pFC333

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相关技术服务:质粒构建    基因合成    质粒大提

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载体基本信息

质粒名称: pFC333
出品公司: Addgene
目录编号: 87844
存储实验室: Uffe Mortensen

载体骨架基本信息
载体名称: custom
完整质粒大小: 15667 bp
载体类型: 细菌表达,CRISPR;真菌表达
筛选标记: Zeocin ; bleomycin, phleomycin
载体抗性: 氨苄霉素
生长温度: 37 ℃
宿主菌: DH5a

插入基因信息
插入基因名称: Cas9
物种来源: 合成的黑曲霉合成基因,密码子优化
插入大小: 4131 bp
GenBank编号 KT031983
启动子: Aspergillus nidulans tef1 promoter
标签/融合蛋白: SV40 NLS(插入件上的C端子)
5’测序引物 CTTCTCTGCTCAGCACCTCTACG

文献引用方法
材料和方法部分: pFC333 was a gift from Uffe Mortensen (Addgene plasmid # 87844 ; http://n2t.net/addgene:87844 ; RRID:Addgene_87844)
参考文献部分: A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. Nodvig CS, Nielsen JB, Kogle ME, Mortensen UH. PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. PONE-D-15-11561 [pii] PubMed 26177455

载体图谱质粒图谱和多克隆位点信息

pFC333载体图谱

载体简介

载体序列

LOCUS       pFC333               15668 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AMA1 plasmid with Aspergillus optimized Cas9 and ble selection 
            marker.
ACCESSION   .
VERSION     .
KEYWORDS    pFC333
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15668)
  AUTHORS   Nodvig CS, Nielsen JB, Kogle ME, Mortensen UH
  TITLE     A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi.
  JOURNAL   PLoS One. 2015 Jul 15;10(7):e0133085. doi: 
            10.1371/journal.pone.0133085. eCollection 2015.
  PUBMED    26177455
REFERENCE   2  (bases 1 to 15668)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..15668
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    complement(63..96)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(566..583)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(566..582)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(681..698)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(852..1440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(932..951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1562..2422)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2185..2204
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2423..2514)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2556..2575
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2581..2600
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    2807..2840
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     rep_origin      3153..8403
                     /label=AMA1
                     /note="replication origin from Aspergillus nidulans 
                     (Aleksenko and Clutterbuck, 1996)"
     CDS             9575..13678
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             13682..13702
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             14528..14902
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
ORIGIN
        1 aggtaaatcc ccactaccgc attaagacct cagcgcggcc gcaaatttaa ataaaatgaa
       61 gtgaagttcc tatactttct agagaatagg aacttctata gtgagtcgaa taagggcgac
      121 acaaaattta ttctaaatgc ataataaata ctgataacat cttatagttt gtattatatt
      181 ttgtattatc gttgacatgt ataattttga tatcaaaaac tgattttccc tttattattt
      241 tcgagattta ttttcttaat tctctttaac aaactagaaa tattgtatat acaaaaaatc
