Foreign gene is carried out the multiple copied Yeast expression carrier of correct secreting, expressing
Technical field
The present invention relates to a kind of Yeast expression carrier and make up primer, particularly relate to a kind of multiple copied Yeast expression carrier and structure primer thereof that foreign gene is carried out correct secreting, expressing.
Background technology
Pichia yeast expression system (Pichia pastoris) is used to one of the most successful expression system of expressing foreign protein, development in recent years is very rapid, at present existing 200 multiple protein are expressed (CreggJM in this expression system, Cereghino JL, Shi JY, et al.Recombinant protein expression in Pichiapastoris[J] .Mol Biotechnol, 2000,16 (1): 23-52.).Pichia spp is a kind of methyl alcohol nutritional type yeast, its growth rapidly, culture condition is simple, can the high-density cultured continuously, genetic manipulation is similar to yeast saccharomyces cerevisiae, technology is maturation quite.
Pichia yeast expression system comprises in the born of the same parents expresses and the secreting, expressing dual mode, and wherein secreting, expressing is owing to its expression amount height, and glycosylation is complete, and disulfide linkage and higher structure form correctly, and especially the later separation purifying is simple and convenient and enjoy favor.Proteic secreting, expressing needs the participation of signal peptide.Had some kinds of signal peptide sequences of the signal peptide sequence that comprises some foreign protein self to be successfully used to the secreting, expressing of foreign protein, but wherein the most successful be the signal peptide sequence that from yeast saccharomyces cerevisiae, separates the alpha factor that obtains.
Below be the partial sequence of signal peptide alpha factor and multiple clone site:
Xho?I?????????Kex2????????????????SnaB?I???EcoR?IATG?AGA?TTT?CCT?…?TCT
CTC?GAG?AAA?AGA↓GAG?GCT??GAA?GCT
TAC?GTA?
GAA?TTC?CCT?…Met?Arg?Phe?Pro?…?Ser?Leu?Glu?Lys?Arg??Glu?Ala↑Glu?Ala↑Tyr?Val?Glu?Phe?Pro?…
Ste13
ATG is a translation initiation codon, and Kex2 is responsible for the shearing of signal peptide, and it is essential that the Glu-Ala-Glu-Ala tumor-necrosis factor glycoproteins is not Kex2 shearing institute, but its existence can improve shear efficiency; Another kind of proteolytic enzyme Ste13 can continue the Glu-Ala-Glu-Ala tumor-necrosis factor glycoproteins is sheared, but shears often also not exclusively.In addition, also have two amino-acid residues can stay the N end (Tyr and Val) of expressing protein at least.
For above-mentioned reasons, existing Yeast expression carrier, when foreign gene is carried out secreting, expressing, expressing protein often exists incorrect N terminal sequence, these amino-acid residues that have more can influence and change expressed proteic 26S Proteasome Structure and Function (Goda S, Takano K, Yamagata Y, et al.Effect of extra N-terminal residueson the stability and folding of human lysozyme expressed in Pichia pastoris[J] .Protein Eng, 2000,13 (4): 299-307.).
Summary of the invention
The purpose of this invention is to provide a kind of multiple copied Yeast expression carrier that can make foreign gene correct secreting, expressing in yeast.
A kind of multiple copied Yeast expression carrier provided by the present invention, its dna sequence dna is the nucleotide sequence of sequence 1 in the sequence table.
A kind of multiple copied Yeast expression carrier provided by the present invention, contain two resistant genes of intestinal bacteria replication orgin Col E1 and penbritin and kantlex, wherein kalamycin resistance gene can make pichia spp that G418 is produced resistance, is used for the screening that multiple copied inserts.
A kind of multiple copied Yeast expression carrier provided by the present invention is an inducible promoter with the promotor (pAOX1) of alcohol oxidase gene, suppressed by glucose, and just transcriptional start, translation is very suitable for expression of exogenous gene behind the methanol induction.
Multiple copied Yeast expression carrier of the present invention is called pMEX9K.
Second purpose of the present invention provides a kind of primer special that makes up above-mentioned multiple copied Yeast expression carrier.
Two pairs of primers provided by the present invention, they are nucleotide sequences of sequence 2 in the sequence table, sequence 3 and sequence 4, sequence 5.
