady temperature, temperature is lower is 37 DEG C, close to normal temperature, and is homogeneous constant temp.
Above-mentioned proteolytic enzyme (engineering enzyme) is multiple through genetic modification, and there is the proteolytic enzyme of high purity and high vigor, having following characteristics: the proteolytic enzyme 1) comprising 4 or 5 kind of Various Functions, is recombinase, single strand binding protein, archaeal dna polymerase, accessory protein and topoisomerase or helicase respectively; 2) all proteolytic enzyme all derives from nature, after to produce through the step such as restructuring, fermentation, cytoclasis, purifying and enzyme activity determination and obtain; 3) recombinase, be responsible for forming initiation complex with primer, the optimal concentration scope in system is 0.5 ~ 0.8mg/ml; 4) single strand binding protein, is responsible for the strand in conjunction with DNA profiling, helps to form Bubble Region, local, so that initiation complex carries out the scanning of matching area in the strand district opened, the optimal concentration scope in system is 0.9 ~ 1.2mg/ml; 5) archaeal dna polymerase, because having strand-displacement activity, therefore except the 3 ' end be responsible for from primer extends and synthesizes new subchain in amplification procedure, also responsiblely substitute a fundamental chain wherein by new subchain, form double-strand with another fundamental chain, the optimal concentration scope in system is 0.3 ~ 0.6mg/ml; 6) accessory protein, help initiation complex affine with DNA single chain better with suitable bonding force, can realize scanning, also well can depart from finding matching area relief recombinase and primer, the optimal concentration scope in system is 0.1 ~ 0.3mg/ml; 7) topoisomerase, help to open DNA superhelix in amplification procedure, in system, belong to optional addO-on therapy, its optimal concentration scope is 0.1 ~ 0.5U/ul; 8) helicase, can assist single strand binding protein to open DNA double chain better, is also optional addO-on therapy.
Second object of the present invention is to provide the reagent for isothermal nucleic acid large fragment amplification in vitros such as normal temperature.
For the reagent of the isothermal nucleic acid large fragment amplification in vitros such as normal temperature, it is characterized in that, described reagent mainly comprises Tris alkali, dNTP, ATP, disodium creatine phosphate, creatine phosphokinase, potassium acetate, trehalose, N.F,USP MANNITOL, polyoxyethylene glycol, dithiothreitol (DTT) and proteolytic enzyme, and described proteolytic enzyme comprises: (1) recombinase; (2) single strand binding protein; (3) archaeal dna polymerase; (4) accessory protein; Preferably also comprise (5) topoisomerase or helicase.
Described reagent is lyophilized powder, the concentration of each component in lyophilized powder is: Tris alkali 20 ~ 30mM, dNTP 200 ~ 250uM, ATP 2 ~ 3mM, disodium creatine phosphate 45 ~ 55mM, creatine phosphokinase 90 ~ 110ng/ μ l, potassium acetate 90 ~ 110mM, trehalose 5.5 ~ 6.5%, N.F,USP MANNITOL 6 ~ 10%, polyoxyethylene glycol 2 ~ 3%, dithiothreitol (DTT) 4 ~ 6mM, recombinase 0.5 ~ 0.8mg/ml, single strand binding protein 0.9 ~ 1.2mg/ml, archaeal dna polymerase 0.3 ~ 0.6mg/ml, accessory protein 0.1 ~ 0.3mg/ml, topoisomerase 0.1 ~ 0.5U/ul, helicase 0.1 ~ 0.5U/ul, described lyophilized powder is made up of following methods: now by reagent lyophilize 4-10 hour at-30 ~-35 DEG C, then 0 ~ 20 DEG C of drying 30 minutes ~ 1 hour.
For the various chemical compositions in the isothermal nucleic acid large fragment amplification in vitro reagent such as normal temperature, a series of for reacting the chemical composition providing the suitableeest buffer environment, there is following characteristics: 1) function of each component in system is different, or provide reaction desired raw material, such as deoxyribonucleoside triphosphate (dNTP); Or provide energy, such as Triphosaden (ATP), disodium creatine phosphate (PCr) and creatine phosphokinase (CK) for reacting; Or provide appropriate salt concentration, such as potassium acetate (KAc); Or composition activity is not lost in guarantee freezing dry process, such as trehalose (Trehalose), N.F,USP MANNITOL (Mannitol) and polyoxyethylene glycol (PEG); 2) optimal concentration of each reactive component in freeze-drying system respectively: PEG 2 ~ 3%, Tris alkali 20 ~ 30mM, KAc90 ~ 110mM, DTT (dithiothreitol (DTT)) 4 ~ 6mM, ATP 2 ~ 3mM, PCr 45 ~ 55mM, CK 90 ~ 110ng/ μ l, dNTP 200 ~ 250uM, trehalose 5.5 ~ 6.5%, N.F,USP MANNITOL 6 ~ 10%.
Proliferation time freezing dry process, is a kind of flow process of reaction system being carried out low temperature drying and other treatment, has following characteristics: 1) comprise dry and dry two steps again of trunk, trunk is dry is-30 ~-35 DEG C, 4 ~ 10 hours, then drying is 0 ~ 20 DEG C, 30 minutes ~ 1 hour; 2) actual temperature often walked and duration depend on the sample total amount of each freeze-drying; 3) reaction system after process can carry out experimental implementation at normal temperatures, can not cause losing or reducing of any detection effect; 4) response sample after frozen dried is white dry powder, is attached to tube wall without any viscous substance, after resuspended with damping fluid, can dissolve completely, exists without any obvious particulate material; 5) after freeze-drying, the detection vigor of reaction system can maintain half a year.
