| 质粒名称: | pCW-Cas9-Blast |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 83481 |
| 存储实验室: | Mohan Babu |
|
载体骨架基本信息 |
|
| 载体名称: | pCW-Cas9 (#50661) |
| 载体出品公司: | Eric Lander, David Sabatini |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 11900 bp |
| 完整质粒大小: | 11687 bp |
| 载体修饰: | 将嘌呤霉素N-乙酰转移酶替换为稻瘟菌素S脱氨酶 |
| 载体类型: | 哺乳动物表达，慢病毒，CRISPR |
| 筛选标记: | 杀稻瘟菌素 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 30 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | SpCas9 |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | 在原质粒上替换嘌呤霉素N-乙酰转移酶 |
| GenBank编号 | -- |
| 启动子 | Tight Tre |
| 标签/融合蛋白: | -- |
| 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: | pCW-Cas9-Blast was a gift from Mohan Babu (Addgene plasmid # 83481 ; http://n2t.net/addgene:83481 ; RRID:Addgene_83481) |
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 pCW-Cas9-Blast 11684 bp ds-DNA circular SYN 16-JUN-2021
DEFINITION Lentiviral vector for mammalian expression of doxycycline-inducible
Cas9 and constitutive expression of Blasticidin S deaminaase -T2A
-rtTA.
ACCESSION .
VERSION .
KEYWORDS pCW-Cas9-Blast
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11684)
AUTHORS Habashy et al
TITLE CRISPR/Cas9-based system for loss-of-function screening
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 11684)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jun 16, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..11684
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 41..158
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 212..526
/label=tight TRE promoter
/note="Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter"
protein_bind 220..238
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 256..274
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 291..309
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 327..345
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 363..381
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 398..416
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 434..452
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 499..523
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 543..552
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 552..617
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 624..644
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 669..4769
/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
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 4770..4817
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
promoter 4827..5337
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
primer_bind 5256..5274
/label=hPGK-F
/note="Human PGK promoter, forward primer"
CDS 5347..5742
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=BSD
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
CDS 5743..5796
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 5797..6543
/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"
primer_bind complement(6583..6603)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(7001..7012)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 7217..7450
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 7522..7643
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(7559..7578)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 7613..7632
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 7683..7818
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 7745..7764
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 7818..7889
/gene="bla"
/label=AmpR promoter
CDS 7890..8750
/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 complement(8108..8127)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 8921..9509
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 9410..9429
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 9663..9680
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 9797..9818
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9833..9863
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9871..9887
/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 9876..9898
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 9895..9911
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 9895..9911
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 9930..9950
/label=T3
/note="T3 promoter, forward primer"
promoter 9932..9950
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 9978..10204
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 10205..10385
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 10432..10557
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 11050..11283
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 11468..11512
/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"
ORIGIN
1 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
181 gagatccact ttggccgcga atcgatatgt cgagtttact ccctatcagt gatagagaac
241 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag
301 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
361 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt
421 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg
481 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattggcta
541 gcgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca
601 aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
661 cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
721 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
781 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
841 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
901 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
961 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
1021 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
1081 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
1141 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
1201 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
1261 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
1321 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
1381 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
1441 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
1501 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
1561 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
1621 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
1681 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
1741 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
1801 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
1861 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
1921 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
1981 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
2041 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
2101 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
2161 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
2221 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
2281 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
2341 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
2401 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
2461 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
2521 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
2581 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
2641 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
2701 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
2761 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
2821 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
2881 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
2941 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
3001 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
3061 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
3121 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
3181 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
3241 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
3301 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
3361 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
3421 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
3481 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
3541 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
3601 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
3661 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
3721 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
3781 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
3841 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
3901 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
3961 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
4021 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
4081 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
4141 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
4201 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
4261 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
4321 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
4381 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
4441 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
4501 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
4561 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
4621 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
4681 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
4741 ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc
4801 aggcaaaaaa gaaaaagtaa ggatccgggg ttggggttgc gccttttcca aggcagccct
4861 gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac
4921 cctgggtctc gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc
4981 cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg
5041 ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac
5101 ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc
5161 tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt
5221 ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg
5281 gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagcaa
5341 ttcaccatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag agcaacggct
5401 acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc tctctctagc
5461 gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa
5521 ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg tatcgtcgcg
5581 atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca ggtgcttctc
5641 gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc gacggcagtt
5701 gggattcgtg aattgctgcc ctctggttat gtgtgggagg gcgaaggtag aggttctctc
5761 ctcacttgtg gtgatgttga agaaaaccct ggtccaatgt ctagactgga caagagcaaa
5821 gtcataaacg gagctctgga attactcaat ggtgtcggta tcgaaggcct gacgacaagg
5881 aaactcgctc aaaagctggg agttgagcag cctaccctgt actggcacgt gaagaacaag
5941 cgggccctgc tcgatgccct gccaatcgag atgctggaca ggcatcatac ccacttctgc
6001 cccctggaag gcgagtcatg gcaagacttt ctgcggaaca acgccaagtc ataccgctgt
6061 gctctcctct cacatcgcga cggggctaaa gtgcatctcg gcacccgccc aacagagaaa
6121 cagtacgaaa ccctggaaaa tcagctcgcg ttcctgtgtc agcaaggctt ctccctggag
6181 aacgcactgt acgctctgtc cgccgtgggc cactttacac tgggctgcgt attggaggaa
6241 caggagcatc aagtagcaaa agaggaaaga gagacaccta ccaccgattc tatgccccca
6301 cttctgagac aagcaattga gctgttcgac cggcagggag ccgaacctgc cttccttttc
6361 ggcctggaac taatcatatg tggcctggag aaacagctaa agtgcgaaag cggcgggccg
6421 accgacgccc ttgacgattt tgacttagac atgctcccag ccgatgccct tgacgacttt
6481 gaccttgata tgctgcctgc tgacgctctt gacgattttg accttgacat gctccccggg
6541 taactaagta aggatcgatc caagatatcg tattcttaac tatgttgctc cttttacgct
6601 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
6661 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
6721 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
6781 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
6841 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
6901 caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg cctgtgttgc
6961 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
7021 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
7081 tcagacgagt cggatctccc tttgggccgc ctccccgcct gtttcgcctc ggcgtccgga
7141 ctagaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
7201 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt
7261 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
7321 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
7381 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
7441 atctctagca gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
7501 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
7561 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
7621 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
7681 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
7741 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
7801 gaggcttttt tggaggcctt tcaaatatgt atccgctcat gagacaataa ccctgataaa
7861 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
7921 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
7981 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
8041 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
8101 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
8161 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
8221 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
8281 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
8341 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
8401 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
8461 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
8521 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
8581 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
8641 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
8701 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
8761 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
8821 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
8881 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
8941 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
9001 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
9061 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
9121 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
9181 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
9241 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
9301 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
9361 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
9421 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
9481 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
9541 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
9601 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
9661 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
9721 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
9781 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
9841 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
9901 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
9961 tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac
10021 gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga
10081 agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg
10141 acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat
10201 aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
10261 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
10321 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
10381 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
10441 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
10501 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
10561 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
10621 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
10681 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
10741 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
10801 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
10861 gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg
10921 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
10981 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
11041 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
11101 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
11161 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
11221 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
11281 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
11341 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
11401 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
11461 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
11521 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
11581 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
11641 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggac
//
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