| 质粒名称: | pAAVS1-NDi-CRISPRi (Gen2) |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 73498 |
| 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: | 13069 bp |
| 载体类型: | 哺乳细胞表达,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 | TRE3G |
| 标签/融合蛋白: |
KRAB（插入件上的N端子）
NLS（插接件上的C端子）
HA（插入件上的C端子）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | PacI(未破坏) |
| 3' 克隆位点: | AgeI (未破坏) |
| 5' 测序引物 | GCTCGTTTAGTGAACCGTCAG |
| 3' 测序引物 | TGTGGAATTGTGAGCGGATA |
|
文献引用方法 |
|
| 材料和方法部分: | pAAVS1-NDi-CRISPRi (Gen2) was a gift from Bruce Conklin (Addgene plasmid # 73498 ; http://n2t.net/addgene:73498 ; RRID:Addgene_73498) |
| 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-NDi-CRISPRi (Gen2) 13069 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Dox-inducible CRISPR interference (CRISPRi) knock in construct into
the AAVS1 locus.
ACCESSION .
VERSION .
KEYWORDS pAAVS1-NDi-CRISPRi (Gen2)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13069)
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 13069)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..13069
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 54..914
/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 complement(272..291)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 1085..1673
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1574..1593
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
misc_feature 1721..2557
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
protein_bind 2590..2608
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2626..2644
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2662..2680
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2698..2716
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2734..2752
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2770..2788
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2806..2824
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 2869..2893
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 2977..2986
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3013..3198
/codon_start=1
/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 3220..7323
/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 7327..7353
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 7372..7392
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7399..7419
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
polyA_signal 7493..7614
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(7530..7549)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 7584..7603
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(7663..7679)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(7663..7679)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(7676..7698)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 7687..7703
/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(7711..7741)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 7756..7777
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(7894..7911)
/label=L4440
/note="L4440 vector, forward primer"
protein_bind complement(7967..8000)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind 8331..8350
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
polyA_signal 8349..8404
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind 8450..8469
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
CDS complement(8559..9305)
/codon_start=1
/product="modified rtTA protein that binds tightly to
promoters containing the tet operator in the presence of
doxycycline"
/label=Tet-On(R) 3G
/translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
primer_bind complement(9337..9356)
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
intron 9364..10381
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind complement(9418..9440)
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
promoter complement(10383..10660)
/label=chicken beta-actin promoter
enhancer 10662..11041
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
polyA_signal 11047..11271
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind 11260..11277
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
CDS complement(11297..12100)
/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(11408..11427)
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind 12018..12037
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
regulatory 12097..12106
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS complement(12122..12175)
/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"
misc_feature 12199..12224
/label=SA
/note="splice acceptor site"
protein_bind complement(12231..12264)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 12266..13069
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