      301 ataaataata gatgaatagt ttaattatag gtgttcatca atcaaaaaag caacgtatct
      361 tatttaaagt gcgttgcttt tttctcattt ataaggttaa ataattctca tatatcaagc
      421 aaagtgacag gcgcccttaa atattctgac aaatgctctt tccctaaact ccccccataa
      481 aaaaacccgc cgaagcgggt ttttacgtta tttgcggatt aacgattact cgttatcaga
      541 accgcccagg gggcccgagc ttaagactgg ccgtcgtttt acaacacaga aagagtttgt
      601 agaaacgcaa aaaggccatc cgtcaggggc cttctgctta gtttgatgcc tggcagttcc
      661 ctactctcgc cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
      721 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
      781 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
      841 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
      901 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
      961 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     1021 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     1081 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1141 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     1201 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1261 aagtggtggg ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     1321 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1381 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     1441 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgacgc gcgcgtaact
     1501 cacgttaagg gattttggtc atgagcttgc gccgtcccgt caagtcagcg taatgctctg
     1561 cttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     1621 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     1681 ccagcgctgc gatgataccg cgagaaccac gctcaccggc tccggattta tcagcaataa
     1741 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     1801 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     1861 acgttgttgc catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     1921 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     1981 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     2041 tcatggttat ggcagcgcta cataattctc ttactgtcat gccatccgta agatgctttt
     2101 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     2161 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     2221 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     2281 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     2341 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     2401 cacggaaatg ttgaatactc atattcttcc tttttcaata ttattgaagc atttatcagg
     2461 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     2521 tcagtgttac aaccaattaa ccaattctga acattatcgc gagcccattt atacctgaat
     2581 atggctcata acaccccttg tttgcctggc ggcagtagcg cggtggtccc acctgacccc
     2641 atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggac tccccatgcg
     2701 agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     2761 tcgcccgggc taattatggg gtgtcgccct tattcgactc tatagtgaag ttcctattct
     2821 ctagaaagta taggaacttc tgaagtgggg atttaaatgc ggccgcgctg agggtttaat
     2881 cgacgaagca gctgacggcc agtgccaagc ttaacgcgta ccgggcccag tatatgttcc
     2941 gcagatgact ggagctctgc catacgtgcc ctctcaagca ccatttgttc catctacaga
     3001 gactagtcac caactagtct atcaagactc acagggtaca ttgctgagac caactgacca