Utilize carrier of the present invention, make foreign protein N hold first amino acid to be close to after Lys-Arg, foreign protein energy secreting, expressing is correct, and N end sequencing result shows that the albumen of secreting, expressing is consistent with the native protein structure.
Description of drawings
Fig. 1 is the structure synoptic diagram of expression vector of the present invention.
Fig. 2 cuts evaluation for the enzyme of expression vector of the present invention.
Fig. 3-Fig. 9 is that foreign gene (people's ω-Interferon, rabbit) is measured figure at the N terminal sequence of the recombinant protein of expression vector pMEX9K secreting, expressing of the present invention
Embodiment
The structure of embodiment 1, carrier pMEX9K of the present invention
As shown in Figure 1, the structure of carrier pMEX9K of the present invention may further comprise the steps:
(1) the plasmid pPIC9K DNA that sells with Invitrogen company is a template, is primer pyrobest enzymatic amplification purpose fragment with sequence in the sequence table 2 and sequence 3.The condition of PCR is: 94 ℃ of pre-sex change 180s; 94 ℃ of sex change 40s; 55 ℃ of annealing 40s; 72 ℃ are extended 60s; 30 circulations; Add general T aq enzyme again and extend 10min;
(2) 1% agarose gel electrophoresis are cut glue and are reclaimed the PCR product and be connected into the pGEMT of TaKaRa company carrier.Behind the transformed into escherichia coli DH5 α, screening positive clone pGEMTXho.
(3) be that primer utilizes point mutation test kit MutanBEST Kit that pGEMTXho is suddenlyd change to remove Xho I restriction enzyme site wherein with sequence in the sequence table 4 and sequence 5.The operation by specification carries out.
(4) the recombinant plasmid pGEMTMuXho after screening and the evaluation sudden change.
(5) downcut the external source fragment MuXho (0.6kb) of sudden change with restriction endonuclease sma I and Sph I from recombinant plasmid pGEMTMuXho, and cut the dna fragmentation (8.7kb) that glue reclaims after being connected into the above-mentioned two kinds of endonuclease digestion pPIC9K of same usefulness.
(6) behind the transformed into escherichia coli DH5 α, screening positive clone pMEX9K and order-checking.Sequencing result shows that sequence is correct.
The enzyme of embodiment 2, expression vector of the present invention is cut evaluation
As shown in Figure 2, confirm that through restriction endonuclease analysis and sequencing vector construction is correct, among the figure: M1.DL15000 Marker; 1.pPIC9K; 2.pMEX9K; 3.pPIC9K/Nae I; 4.pPIC9K/Xho I; 5.pMEX9K/Xho I; M2.DL2000 Marker.Behind the transformed into escherichia coli DH5 α, this carrier makes recipient bacterium obtain penbritin and kalamycin resistance, confirms that the kalamycin resistance gene function is normal.
Embodiment 3, utilize carrier of the present invention to make foreign gene correct secreting, expressing in pichia spp
Adopt the ordinary method in the biotechnology, utilize carrier of the present invention that foreign gene people ω-Interferon, rabbit (Interferon ω) is imported in the pichia spp and carry out secreting, expressing, expressed proteins is carried out N terminal sequence mensuration (utilize 491 sequential analysis of protein instrument of PE company, adopt the automatic edman degradation method), the result shows (Fig. 3): preceding 5 amino-acid residues of target protein N end, its sequence is: LGCDL, and consistent with native protein N terminal amino acid sequence.
