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pPIC9K 构建费用400元/个起

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基本信息

别名:

pPIC9K, pPIC 9K

载体类型:

毕赤酵母蛋白表达载体分泌表达载体

载体抗性:

Ampicillin \ Kanamycin (氨苄青霉素\卡那霉素)

载体标签

N-alpha factor

筛选标记:

HIS4

启动子

AOX1

拷贝/低拷贝:

拷贝

载体大小:

9276 bp

克隆方法:

多克隆位点,限制性内切酶

5' 测序引物:

5´ AOX1:5´-GACTGGTTCCAATTGACAAGC-3´;

alpha-Factor-F: 5´-TACTATTGCCAGCATTGCTGC-3´

3' 测序引物

3´ AOX1:5´-GCAAATGGCATTCTGACATCC-3´

稳定性:

稳表达

组成型:

组成型 Constitutive

备注

利用alpha分泌因子,分泌表达蛋白。


质粒图谱和多克隆位点信息

注意:

客户在我公司选择构建载体服务项目时,请自行提供母源载体。

如要使用本公司保存的母源载体时,本公司不能保证所有母源载体完全符合客户预期,也不承担由此产生的任何责任。

载体描述

pPIC9K载体中存在卡那抗性基因,允许利用卡那抗性在酵母体内筛选多克隆拷贝,载体其余部分与pPIC9载体完全一样。

pPIC9K载体能够使用的毕赤酵母宿主菌是KM71和GS115。

pPIC9K载体详细信息如下:

• pPIC9载体大小9276 bp,是融合表达载体。

• 载体构建过程中,可供使用的单一限制性内切酶位点为SnaB I, EcoR I, Avr II, Not I.

• 利用alpha因子分泌信号肽,分泌表达蛋白基因。

• 在载体构建过程中,你的基因必须保证与信号肽的起始密码子的读码框一致。

• 毕赤酵母中利用HIS4进行筛选。

• 为了将基因插入GS115或KM71的AOX区, 使用SacI限制性内切酶线性化质粒(GS115中产生His+Mut+基因型,KM71中产生His+ MutS基因型)

•为了将基因插入GS115或KM71的His4区, 使用Sal I或者Stu I限制性内切酶线性化质粒(GS115中产生His+ Mut+基因型,KM71中产生His+ MutS基因型)

•将基因插入GS115的AOX1区域,需要使用Bgl II线性化质粒 (产生His+MutS基因型)。

 

载体序列

LOCUS       Exported                9276 bp ds-DNA     circular SYN 02-APR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    pPIC9K

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 9276)

  AUTHORS   BT-Lab(Invitrogen)

  TITLE     Direct Submission

  JOURNAL   Exported Tuesday, Apr 2, 2019 from SnapGene Viewer 4.3.6

FEATURES             Location/Qualifiers

     source          1..9276

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     promoter        2..938

                     /gene="Pichia pastoris AOX1"

                     /label=AOX1 promoter

                     /note="inducible promoter, regulated by methanol"

     primer_bind     855..875

                     /label=5?AOX1

     CDS             949..1215

                     /codon_start=1

                     /gene="MF-alpha-1"

                     /product="N-terminal secretion signal from S. cerevisiae

                     alpha-factor"

                     /label=alpha-factor secretion signal

                     /note="Cleavage by the Kex2 protease occurs after the

                     dibasic KR sequence. The EA dipeptides are then removed by

                     dipeptidyl aminopeptidase A."

                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE

                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA"

     primer_bind     1152..1172

                     /label=alpha-Factor-F

     misc_feature    1216..1241

                     /label=MCS

                     /note="multiple cloning site"

     terminator      1321..1567

                     /gene="Pichia pastoris AOX1"

                     /label=AOX1 terminator

                     /note="transcription terminator for AOX1"

     primer_bind     complement(1327..1347)

                     /label=3?AOX1

     CDS             complement(1980..4514)

                     /codon_start=1

                     /gene="Pichia pastoris HIS4"

                     /product="multifunctional enzyme, required for histidine

                     biosynthesis"

                     /label=PpHIS4

                     /note="auxotrophic marker for Pichia pastoris"

                     /translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE

                     LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL

                     VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII

                     DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI

                     PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER

                     TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED

                     IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP

                     KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE

                     LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVETCP

                     GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE

                     VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA

                     LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED

                     AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV

                     QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLK

                     HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI"

     CDS             complement(4928..5743)

                     /codon_start=1

                     /gene="aph(3')-Ia"

                     /product="aminoglycoside phosphotransferase"

                     /label=KanR

                     /note="confers resistance to kanamycin in bacteria or G418

                     (Geneticin(R)) in eukaryotes"

                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG

                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK

                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA

                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI

                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"

     misc_feature    6122..6878

                     /label=AOX1 3' fragment

                     /note="region downstream of Pichia pastoris AOX1 gene"

     misc_feature    7021..7161

                     /label=bom

                     /note="basis of mobility region from pBR322"

     rep_origin      complement(7347..7935)