     3061 gaggcagggt agcggattga cggctccatc tccttcactt acaaggtcta ttgaaagccc
     3121 tttagcatca ccaagcggag aatagattgt taagcttatt ttttgtatac tgttttgtga
     3181 tagcacgaag tttttccacg gtatcttgtt aaaaatatat atttgtggcg ggcttaccta
     3241 catcaaatta ataagagact aattataaac taaacacaca agcaagctac tttagggtaa
     3301 aagtttataa atgcttttga cgtataaacg ttgcttgtat ttattattac aattaaaggt
     3361 ggatagaaaa cctagagact agttagaaac taatctcagg tttgcgttaa actaaatcag
     3421 agcccgagag gttaacagaa cctagaaggg gactagatat ccgggtaggg aaacaaaaaa
     3481 aaaaaacaag acagccacat attagggaga ctagttagaa gctagttcca ggactaggaa
     3541 aataaaagac aatgatacca cagtctagtt gacaactaga tagattctag attgaggcca
     3601 aagtctctga gatccaggtt agttgcaact aatactagtt agtatctagt ctcctataac
     3661 tctgaagcta gaataactta ctactattat cctcaccact gttcagctgc gcaaacggag
     3721 tgattgcaag gtgttcagag actagttatt gactagtcag tgactagcaa taactaacaa
     3781 ggtattaacc taccatgtct gccatcaccc tgcacttcct cgggctcagc agccttttcc
     3841 tcctcatttt catgctcatt ttccttgttt aagactgtga ctagtcaaag actagtccag
     3901 aaccacaaag gagaaatgtc ttaccacttt cttcattgct tgtctctttt gcattatcca
     3961 tgtctgcaac tagttagagt ctagttagtg actagtccga cgaggacttg cttgtctccg
     4021 gattgttgga ggaactctcc agggcctcaa gatccacaac agagccttct agaagactgg
     4081 tcaataacta gttggtcttt gtctgagtct gacttacgag gttgcatact cgctcccttt
     4141 gcctcgtcaa tcgatgagaa aaagcgccaa aactcgcaat atggctttga accacacggt
     4201 gctgagacta gttagaatct agtcccaaac tagcttggat agcttacctt tgccctttgc
     4261 gttgcgacag gtcttgcagg gtatggttcc tttctcacca gctgatttag ctgccttgct
     4321 accctcacgg cggatctgca taaagagtgg ctagaggtta taaattagca ctgatcctag
     4381 gtacggggct gaatgtaact tgccyttcct ttctcatcgc gcggcaagac aggcttgctc
     4441 aaattcctac cagtcacagg ggtatgcacg gcgtacggac cacttgaact agtcacagat
     4501 tagttagcaa ctagtctgca ttgaatggct gtacttacgg gccctcgcca ttgtcctgat
     4561 catttccagc ttcaccctcg ttgctgcaaa gtagttagtg actagtcaag gactagttga
     4621 aatgggagaa gaaactcacg aattctcgac acccttagta ttgtggtcct tggacttggt
     4681 gctgctatat attagctaat acactagtta gactcacaga aacttacgca gctcgcttgc
     4741 gcttcttggt aggagtcggg gttgggagaa cagtgccttc aaacaagcct tcataccatg
     4801 ctacttgact agtcagggac tagtcaccaa gtaatctaga taggacttgc ctttggcctc
     4861 catcagttcc ttcatagtgg gaggtccatt gtgcaatgta aactccatgc cgtgggagtt
     4921 cttgtccttc aagtgcttga ccaatatgtt tctgttggca gagggaacct gtcaactagt
     4981 taataactag tcagaaacta gtatagcagt agactcactg tacgcttgag gcatcccttc
     5041 actcggcagt agacttcata tggatggata tcaggcacgc cattgtcgtc ctgtggacta
     5101 gtcagtaact aggcttaaag ctagtcgggt cggcttacta tcttgaaatc cggcagcgta
     5161 agctccccgt ccttaactgc ctcgagatag tgacagtact ctggggactt tcggagatcg
     5221 ttatcgcgaa tgctcggcat actaatcgtt gactagtctt ggactagtcc cgagcaaaaa
     5281 ggattggagg aggaggagga aggtgagagt gagacaaaga gcgaaataag agcttcaaag
     5341 gctatctcta agcagtatga aggttaagta tctagttctt gactagattt aaaagagatt
     5401 tcgactagtt atgtacctgg agtttggata taggaatgtg ttgtggtaac gaaatgtaag
     5461 ggggaggaaa gaaaaagtcg gtcaagaggt aactctaagt cggccattcc tttttgggag
     5521 gcgctaacca taaacggcat ggtcgactta gagttagctc agggaattta gggagttatc