ORIGIN
1 aagggcgaat tcaaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
61 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
121 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
181 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
241 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
301 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
361 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
421 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
481 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
541 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
601 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
661 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
721 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
781 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
841 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
901 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
961 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
1021 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
1081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
1141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
1201 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
1261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
1321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
1381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
1441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
1501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
1561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
1621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
1681 gcaacgcggc ctttttacgg ttcctggcga attcgccctt agagcagagc caggaacccc
1741 tgtagggaag gggcaggaga gccaggggca tgagatggtg gacgaggaag ggggacaggg
1801 aagcctgagc gcctctcctg ggcttgccaa ggactcaaac ccagaagccc agagcagggc
1861 cttagggaag cgggaccctg ctctgggcgg aggaatatgt cccagatagc actggggact
1921 ctttaaggaa agaaggatgg agaaagagaa agggagtaga ggcggccacg acctggtgaa
1981 cacctaggac gcaccattct cacaaaggga gttttccaca cggacacccc cctcctcacc
2041 acagccctgc caggacgggg ctggctactg gccttatctc acaggtaaaa ctgacgcacg
2101 gaggaacaat ataaattggg gactagaaag gtgaagagcc aaagttagaa ctcaggacca
2161 acttattctg attttgtttt tccaaactgc ttctcctctt gggaagtgta aggaagctgc
2221 agcaccagga tcagtgaaac gcaccagaca gccgcgtcag agcagctcag gttctgggag
2281 agggtagcgc agggtggcca ctgagaaccg ggcaggtcac gcatcccccc cttccctccc
2341 accccctgcc aagctctccc tcccaggatc ctctctggct ccatcgtaag caaaccttag
2401 aggttctggc aaggagagag atggctccag gaaatggggg tgtgtcacca gataaggaat
2461 ctgcctaaca ggaggtgggg gttagaccca atatcaggag actaggaagg aggaggccta
2521 aggatggggc ttttctgtca ccaatcctgt ccctagtaaa gctgatatct ccaccccaca
2581 gtggtttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
2641 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
2701 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
2761 acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata
2821 gagaacgtat aagctttagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt
2881 gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatacca actttccgta
2941 ccacttccta ccctcgtaaa cttaaggtta attaacgcca ccatggatgc taagtcacta
3001 actgcctggt cccggacact ggtgaccttc aaggatgtat ttgtggactt caccagggag
3061 gagtggaagc tgctggacac tgctcagcag atcgtgtaca gaaatgtgat gctggagaac
3121 tataagaacc tggtttcctt gggttatcag cttactaagc cagatgtgat cctccggttg
3181 gagaagggag aagagcccgg cggttccggc ggagggtcga tggacaagaa gtacagcatc
3241 ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
3301 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
3361 atcggcgccc tgctgttcga cagcggagaa acagccgagg ccacccggct gaagagaacc
3421 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
3481 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
3541 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
3601 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
3661 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
3721 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
3781 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
3841 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
3901 cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc
3961 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
4021 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
4081 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
4141 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
4201 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
4261 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacatcga tggcggagcc
4321 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
4381 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
4441 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