                     /direction=LEFT

                     /label=ori

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of

                     replication"

     CDS             complement(8106..8966)

                     /codon_start=1

                     /gene="bla"

                     /product="beta-lactamase"

                     /label=AmpR

                     /note="confers resistance to ampicillin, carbenicillin, and

                     related antibiotics"

                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS

                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

                     LIKHW"

     promoter        complement(8967..9071)

                     /gene="bla"

                     /label=AmpR promoter

ORIGIN

        1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag

       61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt

      121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc

      181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta

      241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta

      301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg

      361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct

      421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg

      481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcgcca taccgtttgt

      541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct

      601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct

      661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact

      721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat

      781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt

      841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga

      901 caacttgaga agatcaaaaa acaactaatt attcgaagga tccaaacgat gagatttcct

      961 tcaattttta ctgcagtttt attcgcagca tcctccgcat tagctgctcc agtcaacact

     1021 acaacagaag atgaaacggc acaaattccg gctgaagctg tcatcggtta ctcagattta

     1081 gaaggggatt tcgatgttgc tgttttgcca ttttccaaca gcacaaataa cgggttattg

     1141 tttataaata ctactattgc cagcattgct gctaaagaag aaggggtatc tctcgagaaa

     1201 agagaggctg aagcttacgt agaattccct agggcggccg cgaattaatt cgccttagac

     1261 atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa

     1321 tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt

     1381 ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc

     1441 ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa

     1501 tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta

     1561 agtgagaagt tcgtttgtgc aagcttatcg ataagcttta atgcggtagt ttatcacagt

     1621 taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc

     1681 tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc

     1741 tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg ctgctagcgc

     1801 tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg

     1861 gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac gcgatcatgg

     1921 cgaccacacc cgtcctgtgg atctatcgaa tctaaatgta agttaaaatc tctaaataat

     1981 taaataagtc ccagtttctc catacgaacc ttaacagcat tgcggtgagc atctagacct

     2041 tcaacagcag ccagatccat cactgcttgg ccaatatgtt tcagtccctc aggagttacg

     2101 tcttgtgaag tgatgaactt ctggaaggtt gcagtgttaa ctccgctgta ttgacgggca

     2161 tatccgtacg ttggcaaagt gtggttggta ccggaggagt aatctccaca actctctgga

     2221 gagtaggcac caacaaacac agatccagcg tgttgtactt gatcaacata agaagaagca

     2281 ttctcgattt gcaggatcaa gtgttcagga gcgtactgat tggacatttc caaagcctgc

     2341 tcgtaggttg caaccgatag ggttgtagag tgtgcaatac acttgcgtac aatttcaacc

     2401 cttggcaact gcacagcttg gttgtgaaca gcatcttcaa ttctggcaag ctccttgtct

     2461 gtcatatcga cagccaacag aatcacctgg gaatcaatac catgttcagc ttgagacaga

     2521 aggtctgagg caacgaaatc tggatcagcg tatttatcag caataactag aacttcagaa

     2581 ggcccagcag gcatgtcaat actacacagg gctgatgtgt cattttgaac catcatcttg

     2641 gcagcagtaa cgaactggtt tcctggacca aatattttgt cacacttagg aacagtttct

     2701 gttccgtaag ccatagcagc tactgcctgg gcgcctcctg ctagcacgat acacttagca

     2761 ccaaccttgt gggcaacgta gatgacttct ggggtaaggg taccatcctt cttaggtgga

     2821 gatgcaaaaa caatttcttt gcaaccagca actttggcag gaacacccag catcagggaa

     2881 gtggaaggca gaattgcggt tccaccagga atatagaggc caactttctc aataggtctt

     2941 gcaaaacgag agcagactac accagggcaa gtctcaactt gcaacgtctc cgttagttga

     3001 gcttcatgga atttcctgac gttatctata gagagatcaa tggctctctt aacgttatct

     3061 ggcaattgca taagttcctc tgggaaagga gcttctaaca caggtgtctt caaagcgact

     3121 ccatcaaact tggcagttag ttctaaaagg gctttgtcac cattttgacg aacattgtcg

     3181 acaattggtt tgactaattc cataatctgt tccgttttct ggataggacg acgaagggca

     3241 tcttcaattt cttgtgagga ggccttagaa acgtcaattt tgcacaattc aatacgacct

     3301 tcagaaggga cttctttagg tttggattct tctttaggtt gttccttggt gtatcctggc

     3361 ttggcatctc ctttccttct agtgaccttt agggacttca tatccaggtt tctctccacc

     3421 tcgtccaacg tcacaccgta cttggcacat ctaactaatg caaaataaaa taagtcagca

     3481 cattcccagg ctatatcttc cttggattta gcttctgcaa gttcatcagc ttcctcccta

     3541 attttagcgt tcaacaaaac ttcgtcgtca aataaccgtt tggtataaga accttctgga

     3601 gcattgctct tacgatccca caaggtggct tccatggctc taagaccctt tgattggcca

     3661 aaacaggaag tgcgttccaa gtgacagaaa ccaacacctg tttgttcaac cacaaatttc

     3721 aagcagtctc catcacaatc caattcgata cccagcaact tttgagttgc tccagatgta

     3781 gcacctttat accacaaacc gtgacgacga gattggtaga ctccagtttg tgtccttata

     3841 gcctccggaa tagacttttt ggacgagtac accaggccca acgagtaatt agaagagtca

     3901 gccaccaaag tagtgaatag accatcgggg cggtcagtag tcaaagacgc caacaaaatt

     3961 tcactgacag ggaacttttt gacatcttca gaaagttcgt attcagtagt caattgccga

     4021 gcatcaataa tggggattat accagaagca acagtggaag tcacatctac caactttgcg

     4081 gtctcagaaa aagcataaac agttctacta ccgccattag tgaaactttt caaatcgccc

     4141 agtggagaag aaaaaggcac agcgatacta gcattagcgg gcaaggatgc aactttatca

