pC001 - huLshC2C2-MBP for bacterial expression

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载体基本信息

质粒名称: pC001 - huLshC2C2-MBP for bacterial expression 
出品公司: 爱迪基因
目录编号: 79150
存储实验室: Feng Zhang

载体骨架基本信息
载体名称: BPV00356 pET21GG2-His(6)-MBP_Flag-Avi
载体出品公司: Zhang lab
载体抗性: 氨苄青霉素
空载体大小: 6650 bp
完整质粒大小: 10955 bp
载体修饰: 人细胞色素c氧化酶亚基VIII(human cytochrome C oxidase subunit VIII)
载体类型: 哺乳细胞表达
筛选标记: 新霉素G418
高拷贝/低拷贝: 高拷贝
生长条件: 37 ℃

插入基因信息
插入基因名称: C2c2
物种来源: --
插入大小: 4305 bp
基因突变: --
GenBank编号 --
启动子 --
标签/融合蛋白: 人细胞色素c氧化酶亚基VIII(N端)
克隆方法: 限制性内切酶
5' 克隆位点: AgeI(未破坏)
3' 克隆位点: NotI (未破坏)
5' 测序引物 CMV-F
3' 测序引物 SV40pA-R

文献引用方法
材料和方法部分: pC001 - huLshC2C2-MBP for bacterial expression was a gift from Feng Zhang (Addgene plasmid # 79150 ; http://n2t.net/addgene:79150 ; RRID:Addgene_79150)
参考文献部分: C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F. Science. 2016 Jun 2. pii: aaf5573. 10.1126/science.aaf5573 PubMed 27256883

