| 质粒名称: | Lenti-eCas9 |
|---|---|
| 出品公司: | addgene |
| 目录编号: | 140237 |
| 存储实验室: | Urs Greber |
|
载体骨架基本信息 |
|
| 载体名称: | lentiCRISPR v2 |
| 载体出品公司: | Feng Zhang lab |
| 空载体大小: | 10017bp |
| 完整质粒大小: | 14962 bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | Puromycin |
| 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: | enhanced specificity Cas9 (1.1)/eSpCas9(1.1) |
| 物种来源: | Synthetic |
| 插入大小: | 4945 bp |
| 启动子: | EF-1a core |
| 标签/融合蛋白: | 3xFLAG (N terminal on insert) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | XbaI(未破坏) |
| 3’克隆位点: | BamHI(未破坏) |
| 5’测序引物 | gggggagaaccgtatataag |
| 3’测序引物 | WPRE-R |
|
文献引用方法 |
|
| 材料和方法部分: | Lenti-eCas9 was a gift from Urs Greber (Addgene plasmid # 140237 ; http://n2t.net/addgene:140237 ; RRID:Addgene_140237) |
| 参考文献部分: | The E3 Ubiquitin Ligase Mind Bomb 1 Controls Adenovirus Genome Release at the Nuclear Pore Complex. Bauer M, Flatt JW, Seiler D, Cardel B, Emmenlauer M, Boucke K, Suomalainen M, Hemmi S, Greber UF. Cell Rep. 2019 Dec 17;29(12):3785-3795.e8. doi: 10.1016/j.celrep.2019.11.064. 10.1016/j.celrep.2019.11.064 PubMed 31851912 |
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-eCas9 14962 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Lentiviral vector for expression of high-specificity eSpCas9(1.1)
and sgRNA scaffold.
ACCESSION .
VERSION .
KEYWORDS Lenti-eCas9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14962)
AUTHORS Bauer M, Flatt JW, Seiler D, Cardel B, Emmenlauer M, Boucke K,
Suomalainen M, Hemmi S, Greber UF
TITLE The E3 Ubiquitin Ligase Mind Bomb 1 Controls Adenovirus Genome
Release at the Nuclear Pore Complex.
JOURNAL Cell Rep. 2019 Dec 17;29(12):3785-3795.e8. doi:
10.1016/j.celrep.2019.11.064.
PUBMED 31851912
REFERENCE 2 (bases 1 to 14962)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..14962
/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 2371..2611
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 2371..2391
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 2542..2561
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 4501..4576
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 4638..4849
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 4864..4873
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 4873..4938
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 4945..4965
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4990..9090
/codon_start=1
/product="Cas9 endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system, mutated to improve targeting
specificity (Slaymaker et al., 2016)"
/label=eSpCas9(1.1)
/note="carries the mutations K848A, K1003A, and R1060A"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 9091..9138
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 9148..9204
/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 9205..9801
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind 9698..9718
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 9817..10405
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(9870..9890)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(10288..10299)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 10477..10710
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(10736..10753)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 10742..10966
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 11012..11440
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(11099..11118)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 11309..11330
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(11449..11469)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 11454..11783
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 11634..11769
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 11696..11715
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 11831..11878
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 11897..12271
/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 12401..12522
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(12438..12457)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 12492..12511
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(12571..12587)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12571..12587)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12584..12606)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 12595..12611
/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(12619..12649)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 12664..12685
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12802..12819)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(12973..13561)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(13053..13072)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(13732..14592)
/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 14355..14374
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(14593..14697)
/gene="bla"
/label=AmpR promoter
primer_bind complement(14772..14791)
/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 ttaaggtacc gagggcctat ttcccatgat tccttcatat
2401 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
2461 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
2521 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
2581 ttcttggctt tatatatctt gtggaaagga cgaaacaccg gagacggttg taaatgagca
2641 cacaaaatac acatgctaaa atattatatt ctatgacctt tataaaatca accaaaatct
2701 tctttttaat aactttagta tcaataatta gaatttttat gttccttttt gcaaactttt
2761 aataaaaatg agcaaaataa aaaaacgcta gttttagtaa ctcgcgttgt tttcttcacc
2821 tttaataata gctactccac cacttgttcc taagcggtca gctcctgctt caatcatttt
2881 ttgagcatct tcaaatgttc taactccacc agctgcttta actaaagcat tgtctttaac
2941 aactgacttc attagtttaa catcttcaaa tgttgcacct gattttgaaa atcctgttga
3001 tgttttaaca aattctaatc cagcttcaac agctatttca caagctttca tgatttcttc
3061 ttttgttaat aaacaatttt ccataataca tttaacaaca tgtgatccag ctgctttttt
3121 tacagctttc atgtcttcta aaactaattc ataatttttg tcttttaatg caccaatatt
3181 taataccata tcaatttctg ttgcaccatc tttaattgct tcagaaactt cgaatgcttt
3241 tgtagctgtt gtgcatgcac ctagaggaaa acctacaaca tttgttattc ctacatttgt
3301 gccttttaat aattctttac aatagcttgt tcaatatgaa ttaacacaaa ctgttgcaaa
3361 atcaaattca attgcttcat cacataattg tttaatttca gctttcgtag catcttgttt
3421 taataatgtg tgatctatat atttgtttag tttcattttt tctcctatat attcattttt
3481 aattttaatt ctttaataat ttcgtctact ttaactttag cgttttgaac agattcacca
3541 acacctataa aataaatttt tagtttaggt tcagttccac ttgggcgaac agcaaatcat
3601 gacttatctt ctaaataaaa ttttagtaag tcttgtcctg gcatattata cattccatcg
3661 atgtagtctt caacattaac aactttaagt ccagcaattt gagttaaggg tgttgctctc
3721 aatgatttca ttaatggttc aatttttaat ttcttttctt ctggtttaaa attcaagttt
3781 aaagtgaaag tgtaatatgc acccatttct ttaaataaat cttctaaata gtctactaat
3841 gttttatttt gttttttata aaatcaagca gcctctgcta ttaatataga agcttgtatt
3901 ccatctttat ctctagctga gtcatcaatt acatatccat aactttcttc ataagcaaaa
3961 acaaaattta atccgttatc ttcttcttta gcaatttctc tacccattca tttaaatcca
4021 gttaaagttt ttacaatatt aactccatat ttttcatgag cgattctatc acccaaatca
4081 cttgttacaa aacttgaata tagagccgga ttttttggaa tgctatttaa gcgttttaga
4141 tttgataatt ttcaatcaat taaaattggt cctgtttgat ttccatctaa tcttacaaaa
4201 tgaccatcat gttttattgc cattccaaat ctgtcagcat ctgggtcatt cataataata
4261 atatctgcat catgtttaat accatattca agcggtattt ttcatgcagg atcaaattct
4321 ggatttggat ttacaacatt tttaaatgtt tcatcttcaa atgcatgctc ttcaacctca
4381 ataacgttat atcctgattc acgtaatatt tttggggtaa atttagttcc tgttccatta
4441 actgcgctaa aaataatttt taaatctttt ttagcttctt gctctttttt gtacgtctct
4501 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
4561 ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg
4621 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
4681 aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
4741 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
4801 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggttcta
4861 gatgccacca tggactataa ggaccacgac ggagactaca aggatcatga tattgattac
4921 aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc
4981 ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
5041 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
5101 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
5161 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
5221 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
5281 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
5341 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
5401 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
5461 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
5521 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
5581 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
5641 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
5701 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
5761 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
5821 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
5881 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
5941 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