3rd object of the present invention is to provide the isothermal nucleic acid large fragment amplification in vitro method such as a kind of normal temperature.
The isothermal nucleic acid large fragment amplification in vitro method such as a kind of normal temperature, it is characterized in that, described amplification method is after adding template and amplimer with described amplifing reagent, is placed in a temperature control device and hatches 30 minutes to 4 hours, then carry out Visual retrieval by agarose gel electrophoresis; The temperature of described normal temperature is 37 DEG C, keeps homogeneous steady temperature in amplification procedure.Described reagent mainly comprises Tris alkali, dNTP, ATP, disodium creatine phosphate, creatine phosphokinase, potassium acetate, trehalose, N.F,USP MANNITOL, polyoxyethylene glycol, dithiothreitol (DTT) and proteolytic enzyme, and described proteolytic enzyme comprises: (1) recombinase; (2) single strand binding protein; (3) archaeal dna polymerase; (4) accessory protein; Preferably also comprise (5) topoisomerase or helicase.
Temperature control device of the present invention is for reacting the instrument providing appropriate incubation environment, having temperature control modules, set of time function and sample well.Such as metal bath, water-bath, incubator or other small portable incubation equipment etc.
The present invention has following technical characterstic:
1) the present invention utilizes the mutual synergy of multiple engineering enzyme and corresponding chemical ancillary component can realize specific amplification under normal temperature isothermal condition: can under normal temperature isothermal condition (such as 37 DEG C), by amplification and agarose gel electrophoresis after, can the specific location of glue figure observe one special, become clear band.Its light levels is enough to distinguish with other non-specific amplification band.
2) simple fast: break for below 3kb lengthy motion picture, incubation is enough to for 40 minutes; Amplification is greater than the nucleic acid fragment of 3kb, needs to be advisable by 4 hours for 40 minutes.
3) hi-fi: owing to being the situation at utmost simulating the amplification of biological nucleic acid in vivo, the mispairing rate in amplification procedure is very low, should be less than 1/10000kb.
4) difficult expansion sample is applicable to: for difficult extension increasing sequence, the such as sample of high GC content or high AT content, amplification method of the present invention has some superiority.
5) amplification method of the present invention under steady temperature, realizes external rapid amplifying to long segment nucleic acid at normal temperature, can be widely used in order-checking, cyclic plasmid amplification, even in the middle of the field such as whole genome amplification.
Embodiment
Following specific embodiment is further illustrating technique and method scheme provided by the invention, but does not should be understood to limitation of the present invention.
The biological material source used in the present invention:
1. linear die lambda DNA buys in precious biotechnology (Dalian) company limited.
2., containing recombinant plasmid pM18-EF1b and pUC57-A of exogenous sequences, all utilize genetic engineering technique to build in this laboratory, recombinate and complete preparation.
3. all primers are used in amplification, all entrust Shanghai Sheng Gong biotechnology limited-liability company to synthesize.
Embodiment 1
The present embodiment is for illustration of the specificity long segment normal temperature isothermal duplication carried out on linear die lambda DNA.
1. from database NCBI GenBank, finding the gene order of lambda DNA, (Reference sequence number is NC_001416.1, overall length is 48.502kb), recycling software Vector NTI7.0 carries out the design of primers of nucleic acid fragment different in size for gene order.
2. by each primer pair of design, 1132-F/R (SEQ ID No.1 & 2) respectively, 1246F/R (SEQ ID No.3 & 4), 1346F/R (SEQ ID No.5 & 6), 1446F/R (SEQ ID No.7 & 8), 1550F/R (SEQ ID No.9 & 10), 1636F/R (SEQ ID No.11 & 12), 1739F/R (SEQ ID No.13 & 14), 1823F/R (SEQ ID No.15 & 16), 1950F/R (SEQ ID No.17 & 18), 2020F/R (SEQ ID No.19 & 20), 2134F/R (SEQ ID No.21 & 22), 2220F/R (SEQ ID No.23 & 24), 2350F/R (SEQ ID No.25 & 26), 2435F/R (SEQ ID No.27 & 28), 2548F/R (SEQ ID No.29 & 30), Shanghai Sheng Gong biotechnology limited-liability company is entrusted to synthesize.
