pCas-Guide

pCas-Guide

载体基本信息

出品公司:	Ybscience
载体名称:	pCas-Guide
质粒类型:	CRISPR/Cas9载体；基因敲除载体；哺乳动物细胞载体
高拷贝/低拷贝:	高拷贝
插入位点:	限制性内切酶，多克隆位点
启动子:	U6
载体大小:	8033 bp
5' 测序引物及序列:	5’ gatcgATGGGAGGTGGTATGGGAGGg 3’
3' 测序引物及序列:	5’ aaaacCCTCCCATACCACCTCCCATc 3’
载体标签:	Myc(C-ter）
载体抗性:	氨苄青霉素
筛选标记:	--
克隆菌株:	Stbl3
宿主细胞（系）:	哺乳动物细胞
备注:	--
稳定性:	瞬表达
组成型/诱导型:	组成型
病毒/非病毒:	非病毒

 

载体质粒图谱和多克隆位点信息

 

载体简介

 

pCas-Guide载体说明

Cas9 based genome editing has become a popular tool for targeted genome manipulation
because of its simplicity and high cutting efficiency. This system requires a functional cas9
protein and a guide RNA for effective double-stranded breakage at a desired site. OriGene has
developed the pCas-Guide system, a dual-function vector with both guide RNA and Cas9
expression. OriGene also designed a set of donor cassettes for construction of donor vectors.
These include Luciferase-Loxp-Puro-Loxp, tGFP-Loxp-Puro-Loxp and tRFP-Loxp-BSD-Loxp. 

The pCas-Guide vector is designed for cloning a guide RNA insert for genome editing purpose.
The vector also has a CMV-driven codon-optimized Cas9. When co-transfected with a proper
donor DNA, targeted genome editing can be achieved. The vector retains the ampicillin
resistance gene for the selection of E. coli transformants. The vector is supplied as a precut
vector, ready for insert ligation. This system has been successfully validated in multiple cases of
genome editing.

实验操作

I. Design target sequence
OriGene has developed a proprietary Cas9 guide RNA designing tool which is free to use,
http://www.blueheronbio.com/. Follow the instructions below to design your guide RNA:
1. Select your desired Cas9 cutting site from your genomic region of interest.
2. Copy around 100 bp of genomic sequence flanking the cutting site (-50 to +50).
Paste the sequence to the sequence box and click the Search button.
3. The program will return all possible targeting sequences with location and GC
content obtained from searching both the plus and minus strands. If there is no
target returned, expand your genomic region of interest (-100 to +100) and
search again until there is a positive return.
4. Select a few target sequences to Blast against the genomic DNA database to
check sequence specificity.
5. Select 2 to 3 target sequences to clone into pCas-Guide vector

II. Addition of extra bases to the ends of the target sequence
 To facilitate cloning of the 20-bp target sequence, extra bases need to be added to the ends.
1. Select a desired 20-bp sequence as a target. The following is an example sequence:

 Forward sequence: 5’ ATGGGAGGTGGTATGGGAGG 3’
 Reverse complement sequence: 5’ CCTCCCATACCACCTCCCAT 3’
2. Add ‘gatcg’ to the 5’ end of the forward sequence and ‘g’ to its 3’ end. The final
sense oligo in this example will be
5’ gatcgATGGGAGGTGGTATGGGAGGg 3’
3. Add ‘aaaac’ to the 5’ end of reverse complementary sequence and ‘c’ to its 3’
end. The final reverse complementary sequence is
5’ aaaacCCTCCCATACCACCTCCCATc 3’ 

The two oligos should anneal to form the following double strand:
4. Order the two final oligos from a commercial oligo provider, such as IDT.
III. Cloning the double-stranded oligos into the pCas-Guide vector
1. Anneal the oligos to form double-stranded duplexes
In a PCR tube, add the following:
2 μL Forward oligo (100 μM stock)
2 μL Reverse oligo (100 μM stock)
4 μL 10X annealing buffer
32 μL dH2O
Mix the solution and follow the steps to anneal the oligos in a PCR machine:
940C for 4min
750C for 5 min
650C for 15 min
250C for 20 min
After annealing, transfer the solution to a 1.5 mL tube and add 360 μL of dH2O.
The double-stranded oligo DNA is ready for ligation.
2. Ligation and transformation
A. Prepare the ligation according to the following protocol
Component Volume
10x Ligation buffer 1 μL
Precut pCAS-Guide vector (10 ng/ μL) 1 μL
Annealed double-stranded oligos (diluted from step 1) 1 μL
Ligase (0.5 u/ μL, Weiss unit) 0.5 μL
dH2O 6.5 μL
Total Volume 10 μL
B. Mix the solution and incubate the tube at 22 to 370C or room temperature for two
hours according to the manufacturer’s recommendation.
C. Add 1 μL of the ligation mixture to 10 μL of competent cells (efficiency rated > 106
cfu/μg DNA) on ice. Do the transformation according to the manufacturer’s protocol.
 For chemically competent cells, follow steps D-E.
 D. Mix the tube gently and keep it on ice for 25 minutes.
 E. Heat shock the tube for 30 seconds at 420C.

