Disclosure of Invention
In order to solve the problems, the extracellular expression quantity of the pullulanase is further improved, and the extracellular expression of the pullulanase can be improved by synonymous mutation means. Synonymous mutations in genes can leave the amino acid sequence unchanged, but can produce significant changes in gene transcript levels. The invention modifies the coding region of the N end of the gene by synonymous mutation means, which not only can realize the remarkable improvement of the gene expression quantity, but also has almost negligible influence on the enzyme activity. The signal peptide screened by synonymous mutation can obviously improve the extracellular enzyme activity of pullulanase.
The invention provides a kind of signal peptide, the nucleotide sequence of which is shown as any one of SEQ ID NO. 1-3.
In one embodiment of the invention, the nucleotide sequences of the signal peptides are synonymous mutant sequences of the signal peptides AprE, sacB, epr, respectively.
In one embodiment of the invention, the synonymous mutant sequence of the signal peptide AprE is shown in SEQ ID No. 1; the synonymous mutation sequence of the signal peptide SacB is shown in SEQ ID NO. 2; the synonymous mutant sequence of the signal peptide Epr is shown in SEQ ID NO. 3.
The invention provides a recombinant plasmid containing signal peptide with nucleotide sequence shown as SEQ ID NO. 1-3.
In one embodiment of the invention, the heavy plasmid is based on any vector that can be expressed in bacillus subtilis.
In one embodiment of the invention, the departure vector comprises P43NMK.
In one embodiment of the invention, the recombinant plasmid further comprises a constitutive promoter.
In one embodiment of the invention, the constitutive promoter comprises a LytR promoter, the LytR promoter nucleotide sequence of which is shown in SEQ ID NO. 4.
The invention provides recombinant bacteria containing signal peptides with nucleotide sequences shown as SEQ ID NO. 1-3.
In one embodiment of the invention, the recombinant bacterium is a bacillus subtilis host.
In one embodiment of the present invention, the recombinant bacterium is hosted by bacillus subtilis WB600, or bacillus subtilis 168.
The invention provides a method for improving the expression quantity of protein, which takes recombinant bacteria containing signal peptide with nucleotide sequences shown as SEQ ID NO. 1-3 as fermentation strain to produce protein.
In one embodiment of the present invention, the recombinant bacterium is cultured to OD 600 Bacterial liquid not lower than 3.0, and inoculating the bacterial liquid into a reaction system according to the proportion of 1-10 mL/100 mL.
In one embodiment of the present invention, the medium of the culture system is TB medium, and is cultured at 30 to 40℃and 200 to 250rpm for 25 to 35 hours.
The invention provides a method for improving the expression quantity of extracellular proteins of bacillus subtilis, which adds signal peptide with nucleotide sequences shown as SEQ ID NO. 1-3 at the N end of a nucleotide sequence of a coded protein.
In one embodiment of the invention, a signal peptide with a nucleotide sequence shown as SEQ ID NO. 1-3 is added at the N-terminal of a target protein, and a recombinant plasmid is constructed and introduced into bacillus subtilis.
In one embodiment of the present invention, the signal peptide having the nucleotide sequence shown in SEQ ID NO.1 to 3 is inserted after the initiation codon ATG of the nucleotide sequence encoding the target protein.
In one embodiment of the invention, the protein of interest is any protein capable of extracellular expression in bacillus subtilis.
In one embodiment of the invention, the protein of interest comprises pullulanase and/or sfGFP.
In one embodiment of the present invention, the NCBI accession number of the pullulanase amino acid sequence is AMQ67157, and the amino acid sequence is shown in SEQ ID NO. 5.
In one embodiment of the invention, the nucleotide sequence encoding said sfGFP is shown in SEQ ID No. 7.
The invention also protects the application of the signal peptide with the nucleotide sequence shown as SEQ ID NO. 1-3, or the recombinant plasmid containing the signal peptide with the nucleotide sequence shown as SEQ ID NO. 1-3, or the recombinant bacteria containing the signal peptide with the nucleotide sequence shown as SEQ ID NO. 1-3 in improving the expression quantity of extracellular proteins.
The invention also protects the application of the method for improving the protein expression quantity in improving the extracellular protein expression quantity.
