| 质粒名称: | lenti SYN-SVI-DIO-dCas9-KRAB-MeCP2 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 170378 |
| 存储实验室: | Jeremy Day |
|
载体骨架基本信息 |
|
| 载体名称: | lentiCRISPR v2 |
| 载体出品公司: | Feng Zhang Addgene plasmid #52961 |
| 空载体大小: | 7546 bp |
| 完整质粒大小: | 13818 bp |
| 载体类型: | 哺乳动物表达，慢病毒，Cre/Lox，CRISPR |
| 载体抗性: | 氨苄霉素100 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
|
插入基因信息 |
|
| 插入基因名称: | A DIO FLAG-dCas9-KRAB-MeCP2 cassette containing an SV40 intron |
| 物种来源: | Synthetic |
| 插入大小: | 5812 bp |
| 启动子: | 人Synapsin启动子 |
| 标签/融合蛋白: | FLAG（插入件上的N端子） |
| 克隆方法: | Gibson 克隆 |
| 5’测序引物 | GTTTATTACAGGGACAGCAGA |
|
文献引用方法 |
|
| 材料和方法部分: | lenti SYN-SVI-DIO-dCas9-KRAB-MeCP2 was a gift from Jeremy Day (Addgene plasmid # 170378 ; http://n2t.net/addgene:170378 ; RRID:Addgene_170378) |
| 参考文献部分: | A Cre-dependent CRISPR/dCas9 system for gene expression regulation in neurons. Carullo NVN, Hinds JE, Revanna JS, Tuscher JJ, Bauman AJ, Day JJ. eNeuro. 2021 Jul 26. pii: ENEURO.0188-21.2021. doi: 10.1523/ENEURO.0188-21.2021. 10.1523/ENEURO.0188-21.2021 PubMed 34321217 |
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
载体简介
载体序列
LOCUS lenti SYN-SVI-DIO-dCas9-KRAB-MeCP2 13818 bp ds-DNA circular SYN 25-MAY-2021
DEFINITION Expresses a SYN-driven, intron-containing dCas9-KRAB-MeCP2 fusion in
which the part of the dCas9 cassette is double floxed and in
inverted orientation (DIO). Requires Cre-dependent recombination..
ACCESSION .
VERSION .
KEYWORDS lenti SYN-SVI-DIO-dCas9-KRAB-MeCP2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13818)
TITLE A Cre-dependent CRISPR/dCas9 activation system for gene expression
regulation in neurons
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 13818)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 25, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..13818
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 382..580
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 535..555
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 598..778
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 825..950
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1443..1676
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1861..1905
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
misc_feature 2203..2320
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2365..2812
/label=hSyn promoter
/note="human synapsin I promoter; confers neuron-specific
expression (Kugler et al., 2003)"
protein_bind 2825..2858
/label=lox2272
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GGATACTT). lox2272 sites are compatible with each
other, but incompatible with loxP or loxN sites (Lee and
Saito, 1988)."
protein_bind 2909..2942
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS complement(4430..4450)
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS complement(4457..4522)
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
regulatory 4522..4531
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
protein_bind complement(4532..4565)
/label=lox2272
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GGATACTT). lox2272 sites are compatible with each
other, but incompatible with loxP or loxN sites (Lee and
Saito, 1988)."
