| 质粒名称: | PB_tre_dCas9_KRAB |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 126030 |
| 存储实验室: | Mauro Calabrese |
|
载体骨架基本信息 |
|
| 载体名称: | piggyBac cargo vector |
| 载体出品公司: | System Biosciences |
| 空载体大小: | 11669 bp |
| 载体类型: | 哺乳动物表达，CRISPR；piggyBac |
| 筛选标记: | 潮霉素 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 30 ℃ |
| 宿主菌: | DH5a |
|
插入基因信息 |
|
| 插入基因名称: | dCas9 fusion with KRAB,dCas9-KRAB |
| 基因又名: | dCas9-KRAB |
| 插入大小: | 4460 bp |
| 基因突变: | D10A, H840A (catalytically inactive Cas9) |
| 启动子: | TRE |
| 克隆方法: | Gibson克隆 |
| 5’测序引物 | GGGAAAAGGCCTCCACGG |
|
文献引用方法 |
|
| 材料和方法部分: | PB_tre_dCas9_KRAB was a gift from Mauro Calabrese (Addgene plasmid # 126030 ; http://n2t.net/addgene:126030 ; RRID:Addgene_126030) |
| 参考文献部分: | A piggyBac-based toolkit for inducible genome editing in mammalian cells. Schertzer MD, Thulson E, Braceros KCA, Lee DM, Hinkle ER, Murphy RM, Kim SO, Vitucci EM, Calabrese JM. RNA. 2019 May 17. pii: rna.068932.118. doi: 10.1261/rna.068932.118. 10.1261/rna.068932.118 PubMed 31101683 |
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 PB_tre_dCas9_KRAB 11669 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION PiggyBac cargo vector expressing doxycycline inducible dCas9-KRAB
fusion.
ACCESSION .
VERSION .
KEYWORDS PB_tre_dCas9_KRAB
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11669)
AUTHORS Schertzer MD, Thulson E, Braceros KCA, Lee DM, Hinkle ER, Murphy RM,
Kim SO, Vitucci EM, Calabrese JM
TITLE A piggyBac-based toolkit for inducible genome editing in mammalian
cells.
JOURNAL RNA. 2019 May 17. pii: rna.068932.118. doi: 10.1261/rna.068932.118.
PUBMED 31101683
REFERENCE 2 (bases 1 to 11669)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..11669
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..315
/label=tight TRE promoter
/note="Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter"
protein_bind 9..27
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 45..63
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 80..98
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 116..134
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 152..170
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 187..205
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 223..241
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 288..312
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 320..329
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 326..4429
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the H840A mutation in the HNH
catalytic domain"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 4442..4462
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4463..4552
/codon_start=1
/product="three tandem HA epitope tags"
/label=3xHA
/translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
CDS 4589..4783
/codon_start=1
/product="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
/label=KRAB
/translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
QLTKPDVILRLEKGEEPWLV"
polyA_signal 4800..5024
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 5152..5242
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
promoter 5290..5501
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
LTR 5514..5782
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
CDS 5819..6844
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
polyA_signal 6873..6994
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(6910..6929)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 6964..6983
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
repeat_region 7757..7791
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
primer_bind complement(8282..8298)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8282..8298)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8295..8317)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 8306..8322