Sequence Table
<160> 5
<210> 1
<211> 9276
<212> DNA
<213> Artificial Sequence
<220>
<223>
<400> Serial Number
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcgcca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaagga tccaaacgat gagatttcct 960
tcaattttta ctgcagtttt attcgcagca tcctccgcat tagctgctcc agtcaacact 1020
acaacagaag atgaaacggc acaaattccg gctgaagctg tcatcggtta ctcagattta 1080
gaaggggatt tcgatgttgc tgttttgcca ttttccaaca gcacaaataa cgggttattg 1140
tttataaata ctactattgc cagcattgct gctaaagaag aaggggtatc tctcgagaaa 1200
agagaggctg aagcttacgt agaattccct agggcggccg cgaattaatt cgccttagac 1260
atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa 1320
tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt 1380
ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc 1440
ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa 1500
tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta 1560
agtgagaagt tcgtttgtgc aagcttatcg ataagcttta atgcggtagt ttatcacagt 1620
taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc 1680
tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc 1740
tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg ctgctagcgc 1800
tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg 1860
gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac gcgatcatgg 1920
cgaccacacc cgtcctgtgg atctatcgaa tctaaatgta agttaaaatc tctaaataat 1980
taaataagtc ccagtttctc catacgaacc ttaacagcat tgcggtgagc atctagacct 2040
tcaacagcag ccagatccat cactgcttgg ccaatatgtt tcagtccctc aggagttacg 2100
tcttgtgaag tgatgaactt ctggaaggtt gcagtgttaa ctccgctgta ttgacgggca 2160
tatccgtacg ttggcaaagt gtggttggta ccggaggagt aatctccaca actctctgga 2220
gagtaggcac caacaaacac agatccagcg tgttgtactt gatcaacata agaagaagca 2280
ttctcgattt gcaggatcaa gtgttcagga gcgtactgat tggacatttc caaagcctgc 2340
tcgtaggttg caaccgatag ggttgtagag tgtgcaatac acttgcgtac aatttcaacc 2400
cttggcaact gcacagcttg gttgtgaaca gcatcttcaa ttctggcaag ctccttgtct 2460
gtcatatcga cagccaacag aatcacctgg gaatcaatac catgttcagc ttgagacaga 2520
aggtctgagg caacgaaatc tggatcagcg tatttatcag caataactag aacttcagaa 2580
ggcccagcag gcatgtcaat actacacagg gctgatgtgt cattttgaac catcatcttg 2640
gcagcagtaa cgaactggtt tcctggacca aatattttgt cacacttagg aacagtttct 2700
gttccgtaag ccatagcagc tactgcctgg gcgcctcctg ctagcacgat acacttagca 2760
ccaaccttgt gggcaacgta gatgacttct ggggtaaggg taccatcctt cttaggtgga 2820
gatgcaaaaa caatttcttt gcaaccagca actttggcag gaacacccag catcagggaa 2880
gtggaaggca gaattgcggt tccaccagga atatagaggc caactttctc aataggtctt 2940
gcaaaacgag agcagactac accagggcaa gtctcaactt gcaacgtctc cgttagttga 3000
gcttcatgga atttcctgac gttatctata gagagatcaa tggctctctt aacgttatct 3060
ggcaattgca taagttcctc tgggaaagga gcttctaaca caggtgtctt caaagcgact 3120
ccatcaaact tggcagttag ttctaaaagg gctttgtcac cattttgacg aacattgtcg 3180
acaattggtt tgactaattc cataatctgt tccgttttct ggataggacg acgaagggca 3240
tcttcaattt cttgtgagga ggccttagaa acgtcaattt tgcacaattc aatacgacct 3300
tcagaaggga cttctttagg tttggattct tctttaggtt gttccttggt gtatcctggc 3360
ttggcatctc ctttccttct agtgaccttt agggacttca tatccaggtt tctctccacc 3420
tcgtccaacg tcacaccgta cttggcacat ctaactaatg caaaataaaa taagtcagca 3480
cattcccagg ctatatcttc cttggattta gcttctgcaa gttcatcagc ttcctcccta 3540
attttagcgt tcaacaaaac ttcgtcgtca aataaccgtt tggtataaga accttctgga 3600
gcattgctct tacgatccca caaggtggct tccatggctc taagaccctt tgattggcca 3660
aaacaggaag tgcgttccaa gtgacagaaa ccaacacctg tttgttcaac cacaaatttc 3720
aagcagtctc catcacaatc caattcgata cccagcaact tttgagttgc tccagatgta 3780
gcacctttat accacaaacc gtgacgacga gattggtaga ctccagtttg tgtccttata 3840
gcctccggaa tagacttttt ggacgagtac accaggccca acgagtaatt agaagagtca 3900
gccaccaaag tagtgaatag accatcgggg cggtcagtag tcaaagacgc caacaaaatt 3960
tcactgacag ggaacttttt gacatcttca gaaagttcgt attcagtagt caattgccga 4020
gcatcaataa tggggattat accagaagca acagtggaag tcacatctac caactttgcg 4080
gtctcagaaa aagcataaac agttctacta ccgccattag tgaaactttt caaatcgccc 4140
agtggagaag aaaaaggcac agcgatacta gcattagcgg gcaaggatgc aactttatca 4200
accagggtcc tatagataac cctagcgcct gggatcatcc tttggacaac tctttctgcc 4260
aaatctaggt ccaaaatcac ttcattgata ccattattgt acaacttgag caagttgtcg 4320
atcagctcct caaattggtc ctctgtaacg gatgactcaa cttgcacatt aacttgaagc 4380
tcagtcgatt gagtgaactt gatcaggttg tgcagctggt cagcagcata gggaaacacg 4440
gcttttccta ccaaactcaa ggaattatca aactctgcaa cacttgcgta tgcaggtagc 4500
aagggaaatg tcatacttga agtcggacag tgagtgtagt cttgagaaat tctgaagccg 4560
tatttttatt atcagtgagt cagtcatcag gagatcctct acgccggacg catcgtggcc 4620
gacctgcagg gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca 4680
taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga 4740
gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct 4800
gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa 4860
caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac caattaacca 4920
attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat 4980
tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc 5040
agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa 5100
tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag 5160
tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa 5220
caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc 5280
gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag 5340
gaatcgaatg caaccggogc aggaacactg ccagcgcatc aacaatattt tcacctgaat 5400
caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc 5460
atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca 5520
gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt 5580
tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt 5640
gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta 5700
atcgcggcct ctagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac 5760
tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt 5820
aacatcagag attttgagac acaacgtggc tttccccccc ccccctgcag gtcggcatca 5880
ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc 5940
gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg 6000
tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc 6060
tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc 6120
gtcgagtatc tatgattgga agtatgggaa tggtgatacc cgcattcttc agtgtcttga 6180
ggtctcctat cagattatgc ccaactaaag caaccggagg aggagatttc atggtaaatt 6240
tctctgactt ttggtcatca gtagactcga actgtgagac tatctcggtt atgacagcag 6300
aaatgtcctt cttggagaca gtaaatgaag tcccaccaat aaagaaatcc ttgttatcag 6360
gaacaaactt cttgtttcga actttttcgg tgccttgaac tataaaatgt agagtggata 6420
tgtcgggtag gaatggagcg ggcaaatgct taccttctgg accttcaaga ggtatgtagg 6480
gtttgtagat actgatgcca acttcagtga caacgttgct atttcgttca aaccattccg 6540
aatccagaga aatcaaagtt gtttgtctac tattgatcca agccagtgcg gtcttgaaac 6600
tgacaatagt gtgctcgtgt tttgaggtca tctttgtatg aataaatcta gtctttgatc 6660
taaataatct tgacgagcca aggcgataaa tacccaaatc taaaactctt ttaaaacgtt 6720
aaaaggacaa gtatgtctgc ctgtattaaa ccccaaatca gctcgtagtc tgatcctcat 6780
caacttgagg ggcactatct tgttttagag aaatttgcgg agatgcgata tcgagaaaaa 6840
ggtacgctga ttttaaacgt gaaatttatc tcaagatctc tgcctcgcgc gtttcggtga 6900
tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 6960
ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 7020
cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca 7080
tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 7140
aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 7200
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 7260
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 7320
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 7380
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 7440
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 7500
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 7560
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 7620
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 7680
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 7740
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 7800
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 7860
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 7920
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 7980
gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 8040
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 8100
gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 8160
tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 8220
ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 8280
ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 8340
atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 8400
cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct 8460
tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 8520
aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 8580
tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 8640
ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 8700
agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa 8760
gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 8820
agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 8880
accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 8940
gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 9000
cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 9060
ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 9120
atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt 9180
ctcatgtttg acagcttatc atcgataagc tgactcatgt tggtattgtg aaatagacgc 9240
agatcgggaa cactgaaaaa taacagttat tattcg 9276
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>
<400> 2
tacgcgatcg ctgttaaa 18
<210> 3
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223>
<400> 3
gaggccgttg agcac 15
<210> 4
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>
<400> 4
tgctctaggc cgcgatta 18
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>
<400> 5
agacgtttcc cgttgaat 18
...