4501 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
4561 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
4621 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgccagc
4681 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
4741 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt acaacgagct gaccaaagtg
4801 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaagcc
4861 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
4921 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
4981 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
5041 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
5101 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
5161 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
5221 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
5281 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
5341 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
5401 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
5461 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
5521 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
5581 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
5641 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
5701 ctggacatca accggctgtc cgactacgat gtggacgcta tcgtgcctca gagctttctg
5761 aaggacgact ccatcgataa caaagtgctg actcggagcg acaagaaccg gggcaagagc
5821 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg ccagctgctg
5881 aatgccaagc tgattaccca gaggaagttc gacaatctga ccaaggccga gagaggcggc
5941 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
6001 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaacgac
6061 aaactgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
6121 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
6181 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
6241 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
6301 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
6361 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
6421 gaaacaggcg agatcgtgtg ggataagggc cgggactttg ccaccgtgcg gaaagtgctg
6481 tctatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
6541 gagtctatcc tgcccaagag gaacagcgac aagctgatcg ccagaaagaa ggactgggac
6601 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
6661 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
6721 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
6781 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
6841 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
6901 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
6961 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaaaca ctacctggac
7021 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
7081 gacaaggtgc tgagcgccta caacaagcac agagacaagc ctatcagaga gcaggccgag
7141 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
7201 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
7261 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
7321 gacgcctatc cctatgacgt gcccgattat gccagcctgg gcagcggctc ccccaagaaa
7381 aaacgcaagg tggaagatcc taagaaaaag cggaaagtgg acggcattgg tagtgggagc
7441 aacggcagca gcggatccta aaccggtgcg gccgcaatcg atcgcccacc ccaacttgtt
7501 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
7561 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
7621 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
7681 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
7741 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
7801 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
7861 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
7921 cgttcggctg cggcgagcgg tatcagctca ctcaaaggac ggccgcataa cttcgtataa
7981 tgtatgctat acgaagttat aagcttgggc tgcaggtcga gggatctcca taagagaaga
8041 gggacagcta tgactgggag tagtcaggag aggaggaaaa atctggctag taaaacatgt
8101 aaggaaaatt ttagggatgt taaagaaaaa aataacacaa aacaaaatat aaaaaaaatc
8161 taacctcaag tcaaggcttt tctatggaat aaggaatgga cagcaggggg ctgtttcata
8221 tactgatgac ctctttatag ccaacctttg ttcatggcag ccagcatatg ggcatatgtt
8281 gccaaactct aaaccaaata ctcattctga tgttttaaat gatttgccct cccatatgtc
8341 cttccgagtg agagacacaa aaaattccaa cacactattg caatgaaaat aaatttcctt
8401 tattagccag aagtcagatg ctcaaggggc ttcatgatgt ccccataatt tttggcagag
8461 ggaaaaagat ctcagtggta tttgtgagcc agggcattgg ccacaccagc caccaccttc
8521 tgataggcag cctgcacctg aggagtgaat tcacgcgttt agcctggcag catatcgaga
8581 tcgaagtcat cgagagcgtc agcagggagc atatccagat caaaatcatc cagggcatcg
8641 gcgggcagca tgtccaggtc gaagtcgtcc agggcgtcgg tgggtccgcc gctctcgcac
8701 ttcagctgct tctcgaggcc gcagatgatc agttccaggc cgaacaggaa ggcgggctcg
8761 gcgccctgtc tgtcgaacag ctcgatggcc tgcttcagca gggggggcat gctgtcggtg
8821 gtgggtgtct ctctttcctc tttggcgacc tggtgttcct gttcttccag cacgcagccc
8881 agggtgaagt ggcccacggc gctcagggcg tacagggcgt tttccaggct gaagccctgc
