| 质粒名称: | Lenti-iCas9-neo |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 85400 |
| 存储实验室: | Qin Yan |
|
载体骨架基本信息 |
|
| 载体名称: | pInducer-20 |
| 载体出品公司: | -- |
| 空载体大小: | 10000 bp |
| 完整质粒大小: | 15188 bp |
| 载体修饰: | -- |
| 载体类型: | 慢病毒载体 |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | Flag-iCas9-P2A-GFP |
| 物种来源: | -- |
| 插入大小: | 5000 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | -- |
| 标签/融合蛋白: | Flag-iCas9-P2A-GFP |
| 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-iCas9-neo was a gift from Qin Yan (Addgene plasmid # 85400 ; http://n2t.net/addgene:85400 ; RRID:Addgene_85400) |
| 参考文献部分: | An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting. Cao J, Wu L, Zhang SM, Lu M, Cheung WK, Cai W, Gale M, Xu Q, Yan Q. Nucleic Acids Res. 2016 Nov 2;44(19):e149. Epub 2016 Jul 25. 10.1093/nar/gkw660 PubMed 27458201 |
买家导航
Lenti-iCas9-neo载体质粒基本信息
Lenti-iCas9-neo质粒图谱载体图谱和Lenti-iCas9-neo载体序列质粒序列多克隆位点信息
Lenti-iCas9-neo质粒载体简介
Lenti-iCas9-neo质粒序列载体序列
LOCUS Lenti-iCas9-neo 15200 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Lentiviral vector encoding a doxycycline inducible EGFP reporter
downstream of FLAG-tagged spCas9, separated by a P2A self-cleavage
sequence.
ACCESSION .
VERSION .
KEYWORDS Lenti-iCas9-neo
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15200)
AUTHORS Cao J, Wu L, Zhang SM, Lu M, Cheung WK, Cai W, Gale M, Xu Q, Yan Q
TITLE An easy and efficient inducible CRISPR/Cas9 platform with improved
specificity for multiple gene targeting.
JOURNAL Nucleic Acids Res. 2016 Nov 2;44(19):e149. Epub 2016 Jul 25.
PUBMED 27458201
REFERENCE 2 (bases 1 to 15200)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..15200
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 1..634
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1721..1765
/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 2058..2175
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
protein_bind 2230..2500
/label=tetracycline response element
/bound_moiety="tetracycline repressor TetR"
/note="contains seven copies of the tetracycline operator
tetO"
promoter 2533..2571
/label=minimal CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 2568..2592
/label=LNCX
/note="Human CMV promoter, forward primer"
protein_bind 2701..2725
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 2746..2769
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 2776..2796
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2821..6921
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LAEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 6922..6969
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 6985..7041
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 7048..7764
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(7093..7114)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(7354..7373)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 7701..7722
/label=EGFP-C
/note="EGFP, forward primer"
protein_bind complement(7772..7796)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 7849..7875
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
promoter 7961..9172
/label=UbC promoter
/note="human ubiquitin C promoter"
primer_bind 9009..9028
/label=hUBCpro-F
/note="Human Ubiquitin C (UbC) promoter, forward primer"
CDS 9231..9977
/codon_start=1
/product="improved tetracycline-controlled transactivator"
/label=rtTA-Advanced
/note="In the Tet-On(R) system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription only in
the presence of tetracycline or doxycycline."
/translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
misc_feature 10030..10603
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
primer_bind complement(10197..10214)
/label=IRES reverse
/note="IRES internal ribosome entry site, reverse primer.
Also called pCDH-rev"
primer_bind 10424..10443
/label=IRES-F
/note="IRES internal ribosome entry site, forward primer"
CDS 10619..11413
/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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(10673..10692)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 11283..11302
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
misc_feature 11590..12178
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(11643..11663)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(12061..12072)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 12253..12486
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(12605..12623)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 12691..12795
/gene="bla"
/label=AmpR promoter
CDS 12796..13656
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(13014..13033)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 13827..14415
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 14316..14335
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 14569..14586
/label=L4440
/note="L4440 vector, forward primer"
primer_bind 14678..14697
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
protein_bind 14826..14847
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 14862..14892
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 14900..14916
/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)."