protein_bind complement(4616..4649)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS 4914..7412
/codon_start=1
/product="C-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
/label=Cas9(C)
/translation="KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI
SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY
AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH
DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE
NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL
QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV
VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI
LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV
GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL
ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL
PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME
RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP
SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD
KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL
IHQSITGLYETRIDLSQLGGD"
CDS 7413..7460
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 7521..7715
/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
QLTKPDVILRLEKGEEPWLV"
CDS 7749..7769
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7779..8636
/codon_start=1
/product="transcriptional repression domain from rat
methyl-CpG-binding protein 2 (Nan et al., 1997)"
/label=MeCP2
/translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
TERVS"
misc_feature 8673..9261
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(8726..8746)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(9144..9155)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 9333..9566
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(9592..9609)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 9598..9822
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 9868..10296
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(9955..9974)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 10165..10186
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(10305..10325)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 10310..10639
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 10490..10625
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 10552..10571
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 10687..10734
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 10753..11127
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 11257..11378
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(11294..11313)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 11348..11367
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(11427..11443)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11427..11443)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11440..11462)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 11451..11467
/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(11475..11505)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 11520..11541
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11658..11675)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(11829..12417)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(11909..11928)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(12588..13448)
/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 13211..13230
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(13449..13553)
/gene="bla"
/label=AmpR promoter
primer_bind complement(13628..13647)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
2341 cagagatcca gtttggttaa ttaaagtgca agtgggtttt aggaccagga tgaggcgggg
2401 tgggggtgcc tacctgacga ccgaccccga cccactggac aagcacccaa cccccattcc
2461 ccaaattgcg catcccctat cagagagggg gaggggaaac aggatgcggc gaggcgcgtg
2521 cgcactgcca gcttcagcac cgcggacagt gccttcgccc ccgcctggcg gcgcgcgcca
2581 ccgccgcctc agcactgaag gcgcgctgac gtcactcgcc ggtcccccgc aaactcccct
2641 tcccggccac cttggtcgcg tccgcgccgc cgccggccca gccggaccgc accacgcgag
2701 gcgcgagata ggggggcacg ggcgcgacca tctgcgctgc ggcgccggcg actcagcgct
2761 gcctcagtct gcggtgggca gcggaggagt cgtgtcgtgc ctgagagcgc agaccggtgg
2821 taccataact tcgtatagga tactttatac gaagttatgc agaatggtag ctggattgta
2881 gctgctatta gcaatatgaa acctcttaat aacttcgtat agcatacatt atacgaagtt
2941 atactaccca ggctagcccg gtgccaccat caccggatcc gggacctgaa ataaaagaca
3001 aaaagactaa acttacccag gcgaatctgc tgtttcccct ggccagaggg cccacgtagt
3061 aggggatgcg gaaggtcagg atcttctcga tcttttcccg gttgtccttc aggaatgggt
3121 aaaaatcttc ctgccgccgc agaatggcgt gcagctctcc caggtggatc tggtggggga
3181 tgctgccgtt gtcgaaggtc cgctgcttcc gcagcaggtc ctctctgttc agcttcacga
3241 gcagttcctc ggtgccgtcc atcttttcca ggatgggctt gatgaacttg tagaactctt
3301 cctggctggc tccgccgtca atgtagccgg cgtagccgtt cttgctctgg tcgaagaaaa
3361 tctctttgta cttctcaggc agctgctgcc gcacgagagc tttcagcagg gtcaggtcct
3421 ggtggtgctc gtcgtatctc ttgatcatag aggcgctcag gggggccttg gtgatctcgg
3481 tgttcactct caggatgtcg ctcagcagga tggcgtcgga caggttcttg gcggccagaa
3541 acaggtcggc gtactggtcg ccgatctggg ccagcaggtt gtccaggtcg tcgtcgtagg
3601 tgtccttgct cagctgcagt ttggcatcct cggccaggtc gaagttgctc ttgaagttgg
3661 gggtcaggcc caggctcagg gcaatcaggt tgccgaacag gccattcttc ttctcgccgg
3721 gcagctgggc gatcagattt tccagccgtc tgctcttgct cagtctggca gacaggatgg
3781 ccttggcgtc cacgccgctg gcgttgatgg ggttttcctc gaacagctgg ttgtaggtct
3841 gcaccagctg gatgaacagc ttgtccacgt cgctgttgtc ggggttcagg tcgccctcga
3901 tcaggaagtg gccccggaac ttgatcatgt gggccagggc cagatagatc agccgcaggt
3961 cggccttgtc ggtgctgtcc accagtttct ttctcaggtg gtagatggtg gggtacttct
4021 cgtggtaggc cacctcgtcc acgatgttgc cgaagatggg gtgccgctcg tgcttcttat
4081 cctcttccac caggaaggac tcttccagtc tgtggaagaa gctgtcgtcc accttggcca
4141 tctcgttgct gaagatctct tgcagatagc agatccggtt cttccgtctg gtgtatcttc
4201 ttctggcggt tctcttcagc cgggtggcct cggctgtttc gccgctgtcg aacagcaggg
4261 ctccgatcag gttcttcttg atgctgtgcc ggtcggtgtt gcccagcacc ttgaatttct
4321 tgctgggcac cttgtactcg tcggtgatca cggcccagcc cacagagttg gtgccgatgg
4381 ccaggccgat gctgtacttc ttgtcggctg ctgggactcc gtggataccg accttccgct
4441 tcttctttgg ggccatctta tcgtcatcgt ctttgtaatc aatatcatga tccttgtagt
4501 ctccgtcgtg gtccttatag tccatggtgg cataacttcg tataaagtat cctatacgaa
4561 gttatttgcc ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac
4621 ttcgtataat gtatgctata cgaagttatg tggtgcaaat caaagaactg ctcctcagtg
4681 gatgttgcct ttacttctag atgaccagaa agagcgagga aaccatcacc ccctggaact
4741 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact
4801 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
4861 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
4921 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga
4981 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
5041 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
5101 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
5161 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
5221 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
5281 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
5341 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
5401 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
5461 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
5521 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
5581 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
5641 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
5701 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
5761 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
5821 tggacgctat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga
5881 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
5941 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
6001 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
6061 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
6121 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
6181 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
