| 质粒名称: | pAAVS1-NC-CRISPRi (Gen3) |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 73499 |
| 存储实验室: | Bruce Conklin |
|
载体骨架基本信息 |
|
| 载体名称: | pAAVS1 |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 3356 bp |
| 完整质粒大小: | 11437 bp |
| 载体类型: | 哺乳动物表达,CRISPR |
| 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: | dCas9-KRAB |
| 物种来源: | Synthetic; S. pyogenes |
| 插入大小: | 4490 bp |
| 基因突变: | D10A, H840A (catalytically deactivated Cas9) |
| 启动子 | CAG |
| 标签/融合蛋白: |
HA（插入件上的C端子）
NLS（插接件上的C端子）
KRAB（插入件上的N端子）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | SalI（未销毁） |
| 3' 克隆位点: | MluI（未销毁） |
| 5' 测序引物 | CCTCTGCTAACCATGTTCATGC |
| 3' 测序引物 | TTCTGATAGGCAGCCTGCAC |
|
文献引用方法 |
|
| 材料和方法部分: | pAAVS1-NC-CRISPRi (Gen3) was a gift from Bruce Conklin (Addgene plasmid # 73499 ; http://n2t.net/addgene:73499 ; RRID:Addgene_73499) |
| 参考文献部分: | CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs. Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP, Weissman JS, Qi LS, So PL, Conklin BR. Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi: 10.1016/j.stem.2016.01.022. Epub 2016 Mar 10. 10.1016/j.stem.2016.01.022 PubMed 26971820 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
质粒图谱.png)
载体简介
载体序列
LOCUS pAAVS1-NC-CRISPRi (Gen3) 11437 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Constitutive CRISPR interference (CRISPRi) knock in construct into
the AAVS1 locus.
ACCESSION .
VERSION .
KEYWORDS pAAVS1-NC-CRISPRi (Gen3)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11437)
AUTHORS Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan
AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen
TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP,
Weissman JS, Qi LS, So PL, Conklin BR
TITLE CRISPR Interference Efficiently Induces Specific and Reversible Gene
Silencing in Human iPSCs.
JOURNAL Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi:
10.1016/j.stem.2016.01.022. Epub 2016 Mar 10.
PUBMED 26971820
REFERENCE 2 (bases 1 to 11437)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..11437
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 1..804
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
protein_bind 806..839
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 846..871
/label=SA
/note="splice acceptor site"
CDS 895..948
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
regulatory 964..973
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 970..1773
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(1033..1052)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 1643..1662
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(1793..1810)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 1799..2023
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
enhancer 2029..2408
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 2410..2687
/label=chicken beta-actin promoter
intron 2689..3706
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 3630..3652
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 3714..3733
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
regulatory 3765..3774
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3801..3986
/codon_start=1
/gene="ZNF10"
/gene_synonym="KOX1"
/product="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
/label=KRAB
/translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
QLTKPDVILRLEKGEEP"
CDS 4008..8111
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the H840A mutation in the HNH
catalytic domain"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 8115..8141
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 8160..8180
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8187..8207
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
primer_bind complement(8338..8357)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_site 8403..8458
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal"
primer_bind complement(8457..8476)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
protein_bind 8807..8840
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 8881..9717
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
rep_origin complement(9765..10353)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(9845..9864)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(10524..11384)
/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 11147..11166
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
ORIGIN
1 tgctttctct gaccagcatt ctctcccctg ggcctgtgcc gctttctgtc tgcagcttgt
61 ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt
121 cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc
181 cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg
241 tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct
301 gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct
361 tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc
421 gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc
481 ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt
541 catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc
601 ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc
661 ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct
721 gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt acttttatct
781 gtcccctcca ccccacagtg gggcaataac ttcgtatagc atacattata cgaagttata
841 agcttctgac ctcttctctt cctcccacag ggcctcgaga gatctggcag cggagagggc
901 agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gctcgagggt
961 accgccacca tgggatcggc cattgaacaa gatggattgc acgcaggttc tccggccgct
1021 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
1081 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
1141 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
1201 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
1261 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
1321 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
1381 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
1441 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
1501 aaggcgcgca tgcccgacgg cgatgatctc gtcgtgaccc atggcgatgc ctgcttgccg
1561 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
1621 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
1681 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
1741 gccttctatc gccttcttga cgagttcttc tgagtttaaa cccgctgatc agcctcgact
1801 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
1861 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
1921 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
1981 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tgggtctcga cattgattat
2041 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
2101 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
2161 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
2221 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
2281 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
2341 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
2401 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
2461 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
2521 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
2581 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
2641 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcgggg agtcgctgcg
2701 acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
2761 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
2821 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
2881 gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg
2941 cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg
3001 cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc
3061 gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg
3121 gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg
3181 agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct
3241 cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg
3301 ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg
3361 cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc
3421 tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg
3481 gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc
3541 gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg
3601 ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc
3661 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt
3721 gctggttatt gtgctgtctc atcattttgg caaagaatgt cgacgccacc atggatgcta
3781 agtcactaac tgcctggtcc cggacactgg tgaccttcaa ggatgtattt gtggacttca
3841 ccagggagga gtggaagctg ctggacactg ctcagcagat cgtgtacaga aatgtgatgc
3901 tggagaacta taagaacctg gtttccttgg gttatcagct tactaagcca gatgtgatcc
3961 tccggttgga gaagggagaa gagcccggcg gttccggcgg agggtcgatg gacaagaagt
4021 acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
4081 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
4141 agaacctgat cggcgccctg ctgttcgaca gcggagaaac agccgaggcc acccggctga
4201 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
4261 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
4321 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
4381 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
4441 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
4501 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
4561 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
4621 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
4681 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg attgccctga
4741 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
4801 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
4861 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
4921 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
4981 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
5041 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacatcgatg
5101 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
5161 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
5221 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
5281 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
5341 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
5401 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
5461 gcgccagcgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
5521 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtac aacgagctga
5581 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
5641 aaaaagccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
5701 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
5761 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
5821 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
5881 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
5941 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
6001 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
6061 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
6121 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
6181 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
6241 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
6301 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
6361 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
6421 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
6481 accaggaact ggacatcaac cggctgtccg actacgatgt ggacgctatc gtgcctcaga
6541 gctttctgaa ggacgactcc atcgataaca aagtgctgac tcggagcgac aagaaccggg
6601 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcgcc
6661 agctgctgaa tgccaagctg attacccaga ggaagttcga caatctgacc aaggccgaga
6721 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
6781 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
6841 agaacgacaa actgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
6901 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
6961 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
7021 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
7081 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
7141 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
7201 caaacggcga aacaggcgag atcgtgtggg ataagggccg ggactttgcc accgtgcgga
7261 aagtgctgtc tatgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
7321 tcagcaaaga gtctatcctg cccaagagga acagcgacaa gctgatcgcc agaaagaagg
7381 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
7441 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
7501 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
7561 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
7621 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
7681 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
7741 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaaacact
7801 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
7861 ctaatctgga caaggtgctg agcgcctaca acaagcacag agacaagcct atcagagagc
7921 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
7981 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
8041 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
8101 tgggaggcga cgcctatccc tatgacgtgc ccgattatgc cagcctgggc agcggctccc
8161 ccaagaaaaa acgcaaggtg gaagatccta agaaaaagcg gaaagtggac ggcattggta
8221 gtgggagcaa cggcagcagc ggatcctaac gcgtgaattc actcctcagg tgcaggctgc
8281 ctatcagaag gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct
8341 ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg
8401 ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact
8461 cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag
8521 tttggcaaca tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca
8581 tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg
8641 aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt
8701 ttccttacat gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc
8761 tgtccctctt ctcttatgga gatccctcga cctgcagccc aagcttataa cttcgtatag
8821 catacattat acgaagttat gcggccgcac cggtatgcat ttaattaaga tatcagcttt
8881 actagggaca ggattggtga cagaaaagcc ccatccttag gcctcctcct tcctagtctc
8941 ctgatattgg gtctaacccc cacctcctgt taggcagatt ccttatctgg tgacacaccc
9001 ccatttcctg gagccatctc tctccttgcc agaacctcta aggtttgctt acgatggagc
9061 cagagaggat cctgggaggg agagcttggc agggggtggg agggaagggg gggatgcgtg
9121 acctgcccgg ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg agctgctctg
9181 acgcggctgt ctggtgcgtt tcactgatcc tggtgctgca gcttccttac acttcccaag
9241 aggagaagca gtttggaaaa acaaaatcag aataagttgg tcctgagttc taactttggc
9301 tcttcacctt tctagtcccc aatttatatt gttcctccgt gcgtcagttt tacctgtgag
9361 ataaggccag tagccagccc cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt
9421 ggaaaactcc ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct
9481 actccctttc tctttctcca tccttctttc cttaaagagt ccccagtgct atctgggaca
9541 tattcctccg cccagagcag ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt
9601 tgagtccttg gcaagcccag gagaggcgct caggcttccc tgtccccctt cctcgtccac
9661 catctcatgc ccctggctct cctgcccctt ccctacaggg gttcctggct ctgctctaag
9721 ggcgaattcg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
9781 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
9841 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
9901 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
9961 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
10021 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
10081 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
10141 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
10201 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
10261 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
10321 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
10381 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
10441 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
10501 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
10561 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
10621 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
10681 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
10741 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
10801 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
10861 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
10921 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
10981 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
11041 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
11101 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
11161 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
11221 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
11281 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
11341 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
11401 tattattgaa gcatttatca gggttgaatt cgccctt
//