     5581 tgcgaccacc gaggaacggc ggaatgccaa agaatcccga tggagctcta gctggcggtt
     5641 gacaacccca ccttttggcg tttctgcggc gttgcaggcg ggactggata cttcgtagaa
     5701 ccagaaaggc aaggcagaac gcgctcagca agagtgttgg aagtgatagc atgatgtgcc
     5761 ttgttaacta ggtcaaaatc tgcagtatgc ttgatgttat ccgaagtgtg agagaggaag
     5821 gtccaaacat acacgattgg gagagggcct aggtataaga gtttttgagt agaacgcatg
     5881 tgagcccagc catctcgagg agattaaaca cgggccggca tttgatggct atgttagtac
     5941 cccaatggaa acggtgagag tccagtggtc gcagataact ccctaaattc cctgagctaa
     6001 ctctaagtcg accatgccgt ttatggttag cgcctcccaa aaaggaatgg ccgacttaga
     6061 gttacctctt gaccgacttt ttctttcctc ccccttacat ttcgttacca caacacattc
     6121 ctatatccaa actccaggta cataactagt cgaaatctct tttaaatcta gtcaagaact
     6181 agatacttaa ccttcatact gcttagagat agcctttgaa gctcttattt cgctctttgt
     6241 ctcactctca ccttcctcct cctcctccaa tcctttttgc tcgggactag tccaagacta
     6301 gtcaacgatt agtatgccga gcattcgcga taacgatctc cgaaagtccc cagagtactg
     6361 tcactatctc gaggcagtta aggacgggga gcttacgctg ccggatttca agatagtaag
     6421 ccgacccgac tagctttaag cctagttact gactagtcca caggacgaca atggcgtgcc
     6481 tgatatccat ccatatgaag tctactgccg agtgaaggga tgcctcaagc gtacagtgag
     6541 tctactgcta tactagtttc tgactagtta ttaactagtt gacaggttcc ctctgccaac
     6601 agaaacatat tggtcaagca cttgaaggac aagaactccc acggcatgga gtttacattg
     6661 cacaatggac ctcccactat gaaggaactg atggaggcca aaggcaagtc ctatctagat
     6721 tacttggtga ctagtccctg actagtcaag tagcatggta tgaaggcttg tttgaaggca
     6781 ctgttctccc aaccccgact cctaccaaga agcgcaagcg agctgcgtaa gtttctgtga
     6841 gtctaactag tgtattagct aatatatagc agcaccaagt ccaaggacca caatactaag
     6901 ggtgtcgaga attcgtgagt ttcttctccc atttcaacta gtccttgact agtcactaac
     6961 tactttgcag caacgagggt gaagctggaa atgatcagga caatggcgag ggcccgtaag
     7021 tacagccatt caatgcagac tagttgctaa ctaatctgtg actagttcaa gtggtccgta
     7081 cgccgtgcat acccctgtga ctggtaggaa tttgagcaag cctgtcttgc cgcgcgatga
     7141 gaaaggaarg gcaagttaca ttcagccccg tacctaggat cagtgctaat ttataacctc
     7201 tagccactct ttatgcagat ccgccgtgag ggtagcaagg cagctaaatc agctggtgag
     7261 aaaggaacca taccctgcaa gacctgtcgc aacgcaaagg gcaaaggtaa gctatccaag
     7321 ctagtttggg actagattct aactagtctc agcaccgtgt ggttcaaagc catattgcga
     7381 gttttggcgc tttttctcat cgattgacga ggcaaaggga gcgagtatgc aacctcgtaa
     7441 gtcagactca gacaaagacc aactagttat tgaccagtct tctagaaggc tctgttgtgg
     7501 atcttgaggc cctggagagt tcctccaaca atccggagac aagcaagtcc tcgtcggact
     7561 agtcactaac tagactctaa ctagttgcag acatggataa tgcaaaagag acaagcaatg
     7621 aagaaagtgg taagacattt ctcctttgtg gttctggact agtctttgac tagtcacagt
     7681 cttaaacaag gaaaatgagc atgaaaatga ggaggaaaag gctgctgagc ccgaggaagt
     7741 gcagggtgat ggcagacatg gtaggttaat accttgttag ttattgctag tcactgacta
     7801 gtcaataact agtctctgaa caccttgcaa tcactccgtt tgcgcagctg aacagtggtg
     7861 aggataatag tagtaagtta ttctagcttc agagttatag gagactagat actaactagt
     7921 attagttgca actaacctgg atctcagaga ctttggcctc aatctagaat ctatctagtt
     7981 gtcaactaga ctgtggtatc attgtctttt attttcctag tcctggaact agcttctaac
     8041 tagtctccct aatatgtggc tgtcttgttt tttttttttt tgtttcccta cccggatatc