primer_bind 14905..14927
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 14924..14940
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 14924..14940
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 14990..15009
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
ORIGIN
1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactaggatc
2221 ctttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca
2281 gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa
2341 agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc
2401 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
2461 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagctcgg
2521 tacccgggtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg
2581 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg
2641 gaccgatcca gcctccgcgg ccccgaacta gtccagtgtg gtggaattct gcagatatca
2701 acaagtttgt acaaaaaagc aggcttcgaa ggagatagaa ccatggatta caaagacgat
2761 gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc
2821 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
2881 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
2941 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
3001 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
3061 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
3121 gaagagtcct tcctggcgga agaggataag aagcacgagc ggcaccccat cttcggcaac
3181 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
3241 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
3301 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
3361 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
3421 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
3481 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
3541 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
3601 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
3661 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
3721 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
3781 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
3841 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
3901 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
3961 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
4021 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
4081 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
4141 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
4201 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
4261 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
4321 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
4381 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
4441 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
4501 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
4561 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
4621 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
4681 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
4741 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
4801 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
4861 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
4921 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
4981 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
5041 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
5101 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
5161 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
5221 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
5281 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
5341 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
5401 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
5461 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
5521 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
5581 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
5641 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
5701 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
5761 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
5821 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
5881 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
5941 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
6001 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
6061 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
6121 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
6181 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
6241 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
6301 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
6361 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
6421 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
6481 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
6541 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
6601 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
6661 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
6721 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
6781 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
6841 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
6901 ctgtctcagc tgggaggcga caagcgtcct gctgctacta agaaagctgg tcaagctaag
6961 aaaaagaaag ctagcggcag cggcgccacc aacttcagcc tgctgaagca ggccggcgac
7021 gtggaggaga accccggccc cgaattcatg gtgagcaagg gcgaggagct gttcaccggg
7081 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
7141 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
7201 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
7261 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
7321 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
7381 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
7441 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
7501 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
7561 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
7621 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
7681 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
7741 ctcggcatgg acgagctgta caagtgatag gacccagctt tcttgtacaa agtggttgat
7801 atccagcaca gtggcggccg ctcgagtcta gagggcccgc ggttcgaata cccatacgac
7861 gtcccagact acgcttagta atgattaatt aaactagaaa ttctaccggg taggggaggc
7921 gcttttccca aggcagtctg gagtaaccca cccaagatct ggcctccgcg ccgggttttg
7981 gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg ccacgtcaga cgaagggcgc
8041 agcgagcgtc ctgatccttc cgcccggacg ctcaggacag cggcccgctg ctcataagac
8101 tcggccttag aaccccagta tcagcagaag gacattttag gacgggactt gggtgactct
8161 agggcactgg ttttctttcc agagagcgga acaggcgagg aaaagtagtc ccttctcggc
8221 gattctgcgg agggatctcc gtggggcggt gaacgccgat gattatataa ggacgcgccg
8281 ggtgtggcac agctagttcc gtcgcagccg ggatttgggt cgcggttctt gtttgtggat
8341 cgctgtgatc gtcacttggt gagtagcggg ctgctgggct ggccggggct ttcgtggccg
8401 ccgggccgct cggtgggacg gaagcgtgtg gagagaccgc caagggctgt agtctgggtc
8461 cgcgagcaag gttgccctga actgggggtt ggggggagcg cagcaaaatg gcggctgttc
8521 ccgagtcttg aatggaagac gcttgtgagg cgggctgtga ggtcgttgaa acaaggtggg
8581 gggcatggtg ggcggcaaga acccaaggtc ttgaggcctt cgctaatgcg ggaaagctct
8641 tattcgggtg agatgggctg gggcaccatc tggggaccct gacgtgaagt ttgtcactga
8701 ctggagaact cggtttgtcg tctgttgcgg gggcggcagt tatggcggtg ccgttgggca
8761 gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt
8821 ggcttataat gcagggtggg gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg
8881 caggacgcag ggttcgggcc tagggtaggc tctcctgaat cgacaggcgc cggacctctg
8941 gtgaggggag ggataagtga ggcgtcagtt tctttggtcg gttttatgta cctatcttct
9001 taagtagctg aagctccggt tttgaactat gcgctcgggg ttggcgagtg tgttttgtga
9061 agttttttag gcaccttttg aaatgtaatc atttgggtca atatgtaatt ttcagtgtta
9121 gactagtaaa ttgtccgcta aattctggcc gtttttggct tttttgttag acgaagcttg
9181 gtaccgagct cggatctcca ccccgtaccg gtcctgcagt cgaattcacc atgtctagac
9241 tggacaagag caaagtcata aacggagctc tggaattact caatggtgtc ggtatcgaag
9301 gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc ctgtactggc
9361 acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg gacaggcatc
9421 atacccactt ctgccccctg gaaggcgagt catggcaaga ctttctgcgg aacaacgcca
9481 agtcataccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat ctcggcaccc
9541 gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag
9601 gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt acactgggct
9661 gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca cctaccaccg
9721 attctatgcc cccacttctg agacaagcaa ttgagctgtt cgaccggcag ggagccgaac
9781 ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag ctaaagtgcg
9841 aaagcggcgg gccgaccgac gcccttgacg attttgactt agacatgctc ccagccgatg
9901 cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat tttgaccttg
9961 acatgctccc cgggtaacta agtaaggatc cgcggccgca ctagaggaat tccgcccctc
10021 tccccccccc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata
10081 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt
10141 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct
10201 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc
10261 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga
10321 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc
10381 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt
10441 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg
10501 gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc
10561 gaaccacggg gacgtggttt tcctttgaaa aacacgatga taagcttacc ggtccaccat
10621 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
10681 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
10741 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
10801 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
10861 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
10921 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
10981 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
11041 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
11101 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
11161 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
11221 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
11281 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
11341 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
11401 cgagttcttc tgatgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca
11461 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
11521 ataaaatgaa tgcaattgtt gttgtttagt ccctcccaat tcgatatcaa gcttatcgcg
11581 atagatccta atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
11641 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
11701 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
11761 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
11821 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
11881 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
11941 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
12001 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
12061 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
12121 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct
12181 gagatccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
12241 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
12301 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
12361 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
12421 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
12481 ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
12541 tatcagagag tgagaggccc gggttaatta aggaaagggc tagatcattc ttgaagacga
12601 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
12661 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
12721 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
12781 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
12841 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
12901 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
12961 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
13021 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
13081 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
13141 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
13201 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
13261 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
13321 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
13381 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
13441 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
13501 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
13561 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
13621 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
13681 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
13741 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
13801 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
13861 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
13921 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
13981 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
14041 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
14101 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
14161 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
14221 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
14281 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
14341 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
14401 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
14461 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
14521 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
14581 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
14641 cattaatgca gcaagctcat ggctgactaa ttttttttat ttatgcagag gccgaggccg
14701 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
14761 gcaaaaagct ccccgtggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
14821 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
14881 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
14941 atgattacga atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
15001 aaactcatca atgtatctta tcatgtctgg atcaactgga taactcaagc taaccaaaat
15061 catcccaaac ttcccacccc ataccctatt accactgcca attacctgtg gtttcattta
15121 ctctaaacct gtgattcctc tgaattattt tcattttaaa gaaattgtat ttgttaaata
15181 tgtactacaa acttagtagt
//