The invention also provides application of the method for improving the extracellular protein expression quantity of the bacillus subtilis in improving the extracellular protein expression quantity.
The invention also protects the application of the recombinant plasmid, the recombinant bacterium or the signal peptide AprE, sacB and Epr synonymous mutant sequences in improving the expression quantity of extracellular proteins.
The invention has the beneficial effects that:
the invention screens synonymous mutant sequences capable of improving the extracellular expression quantity of protein through synonymous mutation of signal peptides AprE, sacB and Epr, fuses the obtained mutant sequences at the N end of pullulanase, can obviously improve and reduce the extracellular enzyme activity yield of the pullulanase, and can respectively improve the extracellular enzyme activity of the pullulanase by 2.92,1.08,1.37 times in bacillus subtilis transformation production.
Detailed Description
Seed culture medium: peptone 10g/L, yeast extract 5g/L, sodium chloride 5g/L.
The fermentation medium is TB medium: peptone 12g/L, yeast extract 24g/L, glycerol 4mL/L, KH 2 PO 4 0.017mol/L,K 2 HPO 4 0.072mol/L。
Seed culture: and (3) picking a single colony of engineering bacteria, inoculating the single colony of engineering bacteria into a triangular flask (250 mL) with the liquid loading amount of 25mL, and culturing at the culture temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min for 12h.
Fermentation culture: the culture was carried out at 37℃for 48 hours by inoculating the culture medium to a flask (250 mL) having a liquid loading amount of 25mL at an inoculum size of 4% (v/v).
Green fluorescent protein expression amount and biomass measurement: in a 96-well plate, if 200 μl of diluted broth was used, using a Cystation 3 cell imaging microplate detector (Bertoni instruments Co., U.S.) green fluorescence excitation wavelength: 480nm, green fluorescence emission wavelength: 520nm, cell growth OD absorbance wavelength: 600nm.
The enzyme activity determination method of the pullulanase comprises the following steps: 1mL of pullulan substrate and 0.9mL 100mM pH 4.5 acetic acid-sodium acetate buffer solution with 1mg/100mL are uniformly mixed, placed in a water bath kettle with the temperature of 60 ℃ for preheating for 10min, 0.1mL of pullulanase solution is added for reaction for 10min, 3mL of DNS color development solution is added, then the mixture is boiled in a boiling water bath for 7min, placed in ice water for stopping the color development reaction, 10mL of deionized water is added, uniformly mixed, and the light absorption value is measured at 540 nm.
Definition of enzyme activity: the amount of enzyme that produces 1. Mu. Mol of reducing sugar per unit time is one enzyme activity unit.
Example 1: construction of Apre Signal peptide NCS synonymous mutation library
Connecting an LytR promoter (the sequence information of which is shown as SEQ ID NO. 4) to a P43NMK plasmid through a one-step cloning kit (purchased from Nanjinouzan biotechnology Co., ltd.) to construct a recombinant plasmid, transferring the recombinant plasmid into E.coli JM109, coating bacterial liquid on an LB plate containing 100 mug/L ampicillin resistance, culturing at 37 ℃ until a monoclonal grows, and picking up the monoclonal to verify by sequencing to obtain the plasmid P43NMK-LytR; by using the same one-step cloning method, a pullulanase gene (the nucleotide sequence of which is shown as SEQ ID NO. 6) is fused to the downstream of LytR through a one-step cloning kit, and a recombinant plasmid P43NMK-LytR-Pul is constructed; by using the same one-step cloning method, sfGFP fluorescent protein and Apre signal peptide (the nucleotide sequences are shown as SEQ ID NO.7 and 8) are respectively connected to the C end and the N end of pullulanase, and recombinant plasmid P43NMK-Apre-Pul is constructed.
The P43NMK-Apre-Pul is used as a template, and an upstream and downstream degenerate primer is used to obtain a synonymous mutation library (synonymous mutation recombinant plasmid) of the front 30 bases of the N end of Apre through PCR, namely, the front 30 amino acid sequence is unchanged, and the nucleotide sequence is changed.