     8101 tagtcccctt ctaggttctg ttaacctctc gggctctgat ttagtttaac gcaaacctga
     8161 gattagtttc taactagtct ctaggttttc tatccacctt taattgtaat aataaataca
     8221 agcaacgttt atacgtcaaa agcatttata aacttttacc ctaaagtagc ttgcttgtgt
     8281 gtttagttta taattagtct cttattaatt tgatgtaggt aagcccgcca caaatatata
     8341 tttttaacaa gataccgtgg aaaaacttcg tgctatcaca aaacagtata caaaaaataa
     8401 gcttaacaat ctattctccg cttggtgatg ctaaagggct ttcaatagac cttgtaagtg
     8461 aaggagatgg agccgtcaat ccgctaccct gcctctggtc agttggtctc agcaatgtac
     8521 cctgtgagtc ttgatagact agttggtgac tagtctctgt agatggaaca aatggtgctt
     8581 gagagggcac gtatggcaga gctccagtca tctgcggaac atatactggg cccgggaaga
     8641 tctcatggtc atagctgttt ccgctgaggg tttaattaag acctcagccg agacagcaga
     8701 atcaccgccc aagttaagcc tttgtgctga tcatgctctc gaacgggcca agttcgggaa
     8761 aagcaaagga gcgtttagtg aggggcaatt tgactcacct cccaggcaac agatgagggg
     8821 ggcaaaaaga aagaaatttt cgtgagtcaa tatggattcc gagcatcatt ttcttgcggt
     8881 ctatcttgct acgtatgttg atcttgacgc tgtggatcaa gcaacgccac tcgctcgctc
     8941 catcgcaggc tggtcgcaga caaattaaaa ggcggcaaac tcgtacagcc gcggggttgt
     9001 ccgctgcaaa gtacagagtg ataaaagccg ccatgcgacc atcaacgcgt tgatgcccag
     9061 ctttttcgat ccgagaatcc accgtagagg cgatagcaag taaagaaaag ctaaacaaaa
     9121 aaaaatttct gcccctaagc catgaaaacg agatggggtg gagcagaacc aaggaaagag
     9181 tcgcgctggg ctgccgttcc ggaaggtgtt gtaaaggctc gacgcccaag gtgggagtct
     9241 aggagaagaa tttgcatcgg gagtggggcg ggttacccct ccatatccaa tgacagatat
     9301 ctaccagcca agggtttgag cccgcccgct tagtcgtcgt cctcgcttgc ccctccataa
     9361 aaggatttcc cctccccctc ccacaaaatt ttctttccct tcctctcctt gtccgcttca
     9421 gtacgtatat cttcccttcc ctcgcttctc tcctccatcc ttctttcatc catctcctgc
     9481 taacttctct gctcagcacc tctacgcatt actagccgta gtatctgagc acttctccct
     9541 tttatattcc acaaaacata acacaacctt caccatggac aagaagtata gcatcgggct
     9601 ggacattgga acgaactcgg ttggttgggc tgtgattacg gacgaataca aggtgccatc
     9661 caagaagttt aaggtcctgg gaaacaccga ccgtcactca atcaagaaga atctcattgg
     9721 agccctgctc ttcgatagtg gggagaccgc cgaagctact cgactgaagc gaacggctcg
     9781 ccggcgttat acacgacgca agaatcgcat ctgctacctc caggagattt tcagcaacga
     9841 aatggctaag gttgatgact cattctttca tcgactcgaa gaaagtttct tggtcgagga
     9901 ggataagaag cacgagcgcc atccgatctt tggtaacatt gtggatgagg ttgcctatca
     9961 cgaaaagtac ccaactatct atcatcttcg taagaagctg gtcgatagca cggacaaggc
    10021 tgatttgcga cttatctacc tggcactcgc gcacatgatt aagttccgcg gccattttct
    10081 tatcgagggt gacctgaacc ccgataattc tgacgttgat aagctcttca tccagttggt
    10141 ccaaacctac aatcagctgt ttgaggaaaa ccctattaat gcatctggcg tggacgccaa
    10201 ggctatcctt tcggcgcgcc tgtctaagtc gcggcgtttg gagaacctta tcgcacaact
    10261 ccccggcgaa aagaagaacg gcctcttcgg taatttgatt gcgttgtcac ttggtctgac
    10321 tcctaacttc aagagtaatt ttgacctggc agaggatgcg aagctccagt tgtctaagga
    10381 tacgtatgat gacgatctcg acaacttgct tgcccaaatc ggtgaccagt acgctgatct
    10441 tttcctggcc gctaagaatc tctcagatgc aatcctgctc agtgacattt tgcgggtcaa
    10501 caccgagatt actaaggccc ccctgtcagc tagtatgatc aagcggtatg atgagcacca
    10561 tcaggacctc accttgctta aggccctcgt gcgtcagcaa ttgcctgaga agtataagga