载体图谱质粒图谱和多克隆位点信息

pC001 - huLshC2C2-MBP for bacterial expression质粒图谱

载体简介

载体序列

LOCUS       pC001 - huLshC2C2-MBP for bacterial expression               10951 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  Expresses human codon-optimized LshC2c2 for purification in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    pC001 - huLshC2C2-MBP for bacterial expression
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10951)
  AUTHORS   Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox
            DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, 
            Regev A, Lander ES, Koonin EV, Zhang F
  TITLE     C2c2 is a single-component programmable RNA-guided RNA-targeting 
            CRISPR effector.
  JOURNAL   Science. 2016 Jun 2. pii: aaf5573.
  PUBMED    27256883
REFERENCE   2  (bases 1 to 10951)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10951
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1012..1031
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1051..1128)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1334..1353
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1437..1456
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1437..1455
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1456..1480
                     /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             1495..1517
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1531..1548
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2503..2512
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2665..2682
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2689..2709
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2710..6876
                     /codon_start=1
                     /product="type VI CRISPR effector from Leptotrichia shahii 
                     with RNA-guided RNAse activity (Abudayyeh et al., 2016)"
                     /label=LshC2c2
                     /translation="MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNN
                     KEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEE
                     VVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEY
                     TNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYE
                     EHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEK
                     ILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKH
                     EKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTE
                     IFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIE
                     IEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLE
                     LITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFID
                     NKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVS
                     KALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFD
                     TIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIEN
                     YYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDI
                     NDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEY
                     QNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIFNNYY
                     EDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKK
                     DLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYY
                     PKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEI
                     DAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIR
                     DLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAY
                     PKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHF
                     YIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLI
                     GNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL"
     CDS             6877..6924
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6931..7011
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             7035..7058
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7059..7103
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             7119..7136
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7189..7207)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7203..7250
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      7287..7742
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7374..7393)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7584..7605
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7768..7872
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7873..8733
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(8091..8110)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8904..9492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9393..9412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9646..9663
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    9678..9817
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(9743..9762)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9903..9925
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(9919..10110)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(10919..1050)
                     /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"
ORIGIN
        1 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
       61 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
      121 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
      181 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
      241 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
      301 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
      361 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
      421 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
      481 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
      541 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
      601 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
      661 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