/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(8330..8360)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 8375..8396
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(8513..8530)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(8684..9272)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(8764..8783)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(9443..10303)
/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 10066..10085
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(10304..10408)
/gene="bla"
/label=AmpR promoter
rep_origin complement(10434..10889)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(10571..10592)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 10783..10802
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 11016..11038
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 11030..11047
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 11031..11047
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
repeat_region 11177..11239
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
ORIGIN
1 gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag
61 agaacgatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct
121 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg
181 agtttatccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag
241 aacgtatgtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg
301 aaccgtcaga tcgcctggag ccaccatgga caagaagtac tccattgggc tcgctatcgg
361 taccaacagc gtcggctggg ccgtcattac ggacgagtac aaggtgccga gcaaaaaatt
421 caaagttctg ggcaataccg atcgccacag cataaagaag aacctcattg gagccctcct
481 gttcgactcc ggggagacgg ccgaagccac gcggctcaaa agaacagcac ggcgcagata
541 tacccgcaga aagaatcgga tctgctacct gcaggagatc tttagtaatg agatggctaa
601 ggtggatgac tctttcttcc ataggctgga ggagtccttt ttggtggagg aggataaaaa
661 gcacgagcgc cacccaatct ttggcaatat cgtggacgag gtggcgtacc atgaaaagta
721 cccaaccata tatcatctga ggaagaagct ggtagacagt actgataagg ctgacttgcg
781 gttgatctat ctcgcgctgg cgcacatgat caaatttcgg ggacacttcc tcatcgaggg
841 ggacctgaac ccagacaaca gcgatgtcga caaactcttt atccaactgg ttcagactta
901 caatcagctt ttcgaggaga acccgatcaa cgcatccggc gttgacgcca aagcaatcct
961 gagcgctagg ctgtccaaat cccggcggct cgaaaacctc atcgcacagc tccctgggga
1021 gaagaagaac ggcctgtttg gtaatcttat cgccctgtca ctcgggctga cccccaactt
1081 taaatctaac ttcgacctgg ccgaagatgc caagctgcaa ctgagcaaag acacctacga
1141 tgatgatctc gacaatctgc tggcccagat cggcgaccag tacgcagacc tttttttggc
1201 ggcaaagaac ctgtcagacg ccattctgct gagtgatatt ctgcgagtga acacggagat
1261 caccaaagct ccgctgagcg ctagtatgat caagcgctat gatgagcacc accaagactt
1321 gactttgctg aaggcccttg tcagacagca actgcctgag aagtacaagg aaattttctt
1381 cgatcagtct aaaaatggct acgccggata cattgacggc ggagcaagcc aggaggaatt
1441 ttacaaattt attaagccca tcttggaaaa aatggacggc accgaggagc tgctggtaaa
1501 gctgaacaga gaagatctgt tgcgcaaaca gcgcactttc gacaatggaa gcatccccca
1561 ccagattcac ctgggcgaac tgcacgctat cctcaggcgg caagaggatt tctacccctt
1621 tttgaaagat aacagggaaa agattgagaa aatcctcaca tttcggatac cctactatgt
1681 aggccccctc gctcggggaa attccagatt cgcgtggatg actcgcaaat cagaagagac
1741 catcactccc tggaacttcg aggaagtcgt ggataagggg gcctctgccc agtccttcat
1801 cgaaaggatg actaactttg ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc
1861 tctgctgtac gagtacttca cagtttataa cgagctcacc aaggtcaaat acgtcacaga
1921 agggatgaga aagccagcat tcctgtctgg agagcagaag aaagctatcg tggacctcct
1981 cttcaagacg aaccggaaag ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat
2041 tgaatgtttc gactctgttg aaatcagcgg agtggaggat cgcttcaacg catccctggg
2101 aacgtatcac gatctcctga aaatcattaa agacaaggac ttcctggaca atgaggagaa
2161 cgaggacatt cttgaggaca ttgtcctcac ccttacgttg tttgaagata gggagatgat
2221 tgaagaacgc ttgaaaactt acgctcatct cttcgacgac aaagtcatga aacagctcaa
2281 gagacgccga tatacaggat gggggcggct gtcaagaaaa ctgatcaatg gcatccgaga
2341 caagcagagt ggaaagacaa tcctggattt tcttaagtcc gatggatttg ccaaccggaa
2401 cttcatgcag ttgatccatg atgactctct cacctttaag gaggacatcc agaaagcaca