The gene order of lambda DNA: the Reference sequence number in ncbi database is NC_001416.1, overall length is 48.502kb;
1132-Fï¼5â-GTTTCCTTTCTCTGTTTTTGTCCGTGGAAT-3â(SEQ ID No.1)
1132-Rï¼5â-GAGAGCCTTCCTGTTCAATATCATCATCAA-3â(SEQ ID No.2)
1246-Fï¼5â-GTTTAAGGCGTTTCCGTTCTTCTTCGTCAT-3â(SEQ ID No.3)
1246-Rï¼5â-TCGGAGAGCCTTCCTGTTCAATATCATCAT-3â(SEQ ID No.4)
1346-Fï¼5â-AGATCTCCAGCCAGGAACTATTGAGTACGAA-3â(SEQ ID No.5)
1346-Rï¼5â-CGTATCCCCTTTCGTTTTCATCCAGTCTTT-3â(SEQ ID No.6)
1446-Fï¼5â-GTTTCCTTTCTCTGTTTTTGTCCGTGGAAT-3â(SEQ ID No.7)
1446-Rï¼5â-CTTTTCGCAGATATAACGGGCATCAGTAAA-3â(SEQ ID No.8)
1550-Fï¼5â-GCGTTTCCGTTCTTCTTCGTCATAACTTAA-3â(SEQ ID No.9)
1550-Rï¼5â-CTTTTCGCAGATATAACGGGCATCAGTAAA-3â(SEQ ID No.10)
1636-Fï¼5â-CGTTAACGATTTGCTGAACACACCAGTGTAA-3â(SEQ ID No.11)
1636-Rï¼5â-AGTTTCATCCGTGTCATCAAGCTCCTCTTT-3â(SEQ ID No.12)
1739-Fï¼5â-TGGTGTACCGGCTGTCTGGTATGTATGAGTT-3â(SEQ ID No.13)
1739-Rï¼5â-GTAAGCCTTCTGCGCCTCTTCGGTATATTT-3â(SEQ ID No.14)
1823-Fï¼5â-GACGTTTGAGCAGAATAACCATGTGGTGAT-3â(SEQ ID No.15)
1823-Rï¼5â-CTCTCTCGTTTGCTCAGTTGTTCAGGAATA-3â(SEQ ID No.16)
1950-Fï¼5â-ATCAGCGTGGTCTGAGTGTGTTACAGAGGTT-3â(SEQ ID No.17)
1950-Rï¼5â-AGTGGGCTTTTCTGTTCGTTTCATCCATTA-3â(SEQ ID No.18)
2020-Fï¼5â-AATTCCGTTGCAGATGTTCTTGAATACCTT-3â(SEQ ID No.19)
2020-Rï¼5â-GAGCGTCTAAAACTGGTAGATAAGCCTAAA-3â(SEQ ID No.20)
2134-Fï¼5â-TTAAAATTAGAGTTGTGGCTTGGCTCTGCTA-3â(SEQ ID No.21)
2134-Rï¼5â-AGTGCATTTGATCCTTTTACTCCTCCTAAA-3â(SEQ ID No.22)
2220-Fï¼5â-AATGACCTGCCTAGGAATTGGTTAGCAAGTT-3â(SEQ ID No.23)
2220-Rï¼5â-TTGAAAATGAAAGCGTCCTTAACACCTCAT-3â(SEQ ID No.24)
2350-Fï¼5â-ATCCCTCTGAAAAAATCTTCCGAGTTTGCTA-3â(SEQ ID No.25)
2350-Rï¼5â-TATCTTACTGTCTTTGATGAGCATGGTGAA-3â(SEQ ID No.26)
2435-Fï¼5â-CCTGCCTCCAAACGATACCTGTTAGCAATA-3â(SEQ ID No.27)
2435-Rï¼5â-GTGGGTTATCCAAAAGGAAGCAGAAAGCTA-3â(SEQ ID No.28)
2548-Fï¼5â-GTACCTCATCTACTGCGAAAACTTGACCTTT-3â(SEQ ID No.29)
2548-Rï¼5â-ACTACGACCTGCATAACCAGTAAGAAGATA-3â(SEQ ID No.30)
3. use the primer pair of the lyophilized powder reaction system for preparing and synthesis or mix and take primer pair and carry out normal temperature isothermal amplification respectively.Lyophilized powder reaction system is:
Tris-Ac 5ul E-mix 2.5ul CK 2.5ul 20ï¼ PEG35000 12ul Recombinase 0.75mg/ml Accessory protein 0.22mg/ml Single strand binding protein 1.15mg/ml Archaeal dna polymerase 0.51mg/ml Topoisomerase 0.25U/ul Helicase 0.2U/ul dNTP 4ul Trehalose 6ï¼ N.F,USP MANNITOL 7ï¼Wherein, Tris-Ac is dissolved in 10ml distilled water by the 2M DTT of 0.6gTris alkali, 0.98g potassium acetate and 250ul, and pH regulator obtains to 8.3 preparations.E-mix is dissolved in by 0.3g ATP and 2.55g PCr and prepares in 10ml distilled water and obtain.
Complete the preparation of freeze-drying reaction system on ice after, be dispensed in 200ul reaction tubes and carry out follow-up freeze-drying preparation: lyophilize 4-10 hour at-30 ~-35 DEG C, then 0 ~ 20 DEG C of drying 30 minutes ~ 1 hour.
Amplification reaction system is formulated as follows:
Lyophilized powder reaction reagent 1 pipe PEG 25μl Distilled water 17.5μl Primer-F 2.0μl Primer-R 2.0μl Template 1μl Magnesium acetate 2.5μl4. amplified reaction program: 37 DEG C of isothermals, 40 minutes.
5. agarose gel electrophoresis carry out result detection (Fig. 1., Fig. 2., Fig. 3. and Fig. 4 .).
Fig. 1 is using linear lambda DNA as template, and the nucleotide fragment different for length between 1000-1600bp increases, and the bright single band of corresponding size confirms specific amplification.Fig. 2, equally using linear lambda DNA as template, carries out the amplification of Nucleotide large fragment for 1600-2000bp, and the bright single band of same corresponding size confirms its specific amplification.Fig. 3 is the amplification of the long segment for 2000-3400bp, and indivedual amplifications exist the interference of non-specific amplification, but do not affect the difference of specific band.Fig. 4 carries out the result figure tested that increases again after system is carried out frozen dried, clip size contains 1.0-3.3kb, and most swimming lane presents with single bright amplified fragments, confirms the feasibility of the isothermal nucleic acid large fragment amplifications such as normal temperature.