Upstream degenerate primer:
ATGAGRAGYAARAARTTRTGGATHAGYTTRTTRtttgcgttaacgttaatctttacgatgg(SEQ ID NO.12);
downstream degenerate primers:
GTGTACATTTCACCTCCTTTAAATTACTTTCATTAT(SEQ ID NO.11)。
example 2: construction of SacB Signal peptide NCS synonymous mutation library
Specific embodiments are the same as in example 1, except that after P43NMK-LytR-Pul is obtained, a recombinant plasmid P43NMK-SacB-Pul is constructed by connecting sfGFP fluorescent protein and SacB signal peptide (nucleotide sequence shown as SEQ ID NO. 9) to the C-terminus (sfGFP fluorescent protein) and N-terminus (SacB signal peptide) of pullulanase, respectively, by a one-step cloning method.
The P43NMK-SacB-Pul is used as a template, and a synonymous mutation library (synonymous mutation recombinant plasmid) of the front 30 bases of the N end of the SacB is obtained by PCR (polymerase chain reaction) by using upstream and downstream degenerate primers, namely, the front 30 amino acid sequence is unchanged, and the nucleotide sequence is changed.
Upstream degenerate primer:
ATGCTNAARAGRACNTCNTTYGTNTCNTCNTTRttcatcagttcagctgttttactatcaatct(SEQ ID NO.13);
downstream degenerate primers:
GTGTACATTTCACCTCCTTTAAATTACTTTCATTAT(SEQ ID NO.11)。
example 3: construction of Epr Signal peptide NCS synonymous mutation library
Specific embodiments are the same as in example 1, except that after P43NMK-LytR-Pul was obtained, a recombinant plasmid P43NMK-SacB-Pul was constructed by connecting sfGFP fluorescent protein and Epr signal peptide (nucleotide sequence shown in SEQ ID NO. 10) to the C-terminus (sfGFP fluorescent protein) and N-terminus (SacB signal peptide) of pullulanase, respectively, using a one-step cloning method.
And (3) taking P43NMK-Epr-Pul as a template, using upstream and downstream degenerate primers, and obtaining a synonymous mutation library (synonymous mutation recombinant plasmid) of the front 30 bases of the N end of Epr by PCR, wherein the front 30 amino acid sequence is unchanged, and the nucleotide sequence is changed.
Upstream degenerate primer:
ATGTTRAARAARGTNATHTTRGCNGCNTTYATHttagtaggaagtactttgggagctttta(SEQ ID NO.14);
downstream degenerate primers:
GTGTACATTTCACCTCCTTTAAATTACTTTCATTAT(SEQ ID NO.11)。
example 4: screening of NCS synonymous mutant library Signal peptide nucleotide sequence
(1) The synonymous mutant recombinant plasmids constructed in examples 1-3 are respectively transformed into an expression host bacillus subtilis WB600, the transformation solution is coated on an LB plate containing 50 mug/mL of the resistance of the calicheamicin for challenging growth, the culture is carried out at 37 ℃ until monoclonal grows, the monoclonal is picked up to a 96 shallow pore plate containing 200 mug/mL of LB culture medium containing 50 mug/mL of the resistance of the calicheamicin, and the culture is carried out for 8 hours, so as to obtain seed solution;
(2) Inoculating the seed solution into a 96-deep well plate containing 50 mug/mL of a calicheamicin resistant 800 mug TB culture medium according to an inoculum size of 4mL/100mL, and culturing for 24 hours to obtain a fermentation broth;
(3) The fermentation broth was rapidly frozen on ice, centrifuged, the supernatant removed, diluted to a suitable multiple with 100mM PBS buffer (pH 7.2), and the fluorescence (excitation light 480, absorption light 520) and OD were measured by a microplate reader 600 。
Defining the relative fluorescence intensity RFI=fluorescence value/OD, and selecting the cell with the highest relative fluorescence intensity according to the RFI value to send to the Shanghai engineering company for sequencing.
After sequencing and identification, the AprE signal peptide successfully realizes synonymous mutation at the N end, and the nucleotide sequence of a mutant part of the AprE signal peptide is shown as SEQ ID NO. 15; the SacB signal peptide successfully realizes synonymous mutation at the N end, and the nucleotide sequence of a mutant part of the SacB signal peptide is shown as SEQ ID NO. 16; the Epr signal peptide successfully realizes synonymous mutation at the N end, and the nucleotide sequence of the mutant part is shown as SEQ ID NO.17.
