| 出品公司: | ORIGENE |
|---|---|
| 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: | -- |
| 产品目录号: | GE100001V |
| 稳定性: | 瞬表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
买家导航
pCas-Guide载体质粒基本信息
pCas-Guide质粒图谱载体图谱和pCas-Guide载体序列质粒序列多克隆位点信息
pCas-Guide质粒载体简介
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.
pCas-Guide质粒序列载体序列
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
//
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