6001 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
6061 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
6121 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
6181 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
6241 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
6301 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
6361 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
6421 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
6481 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
6541 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
6601 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
6661 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
6721 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
6781 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
6841 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
6901 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
6961 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
7021 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
7081 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
7141 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
7201 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
7261 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
7321 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
7381 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
7441 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
7501 gaccatatcg tgcctcagag ctttctggcc gacgactcca tcgacaacaa ggtgctgacc
7561 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
7621 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
7681 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
7741 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
7801 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
7861 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
7921 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
7981 aaaaagtacc ctgcgctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
8041 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
8101 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
8161 aaggcgcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
8221 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
8281 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
8341 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
8401 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
8461 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
8521 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
8581 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
8641 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
8701 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
8761 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
8821 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
8881 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
8941 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
9001 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
9061 cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc
9121 caggcaaaaa agaaaaaggg atccggcgca acaaacttct ctctgctgaa acaagccgga
9181 gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct cgccacccgc
9241 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
9301 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
9361 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
9421 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
9481 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
9541 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac
9601 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
9661 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
9721 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
9781 acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt
9841 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
9901 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
9961 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
10021 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
10081 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
10141 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
10201 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
10261 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
10321 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
10381 atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg
10441 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
10501 gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct
10561 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
10621 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
10681 ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg atcagcctcg
10741 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
10801 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
10861 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
10921 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
10981 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
11041 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
11101 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
11161 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
11221 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
11281 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
11341 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
11401 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
11461 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
11521 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
11581 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
11641 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
11701 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
11761 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
11821 atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa
11881 ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
11941 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
12001 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
12061 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
12121 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
12181 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
12241 cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc
12301 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
12361 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
12421 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
12481 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
12541 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
12601 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
12661 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
12721 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
12781 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
12841 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
12901 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
12961 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
13021 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
13081 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
13141 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
13201 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
13261 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
13321 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
13381 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
13441 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
13501 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
13561 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
13621 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
13681 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
13741 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
13801 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
13861 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
13921 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
13981 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
14041 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
14101 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
14161 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
14221 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
14281 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
14341 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
14401 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
14461 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
14521 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
14581 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
14641 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
14701 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
14761 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
14821 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
14881 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
14941 gtacgggcca gatatacgcg tt
//