Example 5: application of synonymous mutant signal peptide in improving extracellular expression quantity of pullulanase
Extracting plasmids from sequenced cells having the highest fluorescence intensity obtained in example 4, removing sfGFP fluorescent protein by PCR using primers to obtain recombinant plasmids from which fluorescent protein was removed, transforming recombinant plasmids each containing AprE signal peptide, sacB signal peptide, and Epr signal peptide synonymous mutant sequences into Bacillus subtilis WB600 to obtain recombinant bacteria, inoculating the recombinant bacteria into 250mL shake flask containing 50. Mu.g/mL of calicheamicin-resistant 20mL LB medium, fermenting at 37℃and 220rpm for 8 hours to obtain OD 600 Above 4, the cells were inoculated in a proportion of 4mL/100mL into 250mL shake flasks containing 50. Mu.g/mL of a calicheamicin-resistant 25mL TB medium, and after fermentation at 37℃and 250rpm for 30 hours, the extracellular enzyme activities of pullulanase were measured, and the results are shown in FIG. 3.
Removal of sfGFP upstream primer:
CGGTAAAAAATAAtgagattatcaaaaaggatcttcacctagat(SEQ ID NO.18);
removal of sfGFP downstream primer:
gataatctcaTTATTTTTTACCGTGATCTGGAGAAACttc(SEQ ID NO.19)。
by measuring the extracellular enzyme activity of pullulanase, the synonymous mutant sequences of the Apre, epr, sacB signal peptide are found to respectively increase the extracellular enzyme activity 2.92,1.08,1.37 times compared with the wild type.
TABLE 1 pullulanase extracellular enzyme Activity (U/mL)
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> Jilin grain Biochemical Co., ltd
Jiangnan University
<120> Signal peptide promoting extracellular expression of pullulanase
<160> 19
<170> PatentIn version 3.3
<210> 1
<211> 84
<212> DNA
<213> artificial sequence
<400> 1
agaagtaaaa aattatggat aagtttatta tttgcgttaa cgttaatctt tacgatggcg 60
ttcagcaaca tgtctgcgca ggct 84
<210> 2
<211> 84
<212> DNA
<213> artificial sequence
<400> 2
aatataaaaa aatttgcaaa acaagctaca gtattaacct ttactaccgc actgctggca 60
ggaggcgcaa ctcaagcgtt tgcg 84
<210> 3
<211> 78
<212> DNA
<213> artificial sequence
<400> 3
aaaaatatgt cttgtaaact agttgtatct gtcactctgt ttttcagttt tctcaccata 60
ggccctctcg ctcatgcg 78
<210> 4
<211> 320
<212> DNA
<213> artificial sequence
<400> 4
ctaaccctac ataagtacct tcttttgttt caatgttact gtctggcgat acatcttcac 60
cttgactctt ttgactatta accccgcaac ccgaaagaag caatataaag aacagtaaag 120
caataaattt tttcattttt ttcacctcat tatattttat cgtcaaccta ttttatattt 180
taaagaaaaa ttaagaaaca atgaaacttt tttttataaa aaacgactat tttaggattt 240
cattcttgta ttaaatagag ttgtatttat tggaaattta actcataatg aaagtaattt 300
aaaggaggtg aaatgtacac 320
<210> 5
<211> 724
<212> PRT
<213> artificial sequence
<400> 5
Asp Ala Ala Lys Pro Ala Val Ser Asn Ala Tyr Leu Asp Ala Ser Asn
1 5 10 15
Gln Val Leu Val Lys Leu Ser Gln Pro Leu Thr Leu Gly Glu Gly Ala
20 25 30