     4201 accagggtcc tatagataac cctagcgcct gggatcatcc tttggacaac tctttctgcc

     4261 aaatctaggt ccaaaatcac ttcattgata ccattattgt acaacttgag caagttgtcg

     4321 atcagctcct caaattggtc ctctgtaacg gatgactcaa cttgcacatt aacttgaagc

     4381 tcagtcgatt gagtgaactt gatcaggttg tgcagctggt cagcagcata gggaaacacg

     4441 gcttttccta ccaaactcaa ggaattatca aactctgcaa cacttgcgta tgcaggtagc

     4501 aagggaaatg tcatacttga agtcggacag tgagtgtagt cttgagaaat tctgaagccg

     4561 tatttttatt atcagtgagt cagtcatcag gagatcctct acgccggacg catcgtggcc

     4621 gacctgcagg gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca

     4681 taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga

     4741 gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct

     4801 gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa

     4861 caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac caattaacca

     4921 attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat

     4981 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc

     5041 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa

     5101 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag

     5161 tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa

     5221 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc

     5281 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag

     5341 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat

     5401 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc

     5461 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca

     5521 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt

     5581 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt

     5641 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta

     5701 atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac

     5761 tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt

     5821 aacatcagag attttgagac acaacgtggc tttccccccc ccccctgcag gtcggcatca

     5881 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc

     5941 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg

     6001 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc

     6061 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc

     6121 gtcgagtatc tatgattgga agtatgggaa tggtgatacc cgcattcttc agtgtcttga

     6181 ggtctcctat cagattatgc ccaactaaag caaccggagg aggagatttc atggtaaatt

     6241 tctctgactt ttggtcatca gtagactcga actgtgagac tatctcggtt atgacagcag

     6301 aaatgtcctt cttggagaca gtaaatgaag tcccaccaat aaagaaatcc ttgttatcag

     6361 gaacaaactt cttgtttcga actttttcgg tgccttgaac tataaaatgt agagtggata

     6421 tgtcgggtag gaatggagcg ggcaaatgct taccttctgg accttcaaga ggtatgtagg

     6481 gtttgtagat actgatgcca acttcagtga caacgttgct atttcgttca aaccattccg

     6541 aatccagaga aatcaaagtt gtttgtctac tattgatcca agccagtgcg gtcttgaaac

     6601 tgacaatagt gtgctcgtgt tttgaggtca tctttgtatg aataaatcta gtctttgatc

     6661 taaataatct tgacgagcca aggcgataaa tacccaaatc taaaactctt ttaaaacgtt

     6721 aaaaggacaa gtatgtctgc ctgtattaaa ccccaaatca gctcgtagtc tgatcctcat

     6781 caacttgagg ggcactatct tgttttagag aaatttgcgg agatgcgata tcgagaaaaa

     6841 ggtacgctga ttttaaacgt gaaatttatc tcaagatctc tgcctcgcgc gtttcggtga

     6901 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc

     6961 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg

     7021 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca

     7081 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta

     7141 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg

     7201 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca

     7261 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac

     7321 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac

     7381 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg

     7441 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac

     7501 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat

     7561 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag

     7621 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac

     7681 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt

     7741 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt

     7801 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc

     7861 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga

     7921 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac

     7981 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc

     8041 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct

     8101 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca

     8161 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct

     8221 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca

     8281 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc

     8341 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg

     8401 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct

     8461 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa

     8521 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta

     8581 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc

     8641 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg

     8701 agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa

     8761 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg

     8821 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc

     8881 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg

     8941 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat

     9001 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata

     9061 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc

     9121 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt

     9181 ctcatgtttg acagcttatc atcgataagc tgactcatgt tggtattgtg aaatagacgc

     9241 agatcgggaa cactgaaaaa taacagttat tattcg

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