Embodiment 2
The present embodiment for illustration of in circular template, the normal temperature isothermal duplication that recombinant plasmid pM18-EF1b and pUC57-A carries out.
1. utilize genetic engineering technique to construct two recombinant plasmid pM18-EF1b and pUC57-A in vitro, as annular template.
2. respectively for corresponding templates, carry out the normal temperature isothermal duplication of single primer and decoding for DTMF with the Auele Specific Primer EF1b-F/R (SEQ ID No.31 & 32) designed and A-F/R (SEQ ID No.33 & 34).
EF1b-Fï¼5â-TCACACACACACATATAGAAAGAGAGAGAC-3â(SEQ ID No.31)
EF1b-Rï¼5â-CTGCTCCAGTTAGTTCATATAAGAGATAGG-3â(SEQ ID No.32)
A-Fï¼5â-AGCATCAACTTCTACCGTTCTACATAGCAT-3â(SEQ ID No.33)
A-Rï¼5â-CCGCTATAATACTGCATTTACACTTGGCAT-3â(SEQ ID No.34)
3. lyophilized powder reaction system is:
E-mix 2.5ul CK 2.5ul 20ï¼ PEG35000 12ul Recombinase 0.75mg/ml Accessory protein 0.22mg/ml Single strand binding protein 1.15mg/ml Archaeal dna polymerase 0.51mg/ml Topoisomerase 0.25U/ul Helicase 0.2U/ul dNTP 4ul Trehalose 6ï¼ N.F,USP MANNITOL 7ï¼4. wherein, Tris-Ac is dissolved in 10ml distilled water by the 2M DTT of 0.6gTris alkali, 0.98g potassium acetate and 250ul, and pH regulator obtains to 8.3 preparations.E-mix is dissolved in by 0.3g ATP and 2.55g PCr and prepares in 10ml distilled water and obtain.
5. complete the preparation of freeze-drying reaction system on ice after, be dispensed in 200ul reaction tubes and carry out follow-up freeze-drying preparation: lyophilize 4-10 hour at-30 ~-35 DEG C, then 0 ~ 20 DEG C of drying 30 minutes ~ 1 hour.
6. amplification reaction system is formulated as follows:
Lyophilized powder reaction reagent 1 pipe PEG 25μl Distilled water 17.5μl Primer-F 2.0μl Primer-R 2.0μl Template 1μl Magnesium acetate 2.5μl7. amplified reaction program: 37 DEG C of isothermals, 4 hours.
8. utilize subsequently agarose gel electrophoresis carry out result detection (Fig. 5. with Fig. 6-1,6-2.).
Fig. 5 is the single primer amplification test carried out with linear die lambda DNA, and swimming lane 1 is forward primer, and swimming lane 2 is reverse primers, and swimming lane 3 is positive/negative to primer.Result confirms in linear die, carry out single primer amplification can only unidirectional acquisition long segment, and this may be relevant to the lasting amplification ability of the position of primer, reaction system.Fig. 6 is with circular template, and the single primer amplification test that recombinant plasmid carries out, swimming lane 1 is forward primer, and swimming lane 2 is reverse primers, and swimming lane 3 is positive/negative to primer.Result confirms that in circular template, carry out single primer amplification can all obtain long or annular amplified production in both direction, illustrates the ability tentatively possessing and carry out plasmid amplification in vitro.
SEQUENCE LISTING
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Leu Lys Leu Asp Ala Ser Gly Asn Gly Gln Ala Val Ile Arg Phe Leu
35 40 45
Â
Pro Ala Lys Thr Asp Asp Ala Leu Pro Phe Ala Ile Leu Val Asn His
50 55 60
Â
Gly Phe Lys Lys Asn Gly Lys Trp Tyr Ile Glu Thr Cys Ser Ser Thr
65 70 75 80
Â
His Gly Asp Tyr Asp Ser Cys Pro Val Cys Gln Tyr Ile Ser Lys Asn