      721 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
      781 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
      841 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
      901 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
      961 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     1021 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     1081 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     1141 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     1201 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     1261 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     1321 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     1381 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
     1441 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     1501 taactttaag aaggagatat acatatgggt catcatcatc accatcacaa aatcgaagaa
     1561 ggcaaactgg ttatttggat caatggcgat aaaggctata atggtctggc agaagttggc
     1621 aaaaaattcg aaaaagatac cggcattaaa gtgaccgttg aacatccgga taaactggaa
     1681 gaaaaatttc cgcaggttgc agcaaccggt gatggtccgg atattatctt ttgggcacat
     1741 gatcgttttg gtggttatgc acagagcggt ctgctggcag aaattacacc ggataaagca
     1801 tttcaggaca aactgtatcc gtttacctgg gatgcagttc gctataacgg taaactgatt
     1861 gcatatccga ttgcagttga agcactgagc ctgatctata acaaagatct gctgccgaat
     1921 ccgcctaaaa cctgggaaga aattccggca ctggataaag aactgaaagc aaaaggtaaa
     1981 agcgcactga tgtttaatct gcaagaaccg tattttacct ggcctctgat tgcagcagat
     2041 ggtggctatg cattcaaata tgaaaacggc aaatatgata tcaaagacgt gggtgttgat
     2101 aatgcgggtg caaaagccgg tctgaccttt ctggttgatc tgattaaaaa caaacacatg
     2161 aacgccgata ccgattatag cattgcagaa gcagcattta acaaaggtga aaccgcaatg
     2221 acaattaatg gtccgtgggc atggtcaaat attgatacca gcaaagtgaa ttatggtgtt
     2281 accgttctgc cgacatttaa aggtcagccg agcaaaccgt ttgttggtgt tctgagcgca
     2341 ggtattaatg cagcaagccc gaacaaagaa ctggcaaaag aatttctgga aaactatctg
     2401 ctgaccgatg aaggtctgga agcagtgaat aaagataaac cgctgggtgc agttgcactg
     2461 aaaagctatg aagaagaact ggttaaagat ccgcgtattg cagccaccat ggaaaatgca
     2521 cagaaaggcg aaattatgcc gaatattccg cagatgagcg cattttggta tgccgttcgt
     2581 accgcagtga ttaatgccgc atcaggtcgt cagaccgttg atgaagccct gaaagatgcc
     2641 cagaccggtg caagcaccgg atccctggtt ccgcgtggta gcgctagcga aaatctgtat
     2701 tttcagggaa tggggaacct gttcggacac aagcggtggt acgaggtgcg agataagaaa
     2761 gacttcaaga tcaagcgaaa ggtgaaagtc aagcggaatt atgacgggaa caagtacatt
     2821 ctgaacatca acgaaaacaa caacaaagag aagatcgaca acaacaagtt catcagaaag
     2881 tacatcaact acaagaaaaa cgataatatt ctgaaggagt tcactaggaa atttcatgca
     2941 ggaaatatcc tgttcaaact gaagggcaaa gaagggatca ttagaattga gaataacgac
     3001 gacttcctgg agacagagga agtggtgctg tatatcgagg cctacggcaa gagcgagaag
     3061 ctgaaagcac tggggatcac aaagaaaaag atcattgacg aggccatcag gcagggcatt
     3121 actaaggacg ataaaaagat cgagatcaag cgacaggaga acgaggaaga gatcgaaatt
     3181 gacatccggg atgagtacac taataagacc ctgaacgact gctccatcat tctgcgcatc
     3241 attgaaaacg atgaactgga gacaaagaag agcatctacg agatcttcaa gaacatcaat
     3301 atgagcctgt ataagatcat cgagaagatc atcgaaaacg agacagaaaa ggtgtttgaa
     3361 aatagatact acgaagagca cctgagggag aagctgctga aagacgataa gattgacgtg
     3421 atcctgacca acttcatgga aatccgggag aagatcaagt ctaatctgga gatcctgggc
     3481 ttcgtgaagt tttacctgaa cgtcggcggg gacaaaaaga aaagtaaaaa taagaaaatg
     3541 ctggtggaaa agattctgaa catcaatgtg gatctgaccg tcgaggacat tgccgatttc
     3601 gtgatcaagg agctggaatt ttggaacatc acaaagcgca ttgagaaagt gaagaaagtc
     3661 aataacgagt tcctggagaa gcggagaaat cggacataca tcaagtccta tgtgctgctg
     3721 gacaagcacg aaaagtttaa aatcgagaga gaaaacaaga aggataagat cgtgaagttc
     3781 tttgtcgaga acattaagaa caactctatc aaggaaaaga ttgagaagat cctggctgag
     3841 ttcaagatcg acgagctgat taagaaactg gagaaggaac tgaagaaagg gaactgtgat
     3901 accgagatct tcggaatctt taagaagcat tacaaggtga acttcgacag caagaaattt
     3961 tccaagaaat ctgatgaaga gaaggagctg tataagatca tctacagata cctgaagggc
     4021 agaattgaaa aaatcctggt gaacgagcag aaggtcagac tgaagaaaat ggagaagatc
     4081 gagatcgaaa agattctgaa tgaaagtatc ctgtcagaga aaattctgaa gagagtgaaa
     4141 cagtatacac tggagcacat tatgtacctg gggaagctga ggcataacga catcgatatg
     4201 accacagtga atactgacga tttcagccgc ctgcacgcca aggaagagct ggacctggaa
     4261 ctgatcacct tctttgccag cacaaatatg gagctgaaca agatcttttc ccgagaaaac
     4321 atcaacaacg acgagaacat cgatttcttt ggaggcgacc gggagaagaa ctatgtgctg
     4381 gataagaaaa tcctgaatag taagatcaag atcatccgcg acctggattt catcgataac
     4441 aagaacaaca tcacaaacaa cttcattcga aagtttacaa agatcggcac taatgaaagg
     4501 aaccgcatcc tgcatgccat ttccaaagag agggacctgc aggggactca ggacgattac
     4561 aacaaagtga tcaacatcat tcagaatctg aagatctccg atgaagaggt gagcaaagct
     4621 ctgaacctgg acgtggtctt taaggacaag aaaaacatca tcacaaagat caatgacatc
     4681 aagatctctg aagagaacaa caacgatatc aagtatctgc ccagcttcag caaagtgctg
     4741 cccgaaatcc tgaacctgta ccgcaacaat cccaagaatg agccttttga cacaatcgag
     4801 actgaaaaaa ttgtgctgaa cgctctgatc tacgtcaata aggagctgta taagaaactg
     4861 atcctggagg acgatctgga agagaacgag tccaagaata tcttcctgca ggaactgaag
     4921 aaaaccctgg gcaacattga cgaaatcgat gagaacatca tcgagaacta ctacaagaac
     4981 gcacagattt ctgccagtaa ggggaacaac aaggcaatca agaaatatca gaagaaagtg
     5041 atcgagtgct acattggata tctgcgcaaa aactacgaag agctgttcga cttttcagac
     5101 ttcaagatga acatccagga aatcaagaaa cagattaagg acatcaacga taacaagact
     5161 tatgagcgga tcaccgtgaa aaccagcgac aaaaccattg tcatcaacga cgatttcgag
     5221 tacatcattt ctatctttgc actgctgaac agtaatgccg tgattaataa gatccgaaac
     5281 agattcttcg ccaccagcgt gtggctgaac acctcagaat accagaatat cattgacatc
     5341 ctggatgaga ttatgcagct gaataccctg cggaacgaat gcatcacaga gaactggaat
     5401 ctgaacctgg aagagttcat tcagaagatg aaagagatcg aaaaggattt cgacgacttc
     5461 aagatccaga ctaagaaaga aatcttcaac aactactacg aggacatcaa gaacaacatt
     5521 ctgaccgagt ttaaagacga tatcaacggc tgtgatgtgc tggaaaagaa actggagaag