Ser Gly Phe Thr Val His Asp Asp Thr Ala Asn Lys Asp Ile Pro Val
35 40 45
Thr Ser Val Lys Asp Ala Ser Leu Gly Gln Val Glu Ser Gly Val Lys
50 55 60
Thr Asp Leu Val Thr Val Thr Leu Gly Glu Asp Pro Asp Val Ser His
65 70 75 80
Thr Leu Ser Ile Gln Thr Asp Gly Tyr Gln Ala Lys Gln Val Ile Pro
85 90 95
Arg Asn Val Leu Asn Ser Ser Gln Tyr Tyr Tyr Ser Gly Asp Asp Leu
100 105 110
Gly Asn Thr Tyr Thr Gln Lys Ala Thr Thr Phe Lys Val Trp Ala Pro
115 120 125
Thr Ser Thr Gln Val Asn Val Leu Leu Tyr Asp Ser Ala Thr Gly Ser
130 135 140
Val Thr Lys Ile Val Pro Met Thr Ala Ser Gly His Gly Val Trp Glu
145 150 155 160
Ala Thr Val Asn Gln Asn Leu Glu Asn Trp Tyr Tyr Met Tyr Glu Val
165 170 175
Thr Gly Gln Gly Ser Thr Arg Thr Ala Val Asp Pro Tyr Ala Thr Ala
180 185 190
Ile Ala Pro Asn Gly Thr Arg Gly Met Ile Val Asp Leu Ala Lys Thr
195 200 205
Asp Pro Ala Gly Trp Asn Ser Asp Lys His Ile Thr Pro Lys Asn Ile
210 215 220
Glu Asp Glu Val Ile Tyr Glu Met His Val Arg Asp Phe Ser Ile Asp
225 230 235 240
Pro Asn Ser Gly Met Lys Asn Lys Gly Lys Tyr Leu Ala Leu Thr Glu
245 250 255
Lys Gly Thr Lys Gly Pro Asp Asn Val Lys Thr Gly Ile Asp Ser Leu
260 265 270
Lys Gln Leu Gly Ile Thr His Val Gln Leu Met Pro Val Phe Ala Ser
275 280 285
Asn Ser Val Asp Glu Thr Asp Pro Thr Gln Tyr Asn Trp Gly Tyr Asp
290 295 300
Pro Arg Asn Tyr Asp Val Pro Glu Gly Gln Tyr Ala Thr Asn Ala Asn
305 310 315 320
Gly Asn Ala Arg Ile Lys Glu Phe Lys Glu Met Val Leu Ser Leu His
325 330 335
Arg Glu His Ile Gly Val Asn Met Asp Val Val Tyr Asn His Thr Phe
340 345 350
Ala Thr Gln Ile Ser Asp Phe Asp Lys Ile Val Pro Glu Tyr Tyr Tyr
355 360 365
Arg Thr Asp Asp Ala Gly Asn Tyr Thr Asn Gly Ser Gly Thr Gly Asn
370 375 380
Glu Ile Ala Ala Glu Arg Pro Met Val Gln Lys Phe Ile Ile Asp Ser
385 390 395 400
Leu Lys Tyr Trp Val Asn Glu Tyr His Ile Asp Gly Phe Arg Phe Asp
405 410 415
Leu Met Ala Leu Leu Gly Lys Asp Thr Met Ser Lys Ala Ala Ser Glu
420 425 430
Leu His Ala Ile Asn Pro Gly Ile Ala Leu Tyr Gly Glu Pro Trp Thr
435 440 445
Gly Gly Thr Ser Ala Leu Pro Asp Asp Gln Leu Leu Thr Lys Gly Ala
450 455 460
Gln Lys Gly Met Gly Val Ala Val Phe Asn Asp Asn Leu Arg Asn Ala
465 470 475 480
Leu Asp Gly Asn Val Phe Asp Ser Ser Ala Gln Gly Phe Ala Thr Gly
485 490 495
Ala Thr Gly Leu Thr Asp Ala Ile Lys Asn Gly Val Glu Gly Ser Ile
500 505 510
Asn Asp Phe Thr Ser Ser Pro Gly Glu Thr Ile Asn Tyr Val Thr Ser
515 520 525
His Asp Asn Tyr Thr Leu Trp Asp Lys Ile Ala Leu Ser Asn Pro Asn
530 535 540
Asp Ser Glu Ala Asp Arg Ile Lys Met Asp Glu Leu Ala Gln Ala Val
545 550 555 560
Val Met Thr Ser Gln Gly Val Pro Phe Met Gln Gly Gly Glu Glu Met
565 570 575
Leu Arg Thr Lys Gly Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Ala
580 585 590
Val Asn Glu Phe Asp Trp Ser Arg Lys Ala Gln Tyr Pro Asp Val Phe
595 600 605
Asn Tyr Tyr Ser Gly Leu Ile His Leu Arg Leu Asp His Pro Ala Phe
610 615 620
Arg Met Thr Thr Ala Asn Glu Ile Asn Ser His Leu Gln Phe Leu Asn
625 630 635 640
