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pET-28b-RfxCas13d-His

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pET-28b-RfxCas13d-His載體質粒基本信息

質粒名稱: pET-28b-RfxCas13d-His
出品公司: Addgene
目錄編號: 141322
存儲實驗室: Vladislav Verkhusha

載體骨架基本信息
載體名稱: pET-28b-Cas9-His
載體出品公司: Clontech
空載體大小: 5160bp
完整質粒大小: 8160bp
載體類型: Bacterial Expression, CRISPR
載體抗性: Kanamycin
生長溫度: 37 ℃
宿主菌: DH5a
拷貝數: 未知

插入基因信息
插入基因名稱: Rfx-Cas13d、CasRx
物種來源: Ruminococcus flavefaciens XPD3002
插入大小: 32000bp
啟動子: T7
標簽/融合蛋白: 6xHis (C terminal on insert)
克隆方法: 限制性內切酶
5’克隆位點: NcoI (not destroyed)
3’克隆位點: NotI (unknown if destroyed)
5’測序引物 T7
3’測序引物
GCTAGTTATTGCTCAGCGG
(Common Sequencing Primers)

文獻引用方法
材料和方法部分: pET-28b-RfxCas13d-His was a gift from Ariel Bazzini & Miguel Angel Moreno-Mateos (Addgene plasmid # 141322 ; //n2t.net/addgene:141322 ; RRID:Addgene_141322)
參考文獻部分: CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos. Kushawah G, Hernandez-Huertas L, Abugattas-Nunez Del Prado J, Martinez-Morales JR, DeVore ML, Hassan H, Moreno-Sanchez I, Tomas-Gallardo L, Diaz-Moscoso A, Monges DE, Guelfo JR, Theune WC, Brannan EO, Wang W, Corbin TJ, Moran AM, Sanchez Alvarado A, Malaga-Trillo E, Takacs CM, Bazzini AA, Moreno-Mateos MA. Dev Cell. 2020 Sep 28;54(6):805-817.e7. doi: 10.1016/j.devcel.2020.07.013. Epub 2020 Aug 7. 10.1016/j.devcel.2020.07.013 PubMed 32768421