8941 tggcacagga aggccagctg gttttccagt gtctcgtact gcttctcggt gggtctggtg
9001 cccaggtgca ctttggcgcc gtcccggtgg ctcagcaggg cgcatctgta gctcttggcg
9061 ttgttccgca ggaagtcctg ccagctctcg ccttccagag ggcagctgtg ggtgtggtgc
9121 cggtccagca tctcgatggg cagggcgtcc agcagggccc gcttgttctt cacgtgccag
9181 tacagggtgg gctgttccac gcccagtttc tgggccagct tccgggtggt caggccctcg
9241 atgcccacgc cgttcagcag ttccagggcg ctgttgatca ctttgctctt gtccagccgg
9301 ctcatgtcga cattctttgc caaaatgatg agacagcaca ataaccagca cgttgcccag
9361 gagctgtagg aaaaagaaga aggcatgaac atggttagca gaggctctag agccgccggt
9421 cacacgccag aagccgaacc ccgccctgcc ccgtcccccc cgaaggcagc cgtccccccg
9481 cggacagccc cgaggctgga gagggagaag gggacggcgg cgcggcgacg cacgaaggcc
9541 ctccccgccc atttccttcc tgccggcgcc gcaccgcttc gccccgcgcc cgctagaggg
9601 ggtgcggcgg cgcctcccag atttcggctc cgcacagatt tgggacaaag gaagtccctg
9661 cgccctctcg cacgattacc ataaaaggca atggctgcgg ctcgccgcgc ctcgacagcc
9721 gccggcgctc cgggggccgc cgcgcccctc ccccgagccc tccccggccc gaggcggccc
9781 cgccccgccc ggcaccccca cctgccgcca ccccccgccc ggcacggcga gccccgcgcc
9841 acgccccgta cggagccccg cacccgaagc cgggccgtgc tcagcaactc ggggaggggg
9901 gtgcaggggg ggttgcagcc cgaccgacgc gcccacaccc cctgctcacc cccccacgca
9961 cacaccccgc acgcagcctt tgttcccctc gcagcccccc ccgcaccgcg gggcaccgcc
10021 cccggccgcg ctcccctcgc gcacactgcg gagcgcacaa agccccgcgc cgcgcccgca
10081 gcgctcacag ccgccgggca gcgcggagcc gcacgcggcg ctccccacgc acacacacac
10141 gcacgcaccc cccgagccgc tccccccgca caaagggccc tcccggagcc cctcaaggct
10201 ttcacgcagc cacagaaaag aaacaagccg tcattaaacc aagcgctaat tacagcccgg
10261 aggagaaggg ccgtcccgcc cgctcacctg tgggagtaac gcggtcagtc agagccgggg
10321 cgggcggcgc gaggcggcgg cggagcgggg cacggggcga aggcagcgtc gcagcgactc
10381 cccgcccgcc gcgcgcttcg ctttttatag ggccgccgcc gccgccgcct cgccataaaa
10441 ggaaactttc ggagcgcgcc gctctgattg gctgccgccg cacctctccg cctcgccccg
10501 ccccgcccct cgccccgccc cgccccgcct ggcgcgcgcc cccccccccc ccccgccccc
10561 atcgctgcac aaaataatta aaaaataaat aaatacaaaa ttgggggtgg ggaggggggg
10621 gagatgggga gagtgaagca gaacgtgggg ctcacctcga ccatggtaat agcgatgact
10681 aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc
10741 caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact
10801 tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa
10861 gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg
10921 ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat
10981 gggctatgaa ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt
11041 cgagacccat agagcccacc gcatccccag catgcctgct attgtcttcc caatcctccc
11101 ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc agacaatgcg
11161 atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc agggtcaagg
11221 aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca gtcgaggctg
11281 atcagcgggt ttaaactcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
11341 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
11401 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
11461 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
11521 catcgccatg ggtcacgacg agatcatcgc cgtcgggcat gcgcgccttg agcctggcga
11581 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
11641 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
11701 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
11761 cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc
11821 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
11881 ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg
11941 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
12001 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
12061 aacctgcgtg caatccatct tgttcaatgg ccgatcccat ggtggcggta ccctcgagcc
12121 tagggccggg attctcctcc acgtcaccgc atgttagaag acttcctctg ccctctccgc
12181 tgccagatct ctcgaggccc tgtgggagga agagaagagg tcagaagctt ataacttcgt
12241 ataatgtatg ctatacgaag ttattgcccc actgtggggt ggaggggaca gataaaagta
12301 cccagaacca gagccacatt aaccggccct gggaatataa ggtggtccca gctcggggac
12361 acaggatccc tggaggcagc aaacatgctg tcctgaagtg gacatagggg cccgggttgg
12421 aggaagaaga ctagctgagc tctcggaccc ctggaagatg ccatgacagg gggctggaag
12481 agctagcaca gactagagag gtaagggggg taggggagct gcccaaatga aaggagtgag
12541 aggtgacccg aatccacagg agaacggggt gtccaggcaa agaaagcaag aggatggaga
12601 ggtggctaaa gccagggaga cggggtactt tggggttgtc cagaaaaacg gtgatgatgc
12661 aggcctacaa gaaggggagg cgggacgcaa gggagacatc cgtcggagaa ggccatccta
12721 agaaacgaga gatggcacag gccccagaag gagaaggaaa agggaaccca gcgagtgaag
12781 acggcatggg gttgggtgag ggaggagaga tgcccggaga ggacccagac acggggagga
12841 tccgctcaga ggacatcacg tggtgcagcg ccgagaagga agtgctccgg aaagagcatc
12901 cttgggcagc aacacagcag agagcaaggg gaagagggag tggaggaaga cggaacctga
12961 aggaggcggc agggaaggat ctgggccagc cgtagaggtg acccaggcca caagctgcag
13021 acagaaagcg gcacaggccc aggggagaga atgctggtca gagaaagca
//