Ser Pro Glu Asn Thr Val Ala Tyr Glu Leu Thr Asp His Val Asn Lys
645 650 655
Asp Lys Trp Gly Asn Ile Ile Val Val Tyr Asn Pro Asn Lys Thr Val
660 665 670
Ala Thr Ile Asn Leu Pro Ser Gly Lys Trp Ala Ile Asn Ala Thr Ser
675 680 685
Gly Lys Val Gly Glu Ser Thr Leu Gly Gln Ala Glu Gly Ser Val Gln
690 695 700
Val Pro Gly Ile Ser Met Met Ile Leu His Gln Glu Val Ser Pro Asp
705 710 715 720
His Gly Lys Lys
<210> 6
<211> 2172
<212> DNA
<213> artificial sequence
<400> 6
gatgctgcta aaccagcagt ttctaacgct taccttgacg cttctaacca agttttagtt 60
aaattatctc aaccattaac attaggtgaa ggtgcttctg gtttcactgt acatgatgac 120
actgctaaca aagacatccc agtaacatct gtaaaagacg cttctttagg tcaagttgaa 180
tcaggtgtaa aaactgacct tgttactgtt actttaggcg aagatccaga tgtatctcac 240
actttatcta tccaaacaga cggttaccaa gctaaacaag taatcccacg taacgtactt 300
aactcttctc aatattacta ttctggtgat gatttaggaa acacatacac acaaaaagct 360
actactttca aagtttgggc tcctacatct actcaagtta acgtattgtt atacgattct 420
gctacaggta gcgttacaaa aatcgttcca atgacggctt caggtcacgg tgtttgggag 480
gctactgtta accaaaactt agaaaactgg tactacatgt acgaagtaac tggtcaaggt 540
tctacacgca ctgctgttga tccttacgct actgctatcg ctccaaacgg tacacgcggc 600
atgatcgtag atttagctaa aactgaccca gcaggttgga actctgataa acacattact 660
ccaaaaaaca ttgaagatga agttatctac gaaatgcacg tacgtgattt ctctatcgat 720
ccaaactcag gtatgaaaaa caaaggtaaa tacttagctc taactgaaaa aggcactaaa 780
ggtcctgata acgttaaaac aggtatcgac tctcttaagc aattaggtat tacacatgtt 840
caattaatgc cagttttcgc atctaactca gttgacgaaa ctgatccaac acaatacaac 900
tggggttacg acccacgtaa ctacgatgta ccagaaggtc aatatgcaac taacgctaac 960
ggtaacgcac gtattaaaga attcaaagaa atggttttat cactacaccg tgagcacatc 1020
ggtgttaaca tggacgttgt ttacaaccac acgttcgcta ctcaaatctc tgacttcgat 1080
aaaattgttc cagagtacta ttaccgcact gacgacgcag gtaactacac taacggttct 1140
ggtactggta acgaaattgc tgcagaacgt cctatggtgc aaaaattcat catcgatagc 1200
cttaaatact gggttaacga ataccacatt gacggcttcc gtttcgactt aatggcttta 1260
cttggtaaag acacaatgtc taaggctgct tctgagttac atgctatcaa cccaggtatt 1320
gctttatatg gcgaaccttg gactggtggt acaagcgctc ttcctgacga ccaactttta 1380
actaaaggtg cacaaaaagg catgggagta gctgtattca acgataacct tcgtaacgca 1440
ttagacggaa acgttttcga ttcttctgct caaggattcg caacaggagc tacaggtctg 1500
actgatgcta ttaaaaacgg agttgaagga tcaatcaacg atttcacttc ttctcctggc 1560
gaaacaatta actacgttac atcacacgat aactacactc tttgggacaa aatcgctttg 1620
tctaacccta acgactctga agcagatcgc atcaaaatgg atgagcttgc tcaagctgtt 1680
gttatgactt ctcaaggtgt acctttcatg caaggtggtg aagaaatgtt acgcactaaa 1740
ggtggtaacg ataacagcta taacgcgggt gatgctgtaa acgaattcga ctggtctcgt 1800
aaagctcaat accctgacgt tttcaactac tactcaggtt taatccacct tcgtcttgac 1860
catccagctt tccgtatgac aacagctaac gaaatcaact ctcaccttca attccttaac 1920
tcacctgaaa acacagtagc ttacgaactt actgaccacg taaacaaaga taaatggggt 1980
aacattatcg ttgtttacaa ccctaacaag actgtagcaa ctatcaactt accatctggt 2040
aaatgggcta tcaacgcaac tagcggtaaa gtaggtgaat ctacattagg tcaagctgaa 2100