85 90 95
Â
Asp Leu Tyr Asn Thr Asn Lys Thr Glu Tyr Ser Gln Leu Lys Arg Lys
100 105 110
Â
Thr Ser Tyr Trp Ala Asn Ile Leu Val Val Lys Asp Pro Gln Ala Pro
115 120 125
Â
Asp Asn Glu Gly Lys Val Phe Lys Tyr Arg Phe Gly Lys Lys Ile Trp
130 135 140
Â
Asp Lys Ile Asn Ala Met Ile Ala Val Asp Thr Glu Met Gly Glu Thr
145 150 155 160
Â
Pro Val Asp Val Thr Cys Pro Trp Glu Gly Ala Asn Phe Val Leu Lys
165 170 175
Â
Val Lys Gln Val Ser Gly Phe Ser Asn Tyr Asp Glu Ser Lys Phe Leu
180 185 190
Â
Asn Gln Ser Ala Ile Pro Asn Ile Asp Asp Glu Ser Phe Gln Lys Glu
195 200 205
Â
Leu Phe Glu Gln Met Val Asp Leu Ser Glu Met Thr Ser Lys Asp Lys
210 215 220
Â
Phe Lys Ser Phe Glu Glu Leu Asn Thr Lys Phe Asn Gln Val Leu Gly
225 230 235 240
Â
Thr Ala Ala Leu Gly Gly Ala Ala Ala Ala Ala Ala Ser Val Ala Asp
245 250 255
Â
Lys Val Ala Ser Asp Leu Asp Asp Phe Asp Lys Asp Met Glu Ala Ser
260 265 270
Â
Ser Ala Lys Thr Glu Asp Asp Phe Met Ser Ser Ser Ser Ser Asp Asp
275 280 285
Â
Gly Asp Leu Asp Asp Leu Leu Ala Gly Leu
290 295
Â
<210> 37
<211> 876
<212> PRT
<213> artificial sequence
Â
<400> 37
Â
Val Asn Lys Leu Val Leu Ile Asp Gly Asn Ser Leu Ser Phe Arg Ala
1 5 10 15
Â
Phe Tyr Ala Leu Pro Leu Leu Ser Asn Lys Ala Gly Ile His Thr Asn
20 25 30
Â
Ala Val Tyr Gly Phe Ala Met Leu Leu Glu Lys Ile Leu Lys Glu Glu
35 40 45
Â
Lys Pro Asn His Phe Leu Val Ala Phe Asp Ala Gly Lys Thr Thr Phe
50 55 60
Â
Arg His Glu Lys Tyr Ser Glu Tyr Lys Gly Gly Arg Gln Lys Thr Pro
65 70 75 80
Â
Pro Glu Leu Ser Glu Gln Phe Pro Tyr Ile Arg Gln Leu Leu Asp Ala
85 90 95
Â
Tyr His Ile Lys Arg Tyr Glu Leu Asp Asn Tyr Glu Ala Asp Asp Ile
100 105 110
Â
Ile Gly Thr Leu Ser Lys Glu Ala Asp Lys Ala Gly Phe Gln Thr Ile
115 120 125
Â
Ile Ile Thr Gly Asp Arg Asp Leu Thr Gln Leu Ala Thr Asp Asn Val
130 135 140
Â
Thr Ile Tyr Tyr Thr Lys Lys Gly Val Thr Asp Val Asp His Tyr Thr
145 150 155 160
Â
Â
Po Asp Phe Ile Ala Glu Lys Tyr Asn Gly Lys Thr Pro Asn Gln Ile
165 170 175
Â
Ile Asp Met Lys Gly Leu Met Gly Asp Thr Ser Asp Asn Ile Pro Gly
180 185 190
Â
Val Ala Gly Val Gly Glu Lys Thr Ala Ile Lys Leu Leu Asn Gln Phe
195 200 205
Â
Asp Thr Val Glu Gly Val Tyr Glu His Leu Asp Glu Ile Ser Gly Lys
210 215 220
Â
Lys Leu Lys Glu Lys Leu Gln Asn Ser Lys Glu Asp Ala Leu Met Ser
225 230 235 240
Â
Lys Glu Leu Ala Thr Ile Asn Val Asp Ser Pro Ile Glu Val Lys Leu
245 250 255
Â
Glu Asp Thr Leu Met Thr His Gln Asp Glu Gln Gln Glu Lys Ile Glu
260 265 270
Â
Leu Phe Lys Lys Leu Glu Phe Lys Gln Leu Leu Ala Asp Ile Asp Gln
275 280 285
Â
Ser Ala Ser Val Glu Asp Ala Ile Glu Lys Thr Phe Glu Ile Glu Thr
290 295 300
Â
Ser Phe Asp Asn Ile Asp Phe Thr Ser Leu Lys Glu Ala Ala Ile His
305 310 315 320
Â
Â
Phe Glu Leu Asp Gly Gly Asn Tyr Leu Arg Asn Asn Ile Leu Lys Phe
325 330 335
Â
Ser Leu Phe Thr Gly Glu Lys His Ile Val Ile Asn Ala Asp Asp Ile
340 345 350
Â
Asn Asn Tyr Val Glu Leu Val Ser Trp Leu Glu Asn Pro Asn Ser Lys
355 360 365
Â
Lys Val Val Tyr Asp Ala Lys Lys Thr Tyr Val Ala Ser His Arg Leu
370 375 380
Â
Gly Ile Asp Ile Gln Asn Ile Ser Phe Asp Ile Met Leu Ala Ser Tyr
385 390 395 400
Â
Ile Ile Asp Pro Ser Arg Thr Ile Ser Asp Val Gln Ser Val Val Ser
405 410 415
Â
Leu Tyr Gly Gln Ser Phe Val Lys Asp Asp Val Ser Ile Tyr Gly Lys
420 425 430
Â