    10621 aatcttcttt gaccaatcca agaacggata cgcagggtat attgatggcg gtgcgagcca
    10681 ggaggaattc tacaagttta tcaagccgat tttggagaag atggacggca ctgaggaact
    10741 gctcgtcaag ctgaatcgcg aagatttgct tcgtaagcaa cgaacgttcg acaacggctc
    10801 catcccgcac cagattcatc tgggcgagct ccacgccatc cttcgacgcc aggaagattt
    10861 ctacccattt ctgaaggaca accgtgagaa gatcgaaaag attcttacat tccgaatccc
    10921 ctactatgtg ggacctttgg cccgtgggaa ttcccgattt gcttggatga cccgaaagag
    10981 cgaggaaacc atcactccgt ggaacttcga ggaagtcgtg gacaagggtg catccgcgca
    11041 gagcttcatt gagcggatga ccaattttga taagaacctt ccgaatgaaa aggtcctgcc
    11101 aaagcattcg ctgctctacg agtatttcac cgtgtataac gaactgacta aggtcaagta
    11161 cgtgacggag ggaatgcgga agccagcctt cctctcaggg gaacaaaaga aggctatcgt
    11221 cgatttgctt tttaagacca atcgtaaagt gactgttaag cagctgaagg aggattattt
    11281 caagaagatt gaatgtttcg actccgtcga gatcagcggc gtggaagatc gctttaacgc
    11341 ttccctcggt acctaccacg acctgctcaa gatcattaag gacaaggatt tcctcgataa
    11401 cgaggaaaat gaggacatct tggaagatat tgtcctcacg ttgacacttt ttgaggaccg
    11461 cgaaatgatc gaggaacggc tcaagacata tgcccatttg ttcgacgata aggtgatgaa
    11521 gcagctgaag cggcgtcgat acaccggatg gggtcgcctt agccggaagc tgatcaacgg
    11581 cattcgagat aagcaatctg gtaagactat cttggatttc cttaagtcgg acggcttcgc
    11641 caaccgcaat tttatgcagc ttattcacga cgattccctg acgttcaagg aggacatcca
    11701 gaaggcacaa gtctcaggac aaggggattc cctgcacgag catatcgcca acctggctgg
    11761 atccccggcg atcaagaagg ggattcttca gaccgtcaag gttgtcgacg agctggtcaa
    11821 ggtgatgggc cgtcataagc cagaaaacat cgtgattgag atggcccgag aaaatcagac
    11881 cactcaaaag ggtcagaaga acagccgcga gcggatgaag cggatcgagg aaggcattaa
    11941 ggaacttggt tctcagatcc tgaaggagca ccctgttgaa aacacacagc tccaaaatga
    12001 gaagctgtat ctctactatt tgcaaaatgg acgcgacatg tacgtcgatc aggagctcga
    12061 cattaaccgg ttgtcggact acgatgttga ccatatcgtc ccgcaatcct tccttaagga
    12121 cgatagcatt gataacaagg tgctgactcg ctcagataag aaccggggca agtccgacaa
    12181 tgttccaagc gaggaagtgg ttaagaagat gaagaactac tggcgccaat tgcttaatgc
    12241 caagctcatc acacagcgca agtttgacaa cttgaccaag gccgagcggg gagggctgag
    12301 tgaactcgat aaggctggct tcatcaagcg tcaactcgtg gagacgcgac agatcacaaa
    12361 gcacgttgct cagattctgg actcccggat gaacacaaag tacgacgaga atgataagct
    12421 catccgtgaa gttaaggtca ttaccctcaa gtctaagttg gtgtcggatt tccgcaagga
    12481 cttccaattt tataaggttc gggagatcaa caattatcac catgcacatg atgcgtacct
    12541 caacgcagtc gtgggaactg cgctcatcaa gaagtatccc aagttggagt ccgaattcgt
    12601 ctacggggat tataaggttt acgacgtccg caagatgatc gccaagagtg agcaggaaat
    12661 tggcaaggcc acggctaagt atttctttta ctccaacatc atgaatttct ttaagacgga
    12721 gatcacactc gccaatggag aaatccgtaa gcgacctttg attgagacca acggcgagac
    12781 tggtgaaatc gtttgggata aggggcgcga cttcgctacc gtgcggaagg ttctgagcat
    12841 gccgcaagtc aatatcgtca agaaaaccga ggtgcagaca ggcggtttct ctaaggaatc
    12901 gattcttcca aagcgtaact ctgacaagct gatcgctcga aagaaggatt gggaccccaa
    12961 gaagtatgga gggttcgatt ctcctacagt ggcatactcg gttctcgttg tcgcgaaggt
    13021 tgagaaggga aagtctaaga agctgaagtc ggtcaaggaa ctgctcggga tcaccattat
    13081 ggagcgctcc agcttcgaaa agaatcccat cgactttctc gaggccaagg gctataagga