F. Put the tube on ice for 2 minutes, then add 500 μL LB or SOC medium.
 G. Rock the tube gently at 370C for 1 hour.
 H. Spread 50 μL of the E. Coli cells on an LB ampicillin-agar plate.
 I. Centrifuge the remaining E. Coli cells at 5K rpm for 5 minutes. Discard the majority of
 the supernatant (around 50 μL supernatant left) and resuspend the cell pellet in the
 remaining liquid. Spread all the E. Coli cells on a separate LB ampicillin-agar plate.
 J. Incubate the two plates at 370C for 16 hours to allow colony formation.
3. Screening colonies
In a typical subcloning ligation, at least 95% of the colonies should contain the desired
insert. Pick 6 to 10 colonies into 5 mL LB-ampicillin culture each, and culture overnight.
Perform DNA purification using a mini-prep kit from OriGene,
http://www.origene.com/Other/Plasmid_Purification/ . Sequence the purified DNA and
analyze the sequencing data to identify a correct clone for proper insert identification and
orientation.

 

载体序列

LOCUS       Exported File           8033 bp ds-DNA    circular SYN 23-七月-2016
DEFINITION  Mammalian vector for co-expressing a guide RNA (gRNA) together with 
            Cas9, for genome editing using the S. pyogenes CRISPR/Cas9 system.
ACCESSION   .
VERSION     .
KEYWORDS    pCas-Guide
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8033)
  AUTHORS   OriGene
  TITLE     Direct Submission
  JOURNAL   Exported 2016-07-23 from SnapGene Viewer 1.5.3
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8033
                     /organism="synthetic DNA construct"
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
     promoter        90..330
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #000000; direction: RIGHT"
     misc_RNA        384..459
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: RIGHT"
     terminator      460..465
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
                     /note="color: #ffffff"
     enhancer        511..890
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #c0c0c0"
     promoter        891..1094
                     /note="CMV"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /note="color: #000000; direction: RIGHT"
     promoter        1120..1138
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             1197..5300
                     /codon_start=1
                     /locus_tag="
                     "
                     /product="Cas9 (Csn1) endonuclease from the?Streptococcus 
                     pyogenes?Type II CRISPR/Cas system"
                     /note="Cas9"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /note="color: #993366"
                     /protein_id="
                     "
                     /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             5301..5321
                     /codon_start=1
                     /locus_tag="
                     "
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /note="
                     "
                     /note="color: #993366"
                     /protein_id="
                     "
                     /translation="PKKKRKV"
     CDS             5328..5348
                     /codon_start=1
                     /locus_tag="
                     "
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /note="
                     "
                     /note="color: #993366"
                     /protein_id="
                     "
                     /translation="PKKKRKV"
     CDS             5370..5399
                     /codon_start=1
                     /product="Myc (human c-Myc oncogene) epitope tag"
                     /note="Myc"
                     /note="color: #ff0000"
                     /translation="EQKLISEEDL"
     CDS             5418..5441
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /note="FLAG"
                     /note="color: #cc99b2"
                     /translation="DYKDDDDK"
     polyA_signal    5488..6110
                     /note="hGH poly(A) signal"
                     /note="human growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      complement(6231..6819)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(6994..7854)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                     ???1:?6994?..?7785?/?#ccffcc
                     ???2:?7786?..?7854?/?#ccffcc?/?signal?sequence
                     Cleavage site after base 7785"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 gaattcccca gtggaaagac gcgcaggcaa aacgcaccac gtgacggagc gtgaccgcgc
       61 gccgagcgcg cgccaaggtc gggcaggaag agggcctatt tcccatgatt ccttcatatt
      121 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