Gly Lys Lys Phe Lys Val Pro Glu Asp Asp Val Leu Asn Pro Tyr Val
435 440 445
Â
Ala Ser Ile Thr Asp Ala Ile Tyr Phe Ala Lys Pro Asn Met Asp Lys
450 455 460
Â
Gln Leu Glu Glu Tyr Asn Gln Val Glu Leu Leu Ala Asp Leu Glu Leu
465 470 475 480
Â
Pro Leu Ala Lys Ile Leu Ser Glu Met Glu Glu Ile Gly Ile Phe Thr
485 490 495
Â
Asp Val His Asp Leu Glu Glu Met Glu Lys Glu Ile Gln Glu Lys Leu
500 505 510
Â
Asp Val Leu Ile Arg Asn Ile His Asp Ala Ala Gly Glu Asp Phe Asn
515 520 525
Â
Ile Asn Ser Pro Lys Gln Leu Gly Val Val Leu Phe Glu Thr Leu Gln
530 535 540
Â
Leu Pro Val Ile Lys Lys Thr Lys Thr Gly Tyr Ser Thr Ala Val Asp
545 550 555 560
Â
Val Leu Glu Gln Leu Gln Gly Glu His Pro Ile Ile Asp Tyr Ile Leu
565 570 575
Â
Glu Tyr Arg Gln Leu Ser Lys Leu Gln Ser Thr Tyr Val Glu Gly Leu
580 585 590
Â
Gln Lys Val Ile Ser Asp Asp Gln Arg Ile His Thr Arg Phe Asn Gln
595 600 605
Â
Thr Leu Ala Gln Thr Gly Arg Leu Ser Ser Val Asp Pro Asn Leu Gln
610 615 620
Â
Asn Ile Pro Val Arg Leu Glu Glu Gly Arg Lys Ile Arg Lys Ala Phe
625 630 635 640
Â
Lys Pro Thr Ser Lys Asp Ser Val Ile Leu Ser Ala Asp Tyr Ser Gln
645 650 655
Â
Ile Glu Leu Arg Val Leu Ala His Ile Thr Gln Asp Glu Ser Met Lys
660 665 670
Â
Glu Ala Phe Ile Asn Gly Asp Asp Ile His Thr Ala Thr Ala Met Lys
675 680 685
Â
Val Phe Gly Val Glu Ala Asp Gln Val Asp Ser Leu Met Arg Arg Gln
690 695 700
Â
Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gly Ile Ser Asp Tyr Gly
705 710 715 720
Â
Leu Ser Gln Ser Leu Gly Ile Thr Arg Lys Lys Ala Lys Ala Phe Ile
725 730 735
Â
Asp Asp Tyr Leu Ala Ser Phe Pro Gly Val Lys Gln Tyr Met Ser Asp
740 745 750
Â
Ile Val Lys Asp Ala Lys Ala Leu Gly Tyr Val Glu Thr Leu Leu His
755 760 765
Â
Arg Arg Arg Tyr Ile Pro Asp Ile Thr Ser Arg Asn Phe Asn Leu Arg
770 775 780
Â
Gly Phe Ala Glu Arg Thr Ala Met Asn Thr Pro Ile Gln Gly Ser Ala
785 790 795 800
Â
Ala Asp Ile Ile Lys Leu Ala Met Val Lys Phe Ala Gln Lys Met Lys
805 810 815
Â
Glu Thr Thr Tyr Gln Ala Lys Leu Leu Leu Gln Val His Asp Glu Leu
820 825 830
Â
Ile Phe Glu Val Pro Lys Ser Glu Val Asp Ser Phe Ser Glu Phe Val
835 840 845
Â
Glu Glu Ile Met Glu Asn Ala Leu Gln Leu Asp Val Pro Leu Lys Val
850 855 860
Â
Asp Ser Ser Tyr Gly Ala Thr Trp Tyr Asp Ala Lys
865 870 875
Â
<210> 38
<211> 140
<212> PRT
<213> artificial sequence
Â
<400> 38
Â
Met Arg Leu Glu Asp Leu Gln Glu Glu Leu Lys Lys Asp Val Phe Ile
1 5 10 15
Â
Asp Ser Thr Lys Leu Gln Tyr Glu Ala Ala Asn Asn Val Met Leu Tyr
20 25 30
Â
Ser Lys Trp Leu Asn Lys His Ser Ser Ile Lys Lys Glu Met Leu Arg
35 40 45
Â
Ile Glu Ala Gln Lys Lys Val Ala Leu Lys Ala Arg Leu Asp Tyr Tyr
50 55 60
Â
Ser Gly Arg Gly Asp Gly Asp Glu Phe Ser Met Asp Arg Tyr Glu Lys
65 70 75 80
Â
Ser Glu Met Lys Thr Val Leu Ser Ala Asp Lys Asp Val Leu Lys Val
85 90 95
Â
Asp Thr Ser Leu Gln Tyr Trp Gly Ile Leu Leu Asp Phe Cys Ser Glu
100 105 110
Â
Leu Leu Met Leu Leu Asn His Val Asp Leu Leu Leu Ser Ile Phe Lys
115 120 125
Â
Thr Cys Glu His Leu Arg Leu Gln Asn Asn Glu Ile
130 135 140
Â
<210> 39
<211> 865
<212> PRT
<213> artificial sequence
Â
<400> 39
Â
Met Gly Lys Ala Leu Val Ile Val Glu Ser Pro Ala Lys Ala Lys Thr
1 5 10 15
Â
Ile Asn Lys Tyr Leu Gly Ser Asp Tyr Val Val Lys Ser Ser Val Gly
20 25 30
Â
His Ile Arg Asp Leu Pro Thr Ser Gly Ser Ala Ala Lys Lys Ser Ala
35 40 45