     5581 attgtcatct tcgacgatga aaccaagttc gagatcgaca agaaatccaa catcctgcag
     5641 gatgaacaga gaaagctgtc taacatcaac aagaaggacc tgaagaaaaa ggtggatcag
     5701 tatatcaagg acaaagatca ggagatcaag tctaaaatcc tgtgcaggat cattttcaac
     5761 agtgactttc tgaaaaagta caagaaggaa atcgacaatc tgattgagga tatggagtct
     5821 gaaaatgaga acaagttcca ggagatctac tatcccaagg aacggaagaa cgagctgtat
     5881 atctacaaaa agaatctgtt cctgaacatc ggaaatccta actttgacaa gatctacggc
     5941 ctgattagca acgacatcaa gatggccgat gctaaattcc tgtttaatat cgatggaaag
     6001 aacatcagaa agaacaaaat cagtgagatc gacgctattc tgaagaatct gaacgataaa
     6061 ctgaacggct actcaaagga atacaaggag aagtacatca aaaagctgaa ggagaacgac
     6121 gatttctttg caaagaacat ccagaataag aactacaaat ccttcgaaaa ggactataac
     6181 cgcgtgtctg agtacaaaaa gattcgagat ctggtcgagt tcaactatct gaacaaaatc
     6241 gagtcctacc tgattgacat caactggaag ctggctattc agatggcaag attcgaaagg
     6301 gatatgcact atatcgtgaa tggactgagg gagctgggca tcattaagct gtcaggctat
     6361 aacaccggga tcagcagggc atacccaaag cgcaatggaa gcgacggctt ttacactacc
     6421 acagcctact acaagttctt tgatgaagag tcctacaaga agttcgagaa gatttgctac
     6481 gggtttggaa tcgacctgag cgaaaattcc gagatcaaca agcctgaaaa tgagagcatt
     6541 cggaactata tctcccattt ctacatcgtg agaaatccat ttgccgacta cagtattgct
     6601 gagcagatcg atcgggtgag caacctgctg tcatatagca cacgctacaa caattcaact
     6661 tatgccagcg tgttcgaagt ctttaaaaag gacgtgaatc tggactacga tgagctgaaa
     6721 aagaaattca aactgatcgg caacaatgat attctggagc gcctgatgaa gcccaagaaa
     6781 gtgagcgtcc tggaactgga gtcctacaac agtgactaca ttaagaatct gatcattgaa
     6841 ctgctgacca aaatcgagaa tactaacgat accctgaaaa ggccggcggc cacgaaaaag
     6901 gccggccagg caaaaaagaa aaagggatcc tacccatacg atgttccaga ttacgcttat
     6961 ccctacgacg tgcctgatta tgcataccca tatgatgtcc ccgactatgc ctaagcctcg
     7021 agggtggtgg tagcgattat aaagatgatg atgataaagg cctgaacgat atctttgaag
     7081 cccagaaaat tgaatggcac gagtaaaagc ttgtcgagca ccaccaccac caccactgag
     7141 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     7201 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     7261 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     7321 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     7381 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     7441 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     7501 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     7561 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     7621 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     7681 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     7741 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     7801 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     7861 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     7921 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     7981 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     8041 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     8101 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     8161 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     8221 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     8281 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     8341 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     8401 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     8461 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     8521 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     8581 gagcgtggta gtcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     8641 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     8701 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     8761 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     8821 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     8881 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     8941 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     9001 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     9061 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     9121 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     9181 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     9241 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     9301 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     9361 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     9421 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     9481 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     9541 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     9601 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     9661 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     9721 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     9781 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     9841 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     9901 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     9961 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
    10021 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
    10081 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
    10141 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
    10201 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
    10261 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
    10321 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
    10381 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
    10441 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
    10501 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
    10561 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
    10621 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
    10681 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
    10741 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
    10801 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
    10861 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
    10921 actgcccgct ttccagtcgg gaaacctgtc g
//

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