| 质粒名称: | lenti SYN-SVI-DIO-dCas9-VPR |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 164576 |
| 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: | 14238 bp |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达，慢病毒，Cre/Lox，CRISPR |
| 筛选标记: | -- |
| 载体抗性: | 氨苄霉素100 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | -- |
|
插入基因信息 |
|
| 插入基因名称: | A DIO FLAG-dCas9-VPR cassette containing an SV40 intron |
| 物种来源: | Synthetic |
| 插入大小: | 6692 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | -- |
| 标签/融合蛋白: | FLAG（插入件上的N端子） |
| 克隆方法: | -- |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | -- |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | lenti SYN-SVI-DIO-dCas9-VPR was a gift from Jeremy Day (Addgene plasmid # 164576 ; http://n2t.net/addgene:164576 ; RRID:Addgene_164576) |
| 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-VPR 14238 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses a SYN-driven, intron-containing dCas9-VPR 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-VPR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14238)
TITLE A Cre-dependent CRISPR/dCas9 activation system for gene expression
regulation in neurons
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 14238)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..14238
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(166..1026)
/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 789..808
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(1027..1131)
/gene="bla"
/label=AmpR promoter
primer_bind complement(1206..1225)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 1397..1776
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 1778..1976
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 1931..1951
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 1994..2174
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 2221..2346
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 2839..3072
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 3257..3301
/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 3599..3716
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 3761..4208
/label=hSyn promoter
/note="human synapsin I promoter; confers neuron-specific
expression (Kugler et al., 2003)"
protein_bind 4221..4254
/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 4305..4338
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS complement(5826..5846)
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS complement(5853..5918)
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
regulatory 5918..5927
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
protein_bind complement(5928..5961)
/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(6012..6045)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS 6310..8808
/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 8809..8856
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 8884..9033
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label=VP64
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 9058..9078
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 9508..9864
/codon_start=1
/product="transcriptional activation domain of human RelA,
also known as p65 (O'Shea and Perkins, 2008)"
/label=RelA (p65) AD
/translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
EDFSSIADMDFSALL"
CDS 9883..10452
/codon_start=1
/product="transcriptional activation domain from the human
herpesvirus 4 (Epstein-Barr virus) replication and
transcription activator Rta/BRLF1 (Hardwick et al., 1992;
Chavez et al., 2015)"
/label=Rta AD
/translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
DTFLNDECLLHAMHISTGLSIFDTSLF"
misc_feature 10489..11077
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(10542..10562)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(10960..10971)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 11149..11382
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(11408..11425)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 11414..11638
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 11684..12112
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(11771..11790)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 11981..12002
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(12121..12141)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 12126..12455
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 12306..12441
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 12368..12387
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 12503..12550
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 12569..12943
/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 13073..13194
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(13110..13129)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 13164..13183
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(13243..13259)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(13243..13259)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(13256..13278)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 13267..13283
/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(13291..13321)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 13336..13357
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(13474..13491)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(13645..14233)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(13725..13744)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
61 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
121 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
181 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
241 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
301 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
361 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
421 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
481 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
541 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
601 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
661 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
721 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
781 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
841 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
901 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
961 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
1021 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
1081 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
1141 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
1201 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
1261 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
1321 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