Â
Asp Ser Thr Ser Thr Lys Thr Ala Lys Lys Pro Lys Lys Asp Glu Arg
50 55 60
Â
Gly Ala Leu Val Asn Arg Met Gly Val Asp Pro Trp His Asn Trp Glu
65 70 75 80
Â
Ala His Tyr Glu Val Leu Pro Gly Lys Glu Lys Val Val Ser Glu Leu
85 90 95
Â
Lys Gln Leu Ala Glu Lys Ala Asp His Ile Tyr Leu Ala Thr Asp Leu
100 105 110
Â
Â
Asp Arg Glu Gly Glu Ala Ile Ala Trp His Leu Arg Glu Val Ile Gly
115 120 125
Â
Gly Asp Asp Ala Arg Tyr Ser Arg Val Val Phe Asn Glu Ile Thr Lys
130 135 140
Â
Asn Ala Ile Arg Gln Ala Phe Asn Lys Pro Gly Glu Leu Asn Ile Asp
145 150 155 160
Â
Arg Val Asn Ala Gln Gln Ala Arg Arg Phe Met Asp Arg Val Val Gly
165 170 175
Â
Tyr Met Val Ser Pro Leu Leu Trp Lys Lys Ile Ala Arg Gly Leu Ser
180 185 190
Â
Ala Gly Arg Val Gln Ser Val Ala Val Arg Leu Val Val Glu Arg Glu
195 200 205
Â
Arg Glu Ile Lys Ala Phe Val Pro Glu Glu Phe Trp Glu Val Asp Ala
210 215 220
Â
Ser Thr Thr Thr Pro Ser Gly Glu Ala Leu Ala Leu Gln Val Thr His
225 230 235 240
Â
Gln Asn Asp Lys Pro Phe Arg Pro Val Asn Lys Glu Gln Thr Gln Ala
245 250 255
Â
Ala Val Ser Leu Leu Glu Lys Ala Arg Tyr Ser Val Leu Glu Arg Glu
260 265 270
Â
Asp Lys Pro Thr Thr Ser Lys Pro Gly Ala Pro Phe Ile Thr Ser Thr
275 280 285
Â
Leu Gln Gln Ala Ala Ser Thr Arg Leu Gly Phe Gly Val Lys Lys Thr
290 295 300
Â
Met Met Met Ala Gln Arg Leu Tyr Glu Ala Gly Tyr Ile Thr Tyr Met
305 310 315 320
Â
Arg Thr Asp Ser Thr Asn Leu Ser Gln Asp Ala Val Asn Met Val Arg
325 330 335
Â
Gly Tyr Ile Ser Asp Asn Phe Gly Lys Lys Tyr Leu Pro Glu Ser Pro
340 345 350
Â
Asn Gln Tyr Ala Ser Lys Glu Asn Ser Gln Glu Ala His Glu Ala Ile
355 360 365
Â
Arg Pro Ser Asp Val Asn Val Met Ala Glu Ser Leu Lys Asp Met Glu
370 375 380
Â
Ala Asp Ala Gln Lys Leu Tyr Gln Leu Ile Trp Arg Gln Phe Val Ala
385 390 395 400
Â
Cys Gln Met Thr Pro Ala Lys Tyr Asp Ser Thr Thr Leu Thr Val Gly
405 410 415
Â
Ala Gly Asp Phe Arg Leu Lys Ala Arg Gly Arg Ile Leu Arg Phe Asp
420 425 430
Â
Gly Trp Thr Lys Val Met Pro Ala Leu Arg Lys Gly Asp Glu Asp Arg
435 440 445
Â
Ile Leu Pro Ala Val Asn Lys Gly Asp Ala Leu Thr Leu Val Glu Leu
450 455 460
Â
Thr Pro Ala Gln His Phe Thr Lys Pro Pro Ala Arg Phe Ser Glu Ala
465 470 475 480
Â
Ser Leu Val Lys Glu Leu Glu Lys Arg Gly Ile Gly Arg Pro Ser Thr
485 490 495
Â
Tyr Ala Ser Ile Ile Ser Thr Ile Gln Asp Arg Gly Tyr Val Arg Val
500 505 510
Â
Glu Asn Arg Arg Phe Tyr Ala Glu Lys Met Gly Glu Ile Val Thr Asp
515 520 525
Â
Arg Leu Glu Glu Asn Phe Arg Glu Leu Met Asn Tyr Asp Phe Thr Ala
530 535 540
Â
Gln Met Glu Asn Ser Leu Asp Gln Val Ala Asn His Glu Ala Glu Trp
545 550 555 560
Â
Lys Ala Val Leu Asp His Phe Phe Ser Asp Phe Thr Gln Gln Leu Asp
565 570 575
Â
Lys Ala Glu Lys Asp Pro Glu Glu Gly Gly Met Arg Pro Asn Gln Met
580 585 590
Â
Val Leu Thr Ser Ile Asp Cys Pro Thr Cys Gly Arg Lys Met Gly Ile
595 600 605
Â
Arg Thr Ala Ser Thr Gly Val Phe Leu Gly Cys Ser Gly Tyr Ala Leu
610 615 620
Â
Pro Pro Lys Glu Arg Cys Lys Thr Thr Ile Asn Leu Val Pro Glu Asn
625 630 635 640
Â
Glu Val Leu Asn Val Leu Glu Gly Glu Asp Ala Glu Thr Asn Ala Leu
645 650 655
Â
Arg Ala Lys Arg Arg Cys Pro Lys Cys Gly Thr Ala Met Asp Ser Tyr
660 665 670
Â
Leu Ile Asp Pro Lys Arg Lys Leu His Val Cys Gly Asn Asn Pro Thr
675 680 685
Â
Cys Asp Gly Tyr Glu Ile Glu Glu Gly Glu Phe Arg Ile Lys Gly Tyr
690 695 700
