pCAG-CBE4max-SpRY-P2A-EGFP (RTW5133)

价格:1200元

联系方式:I47-825O-882O

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载体基本信息

质粒名称: pCAG-CBE4max-SpRY-P2A-EGFP (RTW5133) 
出品公司: 爱迪基因
目录编号: 139999
存储实验室: Benjamin Kleinstiver

载体骨架基本信息
载体名称: pRZ33
载体出品公司: Ketih Joung
载体抗性: 氨苄青霉素
空载体大小: --
完整质粒大小: --
载体修饰: --
载体类型: 哺乳细胞表达,CRISPR
筛选标记: --
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: human codon optimized CBE4max SpCas9 variant named SpRY with P2A-EGFP,RTW5133
物种来源: Synthetic
插入大小: --
基因突变: nSpCas9=D10A; SpRY=A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/R1333P/R1335Q/T1337R
GenBank编号 --
启动子 CAG
标签/融合蛋白: P2A-EGFP(插入件上的C端子)
克隆方法: Gibson克隆
5' 克隆位点: --
3' 克隆位点: --
5' 测序引物 oBK280-CCTACAGCTCCTGGGCAACG
3' 测序引物 oBK23-CAGAGGGAAAAAGATCTCAGTGG

文献引用方法
材料和方法部分: pCAG-CBE4max-SpRY-P2A-EGFP (RTW5133) was a gift from Benjamin Kleinstiver (Addgene plasmid # 139999 ; http://n2t.net/addgene:139999 ; RRID:Addgene_139999)
参考文献部分: Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Walton , Russell T., Christie, Kathleen A., Whittaker, Madelynn N., Kleinstiver, Benjamin P.. Science , 26 Mar 2020 10.1126/science.aba8853