1381 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
1441 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
1501 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
1561 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
1621 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
1681 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
1741 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
1801 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
1861 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
1921 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg
1981 ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
2041 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
2101 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
2161 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
2221 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
2281 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
2341 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
2401 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
2461 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
2521 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
2581 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
2641 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag
2701 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
2761 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
2821 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
2881 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
2941 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
3001 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
3061 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
3121 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
3181 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
3241 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
3301 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
3361 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
3421 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
3481 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt
3541 gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc atccacaatt
3601 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
3661 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
3721 tttattacag ggacagcaga gatccagttt ggttaattaa agtgcaagtg ggttttagga
3781 ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc
3841 acccaacccc cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga
3901 tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc
3961 ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc
4021 ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg
4081 gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg
4141 ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga
4201 gagcgcagac cggtggtacc ataacttcgt ataggatact ttatacgaag ttatgcagaa
4261 tggtagctgg attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca
4321 tacattatac gaagttatac tacccaggct agcccggtgc caccatcacc ggatccggga
4381 cctgaaataa aagacaaaaa gactaaactt acccaggcga atctgctgtt tcccctggcc
4441 agagggccca cgtagtaggg gatgcggaag gtcaggatct tctcgatctt ttcccggttg
4501 tccttcagga atgggtaaaa atcttcctgc cgccgcagaa tggcgtgcag ctctcccagg
4561 tggatctggt gggggatgct gccgttgtcg aaggtccgct gcttccgcag caggtcctct
4621 ctgttcagct tcacgagcag ttcctcggtg ccgtccatct tttccaggat gggcttgatg
4681 aacttgtaga actcttcctg gctggctccg ccgtcaatgt agccggcgta gccgttcttg
4741 ctctggtcga agaaaatctc tttgtacttc tcaggcagct gctgccgcac gagagctttc
4801 agcagggtca ggtcctggtg gtgctcgtcg tatctcttga tcatagaggc gctcaggggg
4861 gccttggtga tctcggtgtt cactctcagg atgtcgctca gcaggatggc gtcggacagg
4921 ttcttggcgg ccagaaacag gtcggcgtac tggtcgccga tctgggccag caggttgtcc
4981 aggtcgtcgt cgtaggtgtc cttgctcagc tgcagtttgg catcctcggc caggtcgaag
5041 ttgctcttga agttgggggt caggcccagg ctcagggcaa tcaggttgcc gaacaggcca
5101 ttcttcttct cgccgggcag ctgggcgatc agattttcca gccgtctgct cttgctcagt
5161 ctggcagaca ggatggcctt ggcgtccacg ccgctggcgt tgatggggtt ttcctcgaac
5221 agctggttgt aggtctgcac cagctggatg aacagcttgt ccacgtcgct gttgtcgggg
5281 ttcaggtcgc cctcgatcag gaagtggccc cggaacttga tcatgtgggc cagggccaga
5341 tagatcagcc gcaggtcggc cttgtcggtg ctgtccacca gtttctttct caggtggtag
5401 atggtggggt acttctcgtg gtaggccacc tcgtccacga tgttgccgaa gatggggtgc
5461 cgctcgtgct tcttatcctc ttccaccagg aaggactctt ccagtctgtg gaagaagctg
5521 tcgtccacct tggccatctc gttgctgaag atctcttgca gatagcagat ccggttcttc
5581 cgtctggtgt atcttcttct ggcggttctc ttcagccggg tggcctcggc tgtttcgccg
5641 ctgtcgaaca gcagggctcc gatcaggttc ttcttgatgc tgtgccggtc ggtgttgccc
5701 agcaccttga atttcttgct gggcaccttg tactcgtcgg tgatcacggc ccagcccaca
5761 gagttggtgc cgatggccag gccgatgctg tacttcttgt cggctgctgg gactccgtgg
5821 ataccgacct tccgcttctt ctttggggcc atcttatcgt catcgtcttt gtaatcaata
5881 tcatgatcct tgtagtctcc gtcgtggtcc ttatagtcca tggtggcata acttcgtata
5941 aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta ttctttagaa
6001 tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgtggt gcaaatcaaa
6061 gaactgctcc tcagtggatg ttgcctttac ttctagatga ccagaaagag cgaggaaacc
6121 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
6181 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
6241 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
6301 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
6361 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
6421 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
6481 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
6541 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
6601 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
6661 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
6721 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
6781 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
6841 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
6901 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
6961 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
7021 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
7081 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
7141 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
7201 ctgtccgact acgatgtgga cgctatcgtg cctcagagct ttctgaagga cgactccatc
7261 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
7321 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
7381 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
7441 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
7501 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
7561 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
7621 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
7681 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
7741 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