    13141 agtcaagaag gatcttatca ttaagctgcc taagtactct ttgttcgagc ttgaaaacgg
    13201 tcgaaagcga atgctcgcat cggcaggaga gttgcagaag gggaatgaat tggcacttcc
    13261 ctcaaagtac gtgaacttcc tgtatctcgc gtcccactac gagaagctga agggtagccc
    13321 tgaggacaac gaacagaagc aactttttgt tgagcaacac aagcattatc tggatgagat
    13381 cattgaacag atttcagagt tcagtaagcg cgtcatcctc gccgatgcta atctcgacaa
    13441 ggtgttgtcg gcctacaaca agcaccgtga caagccgatc cgagagcagg ctgaaaatat
    13501 cattcatctg ttcaccctca ctaacttggg agcaccagca gcgttcaagt attttgatac
    13561 gacaatcgac cgtaagcgat acacgtccac aaaggaggtg cttgatgcga ccctgattca
    13621 tcaatccatc actgggctct atgaaacccg tatcgacctt agtcaactgg ggggcgaccc
    13681 tcccaagaag aagcgcaagg tctgagcgga cattcgattt atgccgttat gacttcctta
    13741 aaaaagcctt tacgaatgaa agaaatggaa ttagacttgt tatgtagttg attctacaat
    13801 ggattatgat tcctgaactt caaatccgct gttcattatt aatctcagct cttcccgtaa
    13861 agccaatgtt gaaactattc gtaaatgtac ctcgttttgc gtgtaccttg cttatcacgt
    13921 gatattacat gacctggaca gagttctgcg cgaaagtcat aacgtaaatc ccgggcggta
    13981 ggtgcgtccc gggcggaagg tagttttctc gtccacccca acgcgtttat caacctcaac
    14041 tttcaacaac catcatgcca ccaaaagcgc gtaaaacaaa gcgagatttg attgagcaag
    14101 agggcaggat ccaatgcgcg attcaagaca ttaaaaatgg aaaatttcaa aaaattgcgc
    14161 ccgcagcgcg tgcatacaaa attcatccca atacaagtgt aatgctagtg gaggtcaaca
    14221 catcaatgcc tattttggtt tagtcgtcca ggcggtgagc acaaaatttg tgtcgtttga
    14281 caagatggtt catttaggca actggtcaga tcagccccac ttgtagcagt agcggcggcg
    14341 ctcgaagtgt gactcttatt agcagacagg aacgaggaca ttattatcat ctgctgcttg
    14401 gtgcacgata acttggtgcg tttgtcaagc aaggtaagtg gacgacccgg tcataccttc
    14461 ttaagttcgc ccttcctccc tttatttcag attcaatctg acttacctat tctacccaag
    14521 catccaaatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg
    14581 agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt
    14641 cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt
    14701 gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg
    14761 gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg
    14821 cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt
    14881 cgtggccgag gagcaggact gaccgacgcc gaccaacacc gccggtccga cgcggcccga
    14941 cgggtccgag gagcttgaga tccacttaac gttactgaaa tcatcaaaca gcttgacgaa
    15001 tctggatata agatcgttgg tgtcgatgtc agctccggag ttgagacaaa tggtgttcag
    15061 gatctcgata agatacgttc atttgtccaa gcagcaaaga gtgccttcta gtgatttaat
    15121 agctccatgt caacaagaat aaaacgcgtt tcgggtttac ctcttccaga tacagctcat
    15181 ctgcaatgca ttaatgcatt ggacctcgca accctagtac gcccttcagg ctccggcgaa
    15241 gcagaagaat agcttagcag agtctatttt cattttcggg agacgagatc aagcagatca
    15301 acggtcgtca agagacctac gagactgagg aatccgctct tggctccacg cgactatata
    15361 tttgtctcta attgtacttt gacatgctcc tcttctttac tctgatagct tgactatgaa
    15421 aattccgtca ccagcccctg ggttcgcaaa gataattgca ctgtttcttc cttgaactct
    15481 caagcctaca ggacacacat tcatcgtagg tataaacctc gaaaatcatt cctactaaga
    15541 tgggtataca atagtaacca tgcatggttg cctagtgaat gctccgtaac acccaatacg
    15601 ccggccgaaa cttttttaca actctcctat gagtcgttta cccagaatgc acaggtacac
    15661 ttgtttag
//

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