pET-28b-RfxCas13d-His質粒圖譜載體圖譜和pET-28b-RfxCas13d-His載體序列質粒序列多克隆位點信息

pET-28b-RfxCas13d-His載體圖譜

pET-28b-RfxCas13d-His質粒載體簡介

pET-28b-RfxCas13d-His質粒序列載體序列

LOCUS        pET-28b-RfxCas13d-His        8163 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Plasmid for bacterial expression and purification of RfxCas13d 
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8163)
  AUTHORS   Kushawah G, Hernandez-Huertas L, Abugattas-Nunez Del Prado J, 
            Martinez-Morales JR, DeVore ML, Hassan H, Moreno-Sanchez I, 
            Tomas-Gallardo L, Diaz-Moscoso A, Monges DE, Guelfo JR, Theune WC, 
            Brannan EO, Wang W, Corbin TJ, Moran AM, Sanchez Alvarado A, 
            Malaga-Trillo E, Takacs CM, Bazzini AA, Moreno-Mateos MA
  TITLE     CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos.
  JOURNAL   Dev Cell. 2020 Sep 28;54(6):805-817.e7. doi: 
            10.1016/j.devcel.2020.07.013. Epub 2020 Aug 7.
  PUBMED    32768421
REFERENCE   2  (bases 1 to 8163)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.devcel.2020.07.013"; journalName: "Dev Cell"; date: 
            "2020-09-28- 28"; volume: "54"; issue: "6"; pages: "805-817.e7"
COMMENT     Sequence Label: pET-28b-RfxCas13d-His
FEATURES             Location/Qualifiers
     source          1..8163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..140
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(66..85)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     226..248
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(242..433)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1242..2324)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2286..2305
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2325..2402)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2608..2627
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2711..2730
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2711..2729
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2730..2754
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             2769..2791
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2816..5713
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             5738..5755
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5808..5826)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5822..5869
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5906..6361
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5993..6012)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6203..6224
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6453..7268)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6453..7268)
                     /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"
     primer_bind     7176..7195
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7260..7279
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7390..7978
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7879..7898
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8132..8149
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
       61 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
      121 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
      181 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
      241 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
      301 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
      361 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
      421 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
      481 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
      541 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
      601 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
      661 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
      721 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
      781 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
      841 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
      901 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
      961 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     1021 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     1081 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     1141 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     1201 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     1261 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1321 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     1381 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     1441 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     1501 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     1561 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     1621 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     1681 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     1741 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     1801 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     1861 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     1921 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     1981 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     2041 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     2101 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     2161 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     2221 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     2281 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     2341 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     2401 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     2461 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     2521 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     2581 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     2641 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     2701 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     2761 gaaataattt tgtttaactt taagaaggag atataccatg gcgagcgagg ccagcatcga
     2821 aaaaaaaaag tccttcgcca agggcatggg cgtgaagtcc acactcgtgt ccggctccaa
     2881 agtgtacatg acaaccttcg ccgaaggcag cgacgccagg ctggaaaaga tcgtggaggg
     2941 cgacagcatc aggagcgtga atgagggcga ggccttcagc gctgaaatgg ccgataaaaa
     3001 cgccggctat aagatcggca acgccaaatt cagccatcct aagggctacg ccgtggtggc
     3061 taacaaccct ctgtatacag gacccgtcca gcaggatatg ctcggcctga aggaaactct
     3121 ggaaaagagg tacttcggcg agagcgctga tggcaatgac aatatttgta tccaggtgat
     3181 ccataacatc ctggacattg aaaaaatcct cgccgaatac attaccaacg ccgcctacgc
     3241 cgtcaacaat atctccggcc tggataagga cattattgga ttcggcaagt tctccacagt
     3301 gtatacctac gacgaattca aagaccccga gcaccatagg gccgctttca acaataacga
     3361 taagctcatc aacgccatca aggcccagta tgacgagttc gacaacttcc tcgataaccc
     3421 cagactcggc tatttcggcc aggccttttt cagcaaggag ggcagaaatt acatcatcaa
     3481 ttacggcaac gaatgctatg acattctggc cctcctgagc ggactgaggc actgggtggt
     3541 ccataacaac gaagaagagt ccaggatctc caggacctgg ctctacaacc tcgataagaa
     3601 cctcgacaac gaatacatct ccaccctcaa ctacctctac gacaggatca ccaatgagct
     3661 gaccaactcc ttctccaaga actccgccgc caacgtgaac tatattgccg aaactctggg
     3721 aatcaaccct gccgaattcg ccgaacaata tttcagattc agcattatga aagagcagaa
     3781 aaacctcgga ttcaatatca ccaagctcag ggaagtgatg ctggacagga aggatatgtc
     3841 cgagatcagg aaaaatcata aggtgttcga ctccatcagg accaaggtct acaccatgat
     3901 ggactttgtg atttataggt attacatcga agaggatgcc aaggtggctg ccgccaataa
     3961 gtccctcccc gataatgaga agtccctgag cgagaaggat atctttgtga ttaacctgag
     4021 gggctccttc aacgacgacc agaaggatgc cctctactac gatgaagcta atagaatttg
     4081 gagaaagctc gaaaatatca tgcacaacat caaggaattt aggggaaaca agacaagaga
     4141 gtataagaag aaggacgccc ctagactgcc cagaatcctg cccgctggcc gtgatgtttc
     4201 cgccttcagc aaactcatgt atgccctgac catgttcctg gatggcaagg agatcaacga
     4261 cctcctgacc accctgatta ataaattcga taacatccag agcttcctga aggtgatgcc
     4321 tctcatcgga gtcaacgcta agttcgtgga ggaatacgcc tttttcaaag actccgccaa
     4381 gatcgccgat gagctgaggc tgatcaagtc cttcgctaga atgggagaac ctattgccga
     4441 tgccaggagg gccatgtata tcgacgccat ccgtatttta ggaaccaacc tgtcctatga
     4501 tgagctcaag gccctcgccg acaccttttc cctggacgag aacggaaaca agctcaagaa
     4561 aggcaagcac ggcatgagaa atttcattat taataacgtg atcagcaata aaaggttcca
     4621 ctacctgatc agatacggtg atcctgccca cctccatgag atcgccaaaa acgaggccgt
     4681 ggtgaagttc gtgctcggca ggatcgctga catccagaaa aaacagggcc agaacggcaa
     4741 gaaccagatc gacaggtact acgaaacttg tatcggaaag gataagggca agagcgtgag
     4801 cgaaaaggtg gacgctctca caaagatcat caccggaatg aactacgacc aattcgacaa
     4861 gaaaaggagc gtcattgagg acaccggcag ggaaaacgcc gagagggaga agtttaaaaa
     4921 gatcatcagc ctgtacctca ccgtgatcta ccacatcctc aagaatattg tcaatatcaa
     4981 cgccaggtac gtcatcggat tccattgcgt cgagcgtgat gctcaactgt acaaggagaa
     5041 aggctacgac atcaatctca agaaactgga agagaaggga ttcagctccg tcaccaagct
     5101 ctgcgctggc attgatgaaa ctgcccccga taagagaaag gacgtggaaa aggagatggc
     5161 tgaaagagcc aaggagagca ttgacagcct cgagagcgcc aaccccaagc tgtatgccaa
     5221 ttacatcaaa tacagcgacg agaagaaagc cgaggagttc accaggcaga ttaacaggga
     5281 gaaggccaaa accgccctga acgcctacct gaggaacacc aagtggaatg tgatcatcag
     5341 ggaggacctc ctgagaattg acaacaagac atgtaccctg ttcagaaaca aggccgtcca
     5401 cctggaagtg gccaggtatg tccacgccta tatcaacgac attgccgagg tcaattccta
     5461 cttccaactg taccattaca tcatgcagag aattatcatg aatgagaggt acgagaaaag
     5521 cagcggaaag gtgtccgagt acttcgacgc tgtgaatgac gagaagaagt acaacgatag
     5581 gctcctgaaa ctgctgtgtg tgcctttcgg ctactgtatc cccaggttta agaacctgag
     5641 catcgaggcc ctgttcgata ggaacgaggc cgccaagttc gacaaggaga aaaagaaggt
     5701 gtccggcaat tccggatccg gagcggccgc actcgagcac caccaccacc accactgaga
     5761 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     5821 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     5881 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     5941 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     6001 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     6061 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     6121 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     6181 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     6241 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     6301 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     6361 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     6421 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
     6481 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     6541 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     6601 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     6661 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
     6721 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     6781 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     6841 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     6901 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     6961 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     7021 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     7081 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     7141 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     7201 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     7261 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
     7321 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     7381 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     7441 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     7501 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     7561 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     7621 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     7681 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     7741 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     7801 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7861 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7921 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7981 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     8041 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     8101 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     8161 gcg
//

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