7801 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
7861 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
7921 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
7981 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
8041 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
8101 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
8161 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
8221 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
8281 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
8341 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
8401 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
8461 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
8521 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
8581 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
8641 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
8701 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
8761 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa aaggccggcg
8821 gccacgaaaa aggccggaca ggccaaaaag aaaaagctcg agggcggagg cgggagcgga
8881 tccgacgcat tggacgattt tgatctggat atgctgggaa gtgacgccct cgatgatttt
8941 gaccttgaca tgcttggttc ggatgccctt gatgactttg acctcgacat gctcggcagt
9001 gacgcccttg atgatttcga cctggacatg ctgattaact ctagaagttc cggatctccg
9061 aaaaagaaac gcaaagttgg tagccagtac ctgcccgaca ccgacgaccg gcaccggatc
9121 gaggaaaagc ggaagcggac ctacgagaca ttcaagagca tcatgaagaa gtcccccttc
9181 agcggcccca ccgaccctag acctccacct agaagaatcg ccgtgcccag cagatccagc
9241 gccagcgtgc caaaacctgc cccccagcct taccccttca ccagcagcct gagcaccatc
9301 aactacgacg agttccctac catggtgttc cccagcggcc agatctctca ggcctctgct
9361 ctggctccag cccctcctca ggtgctgcct caggctcctg ctcctgcacc agctccagcc
9421 atggtgtctg cactggctca ggcaccagca cccgtgcctg tgctggctcc tggacctcca
9481 caggctgtgg ctccaccagc ccctaaacct acacaggccg gcgagggcac actgtctgaa
9541 gctctgctgc agctgcagtt cgacgacgag gatctgggag ccctgctggg aaacagcacc
9601 gatcctgccg tgttcaccga cctggccagc gtggacaaca gcgagttcca gcagctgctg
9661 aaccagggca tccctgtggc ccctcacacc accgagccca tgctgatgga ataccccgag
9721 gccatcaccc ggctcgtgac aggcgctcag aggcctcctg atccagctcc tgcccctctg
9781 ggagcaccag gcctgcctaa tggactgctg tctggcgacg aggacttcag ctctatcgcc
9841 gatatggatt tctcagcctt gctgggctct ggcagcggca gccgggattc cagggaaggg
9901 atgtttttgc cgaagcctga ggccggctcc gctattagtg acgtgtttga gggccgcgag
9961 gtgtgccagc caaaacgaat ccggccattt catcctccag gaagtccatg ggccaaccgc
10021 ccactccccg ccagcctcgc accaacacca accggtccag tacatgagcc agtcgggtca
10081 ctgaccccgg caccagtccc tcagccactg gatccagcgc ccgcagtgac tcccgaggcc
10141 agtcacctgt tggaggatcc cgatgaagag acgagccagg ctgtcaaagc ccttcgggag
10201 atggccgata ctgtgattcc ccagaaggaa gaggctgcaa tctgtggcca aatggacctt
10261 tcccatccgc ccccaagggg ccatctggat gagctgacaa ccacacttga gtccatgacc
10321 gaggatctga acctggactc acccctgacc ccggaattga acgagattct ggataccttc
10381 ctgaacgacg agtgcctctt gcatgccatg catatcagca caggactgtc catcttcgac
10441 acatctctgt tttgagaatt caattctaac tagacgcgtt aagtcgacaa tcaacctctg
10501 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
10561 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
10621 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
10681 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
10741 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
10801 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
10861 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
10921 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
10981 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
11041 cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt
11101 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
11161 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
11221 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
11281 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
11341 gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt ttaaacccgc
11401 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
11461 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
11521 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
11581 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
11641 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
11701 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
11761 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
11821 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
11881 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
11941 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
12001 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
12061 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
12121 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
12181 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
12241 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
12301 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
12361 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
12421 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
12481 ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt
12541 ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct
12601 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg
12661 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag
12721 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct
12781 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
12841 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc
12901 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt
12961 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
13021 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
13081 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
13141 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
13201 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
13261 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
13321 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
13381 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
13441 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
13501 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
13561 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
13621 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
13681 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
13741 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
13801 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
13861 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
13921 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
13981 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
14041 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
14101 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
14161 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
14221 aaaaggatct caagaaga
//