      181 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
      241 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
      301 tcttgggttt atatatcttg tggaaaggac gcgggatcca ctggaccagg cagcagcgtc
      361 agaagacttt tttggaacgt ctcgttttag agctagaaat agcaagttaa aataaggcta
      421 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttggtg tacatttata
      481 ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt tattaatagt
      541 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      601 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      661 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      721 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta
      781 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg
      841 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      901 tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      961 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     1021 gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     1081 atataagcag agctcgttta gtgaaccgtc agaattttgt aatacgactc actatagggc
     1141 ggccgggaat tcgtcgactg gaaccggtac cgaggagatc tgccgccgcg atcgccatgg
     1201 ataagaaata ctcaatagga ctggatattg gcacaaatag cgtcggatgg gctgtgatca
     1261 ctgatgaata taaggttcct tctaaaaagt tcaaggttct gggaaataca gaccgccaca
     1321 gtatcaaaaa aaatcttata ggggctcttc tgtttgacag tggagagaca gccgaagcta
     1381 ctagactcaa acggacagct aggagaaggt atacaagacg gaagaatagg atttgttatc
     1441 tccaggagat tttttcaaat gagatggcca aagtggatga tagtttcttt catagacttg
     1501 aagagtcttt tttggtggaa gaagacaaga agcatgaaag acatcctatt tttggaaata
     1561 tagtggatga agttgcttat cacgagaaat atccaactat ctatcatctg agaaaaaaat
     1621 tggtggattc tactgataaa gccgatttgc gcctgatcta tttggccctg gcccacatga
     1681 ttaagtttag aggtcatttt ttgattgagg gcgatctgaa tcctgataat agtgatgtgg
     1741 acaaactgtt tatccagttg gtgcaaacct acaatcaact gtttgaagaa aaccctatta
     1801 acgcaagtgg agtggatgct aaagccattc tttctgcaag attgagtaaa tcaagaagac
     1861 tggaaaatct cattgctcag ctccccggtg agaagaaaaa tggcctgttt gggaatctca
     1921 ttgctttgtc attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg
     1981 ctaaactcca gctttcaaaa gatacttacg atgatgatct ggataatctg ttggctcaaa
     2041 ttggagatca atatgctgat ttgtttttgg cagctaagaa tctgtcagat gctattctgc
     2101 tttcagacat cctgagagtg aatactgaaa taactaaggc tcccctgtca gcttcaatga
     2161 ttaaacgcta cgatgaacat catcaagact tgactcttct gaaagccctg gttagacaac
     2221 aacttccaga aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt
     2281 atattgatgg cggcgcaagc caagaagaat tttataaatt tatcaaacca attctggaaa
     2341 aaatggatgg tactgaggaa ctgttggtga aactgaatag agaagatttg ctgcgcaagc
     2401 aacggacctt tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta
     2461 ttttgagaag acaagaagac ttttatccat ttctgaaaga caatagagag aagattgaaa
     2521 aaatcttgac ttttaggatt ccttattatg ttggtccatt ggccagaggc aatagtaggt
     2581 ttgcatggat gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg
     2641 tcgataaagg tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc
     2701 ttccaaatga aaaagtgctg ccaaaacata gtttgcttta tgagtatttt accgtttata
     2761 acgaattgac aaaggtcaaa tatgttactg aaggaatgag aaaaccagca tttctttcag
     2821 gtgaacagaa gaaagccatt gttgatctgc tcttcaaaac aaataggaaa gtgaccgtta
     2881 agcaactgaa agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag
     2941 gagttgaaga tagatttaat gcttcactgg gtacatacca tgatttgctg aaaattatta
     3001 aagataaaga ttttttggat aatgaagaaa atgaagacat cctggaggat attgttctga
     3061 cattgaccct gtttgaagat agggagatga ttgaggaaag acttaaaaca tacgctcacc
     3121 tctttgatga taaggtgatg aaacagctta aaagacgcag atatactggt tggggaaggt
     3181 tgtccagaaa attgattaat ggtattaggg ataagcaatc tggcaaaaca atactggatt
     3241 ttttgaaatc agatggtttt gccaatcgca attttatgca gctcatccat gatgatagtt
     3301 tgacatttaa agaagacatc caaaaagcac aagtgtctgg acaaggcgat agtctgcatg
     3361 aacatattgc aaatctggct ggtagccctg ctattaaaaa aggtattctc cagactgtga
     3421 aagttgttga tgaattggtc aaagtgatgg ggcggcataa gccagaaaat atcgttattg
     3481 aaatggcaag agaaaatcag acaactcaaa agggccagaa aaattccaga gagaggatga
     3541 aaagaatcga agaaggtatc aaagaactgg gaagtcagat tcttaaagag catcctgttg
     3601 aaaatactca attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagata
     3661 tgtatgtgga ccaagaactg gatattaata ggctgagtga ttatgatgtc gatcacattg
     3721 ttccacaaag tttccttaaa gacgattcaa tagacaataa ggtcctgacc aggtctgata
     3781 aaaatagagg taaatccgat aacgttccaa gtgaagaagt ggtcaaaaag atgaaaaact
     3841 attggagaca acttctgaac gccaagctga tcactcaaag gaagtttgat aatctgacca
     3901 aagctgaaag aggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg
     3961 ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta
     4021 aatacgatga aaatgataaa cttattagag aggttaaagt gattaccctg aaatctaaac
     4081 tggtttctga cttcagaaaa gatttccaat tctataaagt gagagagatt aacaattacc
     4141 atcatgccca tgatgcctat ctgaatgccg tcgttggaac tgctttgatt aagaaatatc
     4201 caaaacttga aagcgagttt gtctatggtg attataaagt ttatgatgtt aggaaaatga
     4261 ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa gtatttcttt tactctaata
     4321 tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc
     4381 tgatcgaaac taatggggaa actggagaaa ttgtctggga taaagggaga gattttgcca
     4441 cagtgcgcaa agtgttgtcc atgccccaag tcaatatcgt caagaaaaca gaagtgcaga
     4501 caggcggatt ctctaaggag tcaattctgc caaaaagaaa ttccgacaag ctgattgcta
     4561 ggaaaaaaga ctgggaccca aaaaaatatg gtggttttga tagtccaacc gtggcttatt
     4621 cagtcctggt ggttgctaag gtggaaaaag ggaaatccaa gaagctgaaa tccgttaaag
     4681 agctgctggg gatcacaatt atggaaagaa gttcctttga aaaaaatccc attgactttc
     4741 tggaagctaa aggatataag gaagttaaaa aagacctgat cattaaactg cctaaatata
     4801 gtctttttga gctggaaaac ggtaggaaac ggatgctggc tagtgccgga gaactgcaaa
     4861 aaggaaatga gctggctctg ccaagcaaat atgtgaattt tctgtatctg gctagtcatt
     4921 atgaaaagtt gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc
     4981 ataagcatta tctggatgag attattgagc aaatcagtga attttctaag agagttattc
     5041 tggcagatgc caatctggat aaagttctta gtgcatataa caaacataga gacaaaccaa
     5101 taagagaaca agcagaaaat atcattcatc tgtttacctt gaccaatctt ggagcacccg
     5161 ctgcttttaa atactttgat acaacaattg ataggaaaag atatacctct acaaaagaag
     5221 ttctggatgc cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt
     5281 tgagtcagct gggaggtgac cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc
     5341 ggaaagtgga cacgcgtacg cggccgctcg agcagaaact catctcagaa gaggatctgg
     5401 cagcaaatga tatcctggat tacaaggatg acgacgataa ggtttaaacg gccggccgcg
     5461 gtcatagctg tttcctgaac agatcccggg tggcatccct gtgacccctc cccagtgcct
     5521 ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt
     5581 tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta
     5641 tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca
     5701 agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg
     5761 attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc
     5821 taatttttgt ttttttggta gaggcggggt ttcaccatat tggccaggct ggtctccaac
     5881 tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga
     5941 accactgctc ccttccctgt ccttctgatt ttaaaataac tataccagca ggaggacgtc
     6001 cagacacagc ataggctacc tggccatgcc caaccggtgg gacatttgag ttgcttgctt
     6061 ggcactgtcc tctcatgcgt tgggtccact cagtagatgc ctgttgaatt gggtacgcgg
     6121 ccagcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     6181 ggataacgca ggaaagaaca tgtccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6241 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6301 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6361 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6421 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6481 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6541 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6601 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6661 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6721 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6781 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6841 acggggtctg acgctcagtg gaacgacgcg taactcacgt taagggattt tggtcatgag
     6901 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6961 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     7021 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7081 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7141 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7201 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7261 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     7321 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7381 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7441 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7501 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7561 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7621 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7681 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7741 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7801 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7861 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgatga tattatttta
     7921 tcttgtgcaa tgtaacatca gagattttga gacacgggcc agagctgcca ggaaacagct
     7981 atgaccatgt aatacgactc actatagggg atatcagctg gatggcagtt aac