载体质粒图谱和多克隆位点信息

pCAG-CBE4max-SpRY-P2A-EGFP (RTW5133)质粒图谱

载体简介

载体序列

LOCUS       pCAG-CBE4max-SpRY-P2A-EGFP (RTW5133)               11195 bp ds-DNA     circular SYN 20-MAR-2020
DEFINITION  CAG promoter expression plasmid for human codon optimized BE4max 
            C-to-T base editor with 
            SpRY(D10A/A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/R133
            3P/R1335Q/T1337R) and P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    pCAG-CBE4max-SpRY-P2A-EGFP (RTW5133)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11195)
  AUTHORS   Kleinstiver
  TITLE     Kleinstiver lab CRISPR plasmids
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 11195)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 20, 2020 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..11195
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        37..416
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        418..693
                     /label=chicken beta-actin promoter
     intron          694..1710
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1634..1656
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1718..1737
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1845..1865
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1866..2549
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             2646..4361
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             6777..7025
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             7056..7304
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             7347..7367
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7386..7442
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7443..8162
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7488..7509)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7749..7768)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8096..8117
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(8249..8268)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_site      8314..8369
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     primer_bind     complement(8368..8387)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(8730..8746)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8730..8746)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8743..8765)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8754..8770
                     /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)."
     promoter        complement(8778..8808)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8823..8844
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8903..9098
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      8949..9084
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9011..9030
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    9104..9238
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9141..9160)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9195..9214
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(9306..9323)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9477..10065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9557..9576)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(10236..11096)
                     /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     10859..10878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11097..6)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca ttgattattg actagttatt
       61 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      121 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      181 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      241 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      301 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      361 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
      421 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
      481 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggcgc
      541 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg
      601 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
      661 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
      721 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
      781 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa
      841 tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct
      901 ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg
      961 tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg
     1021 ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc
     1081 tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg
     1141 ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc
     1201 ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg
     1261 gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg
     1321 ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc
     1381 cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt
     1441 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg
     1501 tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt
     1561 ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga
     1621 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac
     1681 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg
     1741 tctcatcatt ttggcaaaga attctgcagt cgacggtacc gcgggcccgg gatccaccgg
     1801 tcgccaccat gaaacggaca gccgacggaa gcgagttcga gtcaccaaag aagaagcgga
     1861 aagtctcctc agagactggg cctgtcgccg tcgatccaac cctgcgccgc cggattgaac
     1921 ctcacgagtt tgaagtgttc tttgaccccc gggagctgag aaaggagaca tgcctgctgt
     1981 acgagatcaa ctggggaggc aggcactcca tctggaggca cacctctcag aacacaaata
     2041 agcacgtgga ggtgaacttc atcgagaagt ttaccacaga gcggtacttc tgccccaata
     2101 ccagatgtag catcacatgg tttctgagct ggtccccttg cggagagtgt agcagggcca
     2161 tcaccgagtt cctgtccaga tatccacacg tgacactgtt tatctacatc gccaggctgt
     2221 atcaccacgc agacccaagg aataggcagg gcctgcgcga tctgatcagc tccggcgtga
     2281 ccatccagat catgacagag caggagtccg gctactgctg gcggaacttc gtgaattatt
     2341 ctcctagcaa cgaggcccac tggcctaggt acccacacct gtgggtgcgc ctgtacgtgc
     2401 tggagctgta ttgcatcatc ctgggcctgc ccccttgtct gaatatcctg cggagaaagc
     2461 agccccagct gaccttcttt acaatcgccc tgcagtcttg tcactatcag aggctgccac
     2521 cccacatcct gtgggccaca ggcctgaagt ctggaggatc tagcggagga tcctctggca
     2581 gcgagacacc aggaacaagc gagtcagcaa caccagagag cagtggcggc agcagcggcg
     2641 gcagcgacaa gaagtacagc atcggcctgg ccatcggcac caactctgtg ggctgggccg
     2701 tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc
     2761 ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg
     2821 agagaacccg gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct
     2881 gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca
     2941 gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg
     3001 gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa
     3061 agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc
     3121 acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg
     3181 acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc
     3241 ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca
     3301 gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa
     3361 acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg
     3421 aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg
     3481 cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca
     3541 tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct
     3601 ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc
     3661 ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg
     3721 ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc
     3781 tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc
     3841 ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc
     3901 acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga
     3961 tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca
     4021 gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg
     4081 aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata
     4141 agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg
     4201 tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc
     4261 tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga
     4321 ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa
     4381 tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa
     4441 ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg
     4501 tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg
     4561 cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg
     4621 gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc
     4681 tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg
     4741 acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc
     4801 tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga
     4861 cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg
     4921 tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga
     4981 gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc
     5041 ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc
     5101 gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc
     5161 atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa
     5221 gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga
     5281 agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc
     5341 tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac
     5401 agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga
     5461 acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt
     5521 ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca
     5581 actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa
     5641 agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga
     5701 agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca
     5761 gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc
     5821 ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt
     5881 ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg
     5941 tgcagacagg cggcttcagc aaagagtcta tcagacccaa gaggaacagc gataagctga
     6001 tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcctgtgg cccaccgtgg
     6061 cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg
     6121 tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg
     6181 actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta
     6241 agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccaagcagc
     6301 tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca
     6361 gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg
     6421 aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag
     6481 tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata
     6541 agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc agactgggag
     6601 cccctagagc cttcaagtac tttgacacca ccatcgaccc caagcagtac agaagcacca
     6661 aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga
     6721 tcgacctgtc tcagctggga ggtgacagcg gcgggagcgg cgggagcggg gggagcacta
     6781 atctgagcga catcattgag aaggagactg ggaaacagct ggtcattcag gagtccatcc
     6841 tgatgctgcc tgaggaggtg gaggaagtga tcggcaacaa gccagagtct gacatcctgg
     6901 tgcacaccgc ctacgacgag tccacagatg agaatgtgat gctgctgacc tctgacgccc
     6961 ccgagtataa gccttgggcc ctggtcatcc aggattctaa cggcgagaat aagatcaaga
     7021 tgctgagcgg aggatccgga ggatctggag gcagcaccaa cctgtctgac atcatcgaga
     7081 aggagacagg caagcagctg gtcatccagg agagcatcct gatgctgccc gaagaagtcg
     7141 aagaagtgat cggaaacaag cctgagagcg atatcctggt ccataccgcc tacgacgaga
     7201 gtaccgacga aaatgtgatg ctgctgacat ccgacgcccc agagtataag ccctgggctc
     7261 tggtcatcca ggattccaac ggagagaaca aaatcaaaat gctgtctggc ggctcaaaaa
     7321 gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtcgga ggcggtggaa
     7381 gcggcgcaac aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac
     7441 cgatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     7501 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     7561 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     7621 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     7681 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     7741 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     7801 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     7861 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     7921 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     7981 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     8041 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     8101 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     8161 gaagcggccg cactcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca
     8221 atgccctggc tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca
     8281 tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa
     8341 tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt
     8401 taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg
     8461 ccatgaacaa aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc
     8521 attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg
     8581 tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt
     8641 tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatgg agatccctcg
     8701 acctgcagcc caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     8761 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     8821 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     8881 aacctgtcgt gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa
     8941 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     9001 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     9061 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc
     9121 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     9181 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg
     9241 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     9301 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     9361 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     9421 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     9481 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     9541 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     9601 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     9661 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     9721 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     9781 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     9841 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     9901 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     9961 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
    10021 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
    10081 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
    10141 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
    10201 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
    10261 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
    10321 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
    10381 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
    10441 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
    10501 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
    10561 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
    10621 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
    10681 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
    10741 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
    10801 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
    10861 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
    10921 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
    10981 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
    11041 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
    11101 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
    11161 tttgaatgta tttagaaaaa taaacaaata ggggt
//

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