ggatctgtac aagttcctgg tatttctatg atgatccttc accaagaagt ttctccagat 2160
cacggtaaaa aa 2172
<210> 7
<211> 717
<212> DNA
<213> artificial sequence
<400> 7
gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60
gacgtaaacg gccacaagtt cagcgtgaga ggcgagggcg agggcgatgc caccaatggc 120
aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180
gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcgc 240
cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcagtttc 300
aaggacgacg gcacatacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360
aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420
ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca gaagaacggc 480
atcaaggcca acttcaagat ccgccacaac gtggaggacg gcagcgtgca gctcgccgac 540
cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600
ctgagcaccc agtccgtgct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660
ctggagttcg tgaccgccgc cgggatcact cacggcatgg acgagctgta caagtaa 717
<210> 8
<211> 84
<212> DNA
<213> artificial sequence
<400> 8
agaagcaaaa aattgtggat cagcttgttg tttgcgttaa cgttaatctt tacgatggcg 60
ttcagcaaca tgtctgcgca ggct 84
<210> 9
<211> 84
<212> DNA
<213> artificial sequence
<400> 9
aacatcaaaa agtttgcaaa acaagcaaca gtattaacct ttactaccgc actgctggca 60
ggaggcgcaa ctcaagcgtt tgcg 84
<210> 10
<211> 78
<212> DNA
<213> artificial sequence
<400> 10
aaaaacatgt cttgcaaact tgttgtatca gtcactctgt ttttcagttt tctcaccata 60
ggccctctcg ctcatgcg 78
<210> 11
<211> 36
<212> DNA
<213> artificial sequence
<400> 11
gtgtacattt cacctccttt aaattacttt cattat 36
<210> 12
<211> 61
<212> DNA
<213> artificial sequence
<400> 12
atgagragya araarttrtg gathagyttr ttrtttgcgt taacgttaat ctttacgatg 60
g 61
<210> 13
<211> 64
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (6)..(6)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (15)..(15)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (18)..(18)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (24)..(24)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, or t
<400> 13
atgctnaara gracntcntt ygtntcntcn ttrttcatca gttcagctgt tttactatca 60
atct 64
<210> 14
<211> 61
<212> DNA
<213> artificial sequence
<220>
<221> misc_feature
<222> (15)..(15)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (24)..(24)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, or t
<400> 14
atgttraara argtnathtt rgcngcntty athttagtag gaagtacttt gggagctttt 60
a 61
<210> 15
<211> 30
<212> DNA
<213> artificial sequence
<400> 15
agaagtaaaa aattatggat aagtttatta 30
<210> 16
<211> 30
<212> DNA
<213> artificial sequence
<400> 16
aatataaaaa aatttgcaaa acaagctaca 30
<210> 17
<211> 30
<212> DNA
<213> artificial sequence
<400> 17
aaaaatatgt cttgtaaact agttgtatct 30
<210> 18
<211> 44
<212> DNA
<213> artificial sequence
<400> 18
cggtaaaaaa taatgagatt atcaaaaagg atcttcacct agat 44
<210> 19
<211> 40
<212> DNA
<213> artificial sequence
<400> 19
gataatctca ttatttttta ccgtgatctg gagaaacttc 40