Â
Asp Gly Pro Ile Val Glu Cys Glu Lys Cys Gly Ser Glu Met His Leu
705 710 715 720
Â
Lys Met Gly Arg Phe Gly Lys Tyr Met Ala Cys Thr Asn Glu Glu Cys
725 730 735
Â
Lys Asn Thr Arg Lys Ile Leu Arg Asn Gly Glu Val Ala Pro Pro Lys
740 745 750
Â
Glu Asp Pro Val Pro Leu Pro Glu Leu Pro Cys Glu Lys Ser Asp Ala
755 760 765
Â
Tyr Phe Val Leu Arg Asp Gly Ala Ala Gly Val Phe Leu Ala Ala Asn
770 775 780
Â
Thr Phe Pro Lys Ser Arg Glu Thr Arg Ala Pro Leu Val Glu Glu Leu
785 790 795 800
Â
Tyr Arg Phe Arg Asp Arg Leu Pro Glu Lys Leu Arg Tyr Leu Ala Asp
805 810 815
Â
Ala Pro Gln Gln Asp Pro Glu Gly Asn Lys Thr Met Val Arg Phe Ser
820 825 830
Â
Arg Lys Thr Lys Gln Gln Tyr Val Ser Ser Glu Lys Asp Gly Lys Ala
835 840 845
Â
Thr Gly Trp Ser Ala Phe Tyr Val Asp Gly Lys Trp Val Glu Gly Lys
850 855 860
Â
Lys
865
Â
<210> 40
<211> 471
<212> PRT
<213> artificial sequence
Â
<400> 40
Â
Met Ala Gly Asn Lys Pro Phe Asn Lys Gln Gln Ala Glu Pro Arg Glu
1 5 10 15
Â
Arg Asp Pro Gln Val Ala Gly Leu Lys Val Pro Pro His Ser Ile Glu
20 25 30
Â
Ala Glu Gln Ser Val Leu Gly Gly Leu Met Leu Asp Asn Glu Arg Trp
35 40 45
Â
Asp Asp Val Ala Glu Arg Val Val Ala Asp Asp Phe Tyr Thr Arg Pro
50 55 60
Â
His Arg His Ile Phe Thr Glu Met Ala Arg Leu Gln Glu Ser Gly Ser
65 70 75 80
Â
Pro Ile Asp Leu Ile Thr Leu Ala Glu Ser Leu Glu Arg Gln Gly Gln
85 90 95
Â
Leu Asp Ser Val Gly Gly Phe Ala Tyr Leu Ala Glu Leu Ser Lys Asn
100 105 110
Â
Thr Pro Ser Ala Ala Asn Ile Ser Ala Tyr Ala Asp Ile Val Arg Glu
115 120 125
Â
Arg Ala Val Val Arg Glu Met Ile Ser Val Ala Asn Glu Ile Ala Glu
130 135 140
Â
Ala Gly Phe Asp Pro Gln Gly Arg Thr Ser Glu Asp Leu Leu Asp Leu
145 150 155 160
Â
Ala Glu Ser Arg Val Phe Lys Ile Ala Glu Ser Arg Ala Asn Lys Asp
165 170 175
Â
Glu Gly Pro Lys Asn Ile Ala Asp Val Leu Asp Ala Thr Val Ala Arg
180 185 190
Â
Ile Glu Gln Leu Phe Gln Gln Pro His Asp Gly Val Thr Gly Val Asn
195 200 205
Â
Thr Gly Tyr Asp Asp Leu Asn Lys Lys Thr Ala Gly Leu Gln Pro Ser
210 215 220
Â
Asp Leu Ile Ile Val Ala Ala Arg Pro Ser Met Gly Lys Thr Thr Phe
225 230 235 240
Â
Ala Met Asn Leu Val Glu Asn Ala Ala Met Leu Gln Asp Lys Pro Val
245 250 255
Â
Leu Ile Phe Ser Leu Glu Met Pro Ser Glu Gln Ile Met Met Arg Ser
260 265 270
Â
Leu Ala Ser Leu Ser Arg Val Asp Gln Thr Lys Ile Arg Thr Gly Gln
275 280 285
Â
Leu Asp Asp Glu Asp Trp Ala Arg Ile Ser Gly Thr Met Gly Ile Leu
290 295 300
Â
Leu Glu Lys Arg Asn Ile Tyr Ile Asp Asp Ser Ser Gly Leu Thr Pro
305 310 315 320
Â
Thr Glu Val Arg Ser Arg Ala Arg Arg Ile Ala Arg Glu His Gly Gly
325 330 335
Â
Ile Gly Leu Ile Met Ile Asp Tyr Leu Gln Leu Met Arg Val Pro Ala
340 345 350
Â
Leu Ser Asp Asn Arg Thr Leu Glu Ile Ala Glu Ile Ser Arg Ser Leu
355 360 365
Â
Lys Ala Leu Ala Lys Glu Leu Asn Val Pro Val Val Ala Leu Ser Gln
370 375 380
Â
Leu Asn Arg Ser Leu Glu Gln Arg Ala Asp Lys Arg Pro Val Asn Ser
385 390 395 400
Â
Asp Leu Arg Glu Ser Gly Ser Ile Glu Gln Asp Ala Asp Leu Ile Met
405 410 415
Â
Phe Ile Tyr Arg Asp Glu Val Tyr His Glu Asn Ser Asp Leu Lys Gly
420 425 430
Â
Ile Ala Glu Ile Ile Ile Gly Lys Gln Arg Asn Gly Pro Ile Gly Thr
435 440 445
Â
Val Arg Leu Thr Phe Asn Gly Gln Trp Ser Arg Phe Asp Asn Tyr Ala
450 455 460
Â
Gly Pro Gln Tyr Asp Asp Glu
465 470
Â
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