| 质粒名称: | lentiCRISPR_DKO |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 183192 |
| 存储实验室: | Lang Li |
|
载体骨架基本信息 |
|
| 载体名称: | lentiCRISPR v2 |
| 载体出品公司: | Plasmid #52961 |
| 空载体大小: | 14873 bp |
| 完整质粒大小: | 13479bp |
| 载体类型: | 哺乳细胞表达, Lentiviral, CRISPR |
| 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 |
|
| 启动子: | hU6, mU6 |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | hU6-F |
|
文献引用方法 |
|
| 材料和方法部分: | lentiCRISPR_DKO was a gift from Lang Li (Addgene plasmid # 183192 ; http://n2t.net/addgene:183192 ; RRID:Addgene_183192) |
| 参考文献部分: | Generation of dual-gRNA library for combinatorial CRISPR screening of synthetic lethal gene pairs. Tang S, Wu X, Liu J, Zhang Q, Wang X, Shao S, Gokbag B, Fan K, Liu X, Li F, Cheng L, Li L. STAR Protoc. 2022 Aug 24;3(3):101556. doi: 10.1016/j.xpro.2022.101556. eCollection 2022 Sep 16. 10.1016/j.xpro.2022.101556 PubMed 36060092 |
买家导航
lentiCRISPR_DKO载体质粒基本信息
lentiCRISPR_DKO质粒图谱载体图谱和lentiCRISPR_DKO载体序列质粒序列多克隆位点信息
lentiCRISPR_DKO质粒载体简介
lentiCRISPR_DKO质粒序列载体序列
LOCUS lentiCRISPR_DKO 13479 bp ds-DNA circular SYN 21-APR-2022
DEFINITION Plasmid with Cas9, two U6 promoters and two gRNA scaffolds that
allows for inserting two gRNAs for combinatorial CRISPR Screen or
double-knock-out (DKO) screen.
ACCESSION .
VERSION .
KEYWORDS lentiCRISPR_DKO
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13479)
TITLE Fast and Efficient Generation of Paired-gRNA Library Facilitates
Combinatorial CRISPR Screen for Discovering Synthetic Lethal Gene
Pairs
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 13479)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 21, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..13479
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 36..234
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 189..209
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 252..432
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 479..604
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1097..1330
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1515..1559
/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 1857..1974
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2025..2265
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 2025..2045
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 2196..2215
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
promoter 2409..2722
/label=U6 promoter
/note="RNA polymerase III promoter for mouse U6 snRNA (Das
et al., 1988)"
primer_bind 2641..2661
/label=mU6-F
/note="Mouse U6 promoter, forward primer"
misc_RNA 2761..2836
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 2898..3109
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 3128..3137
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3134..7237
/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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 7238..7285
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 7286..7309
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 7319..7375
/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 7376..7972
/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 7869..7889
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 7988..8576
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(8041..8061)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(8459..8470)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 8648..8881
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(8907..8924)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 8913..9137
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 9183..9611
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(9270..9289)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 9480..9501
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(9620..9640)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 9625..9954
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 9805..9940
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 9867..9886
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 10002..10049
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 10068..10442
/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 10572..10693
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(10609..10628)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 10663..10682
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(10742..10758)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10742..10758)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(10755..10777)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 10766..10782
/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(10790..10820)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 10835..10856
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(10973..10990)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(11144..11732)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(11224..11243)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(11903..12763)
/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 12526..12545
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(12764..12868)
/gene="bla"
/label=AmpR promoter
primer_bind complement(12943..12962)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 13134..34
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
ORIGIN
1 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
61 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
121 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
181 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt
241 ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
301 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
361 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
421 aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct
481 ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact
541 ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag
601 cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg
661 gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt
721 cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct
781 acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac
841 cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat
901 agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac
961 ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa
1021 aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa
1081 aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta
1141 tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc
1201 agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag
1261 tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc
1321 aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt
1381 ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg
1441 agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc
1501 aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt
1561 ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag
1621 gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc
1681 agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc
1741 ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga
1801 acggatcggc actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt
1861 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
1921 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
1981 tattacaggg acagcagaga tccagtttgg ttaattaagg taccgagggc ctatttccca
2041 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt
2101 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
2161 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
2221 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggactctag aaccaggatg
2281 ggcaccaccc gtttaagagc taagctggaa acagcatagc aagtttaaat aaggctagtc
2341 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttgtactga gtcgcccagt
2401 ctcagataga tccgacgccg ccatctctag gcccgcgccg gccccctcgc acagacttgt
2461 gggagaagct cggctactcc cctgccccgg ttaatttgca tataatattt cctagtaact
2521 atagaggctt aatgtgcgat aaaagacaga taatctgttc tttttaatac tagctacatt
2581 ttacatgata ggcttggatt tctataagag atacaaatac taaattatta ttttaaaaaa
2641 cagcacaaaa ggaaactcac cctaactgta aagtaattgt gtgttttgag actataaata
2701 tcccttggag aaaagccttg ttcaccggag acggcttctt gctctttttt gtacgtctct
2761 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
2821 ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg
2881 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
2941 aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
3001 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
3061 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggttcta
3121 gagcgctgcc accatggaca agaagtacag catcggcctg gacatcggca ccaactctgt
3181 gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg
3241 caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg
3301 cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa
3361 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag
3421 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
3481 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta
3541 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct
3601 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc
3661 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt
3721 cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact
3781 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg
3841 cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt
3901 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga
3961 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct
4021 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
4081 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa
4141 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa
4201 gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat
4261 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga
4321 ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct
4381 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa
4441 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc
4501 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg
4561 gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac
4621 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
4681 gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa
4741 gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa
4801 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga
4861 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga
4921 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
4981 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct
5041 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata
5101 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg
5161 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct
5221 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
5281 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg
5341 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc
5401 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa
5461 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct
5521 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
5581 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta
5641 cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt
5701 gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt
5761 gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa
5821 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
5881 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga
5941 ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat
6001 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg
6061 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc
6121 cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta
6181 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta
6241 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga
6301 gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa
6361 gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa
6421 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
6481 cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag
6541 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa
6601 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa
6661 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat
6721 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
6781 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct
6841 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca
6901 gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt
6961 ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa
7021 gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
7081 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta
7141 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta
7201 cgagacacgg atcgacctgt ctcagctggg aggcgacaag cgacctgccg ccacaaagaa
7261 ggctggacag gctaagaaga agaaagatta caaagacgat gacgataagg gatccggcgc
7321 aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccgaccga
7381 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
7441 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
7501 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
7561 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
7621 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
7681 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
7741 gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
7801 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
7861 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
7921 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta
7981 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
8041 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
8101 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
8161 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
8221 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
8281 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
8341 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
8401 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
8461 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
8521 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
8581 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
8641 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
8701 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
8761 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
8821 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
8881 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
8941 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
9001 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
9061 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
9121 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
9181 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
9241 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
9301 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
9361 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
9421 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
9481 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
9541 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
9601 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
9661 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
9721 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
9781 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
9841 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
9901 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
9961 ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg
10021 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga
10081 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
10141 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
10201 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
10261 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
10321 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
10381 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
10441 gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
10501 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
10561 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
10621 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
10681 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
10741 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
10801 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
10861 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
10921 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
10981 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
11041 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
11101 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
11161 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
11221 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
11281 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
11341 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
11401 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
11461 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
11521 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
11581 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
11641 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
11701 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
11761 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
11821 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
11881 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
11941 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
12001 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
12061 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
12121 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
12181 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
12241 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
12301 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
12361 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
12421 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
12481 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
12541 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
12601 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
12661 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
12721 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
12781 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
12841 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
12901 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
12961 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
13021 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
13081 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
13141 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
13201 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
13261 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
13321 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
13381 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
13441 tgcccagtac atgaccttat gggactttcc tacttggca
//