6241 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
6301 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
6361 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
6421 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
6481 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
6541 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
6601 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
6661 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
6721 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
6781 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
6841 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
6901 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
6961 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
7021 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
7081 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
7141 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
7201 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
7261 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
7321 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
7381 cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg
7441 gacaggccaa aaagaaaaag agtggtggag gaagtggcgg gtcagggtcg atggacgcga
7501 aatcacttac ggcatggtcg agaacactgg ttacgttcaa ggacgtgttt gtggacttta
7561 cacgtgagga gtggaaattg ctggatactg cgcaacaaat tgtgtatcga aatgtcatgc
7621 ttgagaatta caagaacctc gtcagtctcg gataccagtt gacgaaaccg gatgtgatcc
7681 ttaggctcga aaagggggaa gaaccttggc tggtatcggg aggtggttcg ggtggctctg
7741 gatcaagccc aaagaagaaa cggaaggtgg aagcctcagt gcaggtgaaa agggtgctgg
7801 aaaaatcccc cggcaaactc ctcgtgaaga tgcccttcca ggcttcccct ggcggaaaag
7861 gtgaaggggg tggcgcaacc acatctgccc aggtcatggt catcaagcga cctggaagga
7921 aaagaaaggc cgaggctgac cctcaggcca ttccaaagaa acggggacgc aagccagggt
7981 ccgtggtcgc agctgcagca gctgaggcta agaaaaaggc agtgaaggaa agctccatcc
8041 gcagtgtgca ggagactgtc ctgcccatca agaagaggaa gactagggag accgtgtcca
8101 tcgaggtcaa agaagtggtc aagcccctgc tcgtgtccac cctgggcgaa aaatctggaa
8161 aggggctcaa aacatgcaag tcacctggac ggaaaagcaa ggagtctagt ccaaaggggc
8221 gctcaagctc cgcttctagt ccccctaaaa aggaacacca tcaccatcac catcacgccg
8281 agtctcctaa ggctcctatg ccactgctcc caccacctcc accacctgag ccacagtcaa
8341 gcgaagaccc catcagccca cccgagcctc aggatctgtc ctctagtatt tgcaaagagg
8401 aaaagatgcc cagagcaggc agcctggaga gtgatggctg tccaaaagaa cccgccaaga
8461 cccagcctat ggtggcagcc gctgcaacta ccaccacaac cacaactacc acagtggccg
8521 aaaaatacaa gcatcgcggc gagggcgaac gaaaggacat tgtgtcaagc tccatgccca
8581 gacctaaccg ggaggaacca gtcgatagta ggacacccgt gactgagaga gtctcatagg
8641 aattcaattc taactagacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg
8701 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
8761 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
8821 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
8881 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
8941 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
9001 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
9061 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
9121 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
9181 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
9241 ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga
9301 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
9361 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
9421 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
9481 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
9541 ttagtcagtg tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg
9601 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
9661 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
9721 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
9781 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
9841 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
9901 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
9961 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
10021 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
10081 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
10141 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
10201 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
10261 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
10321 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
10381 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
10441 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
10501 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
10561 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
10621 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
10681 gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
10741 aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc
10801 gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac
10861 gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag
10921 gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc
10981 gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag
11041 atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg
11101 cacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc
11161 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
11221 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
11281 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
11341 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
11401 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
11461 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
11521 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
11581 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
11641 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
11701 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
11761 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
11821 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
11881 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
11941 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
12001 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
12061 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
12121 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
12181 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
12241 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
12301 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
12361 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
12421 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
12481 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
12541 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
12601 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
12661 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
12721 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
12781 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
12841 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
12901 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
12961 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
13021 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
13081 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
13141 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
13201 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
13261 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
13321 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
13381 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
13441 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
13501 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
13561 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
13621 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
13681 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
13741 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
13801 gggccagata tacgcgtt
//