2461 agtttctggc cagggggaca gtcttcacga gcacatcgct aatcttgcag gtagcccagc
2521 tatcaaaaag ggaatactgc agaccgttaa ggtcgtggat gaactcgtca aagtaatggg
2581 aaggcataag cccgagaata tcgttatcga gatggcccga gagaaccaaa ctacccagaa
2641 gggacagaag aacagtaggg aaaggatgaa gaggattgaa gagggtataa aagaactggg
2701 gtcccaaatc cttaaggaac acccagttga aaacacccag cttcagaatg agaagctcta
2761 cctgtactac ctgcagaacg gcagggacat gtacgtggat caggaactgg acatcaaccg
2821 gttgtccgac tacgacgtgg atgctatcgt gccccaaagc tttctcaaag atgattctat
2881 tgataataaa gtgttgacaa gatccgataa aaatagaggg aagagtgata acgtcccctc
2941 agaagaagtt gtcaagaaaa tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat
3001 cacacaacgg aagttcgata atctgactaa ggctgaacga ggtggcctgt ctgagttgga
3061 taaagccggc ttcatcaaaa ggcagcttgt tgagacacgc cagatcacca agcacgtggc
3121 ccaaattctc gattcacgca tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga
3181 ggtgaaagtt attactctga agtctaagct ggtctcagat ttcagaaagg actttcagtt
3241 ttataaggtg agagagatca acaattacca ccatgcgcat gatgcctacc tgaatgcagt
3301 ggtaggcact gcacttatca aaaaatatcc caagctggaa tctgaatttg tttacggaga
3361 ctataaagtg tacgatgtta ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc
3421 caccgctaag tacttctttt acagcaatat tatgaatttt ttcaagaccg agattacact
3481 ggccaatgga gagattcgga agcgaccact tatcgaaaca aacggagaaa caggagaaat
3541 cgtgtgggac aagggtaggg atttcgcgac agtccgcaag gtcctgtcca tgccgcaggt
3601 gaacatcgtt aaaaagaccg aagtacagac cggaggcttc tccaaggaaa gtatcctccc
3661 gaaaaggaac agcgacaagc tgatcgcacg caaaaaagat tgggacccca agaaatacgg
3721 cggattcgat tctcctacag tcgcttacag tgtactggtt gtggccaaag tggagaaagg
3781 gaagtctaaa aaactcaaaa gcgtcaagga actgctgggc atcacaatca tggagcgatc
3841 cagcttcgag aaaaacccca tcgactttct cgaagcgaaa ggatataaag aggtcaaaaa
3901 agacctcatc attaagctgc ccaagtactc tctctttgag cttgaaaacg gccggaaacg
3961 aatgctcgct agtgcgggcg agctgcagaa aggtaacgag ctggcactgc cctctaaata
4021 cgttaatttc ttgtatctgg ccagccacta tgaaaagctc aaagggtctc ccgaagataa
4081 tgagcagaag cagctgttcg tggaacaaca caaacactac cttgatgaga tcatcgagca
4141 aataagcgag ttctccaaaa gagtgatcct cgccgacgct aacctcgata aggtgctttc
4201 tgcttacaat aagcacaggg ataagcccat cagggagcag gcagaaaaca ttatccactt
4261 gtttactctg accaacttgg gcgcgcctgc agccttcaag tacttcgaca ccaccataga
4321 cagaaagcgg tacacctcta caaaggaggt cctggacgcc acactgattc atcagtcaat
4381 tacggggctc tatgaaacaa gaatcgacct ctctcagctc ggtggagacg gcaccggcgg
4441 gcccaagaag aagaggaagg tatacccata cgatgttcct gactatgcgg gctatcccta
4501 tgacgtcccg gactatgcag gatcgtatcc ttatgacgtt ccagattacg ctggatccat
4561 ggacgcgaaa tcacttacgg catggtcgag aacactggtt acgttcaagg acgtgtttgt
4621 ggactttaca cgtgaggagt ggaaattgct ggatactgcg caacaaattg tgtatcgaaa
4681 tgtcatgctt gagaattaca agaacctcgt cagtctcgga taccagttga cgaaaccgga
4741 tgtgatcctt aggctcgaaa agggggaaga accttggctg gtataacaat gcgtttcgac
4801 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
4861 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
4921 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
4981 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggaaacac gcggcgacgc
5041 gttcgaccga ataaaacctg tgacggaaga tcacttcgca gaataaataa atcctggtgt
5101 ccctgttgat accgggaagc cctgggccaa cttttggcga aaatgagacg ttgatcggca
5161 cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg tattttttga
5221 gttgtcgaga ttttcaggag ctaaagatct ggaagctaag gatctgcgat cgctccggtg
5281 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
5341 gcaattgaac gggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
5401 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
5461 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca gctgaagctt cgaggggctc
5521 gcatctctcc ttcacgcgcc cgccgcccta cctgaggccg ccatccacgc cggttgagtc
5581 gcgttctgcc gcctcccgcc tgtggtgcct cctgaactgc gtccgccgtc taggtaagtt
5641 taaagctcag gtcgagaccg ggcctttgtc cggcgctccc ttggagccta cctagactca
5701 gccggctctc cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt
5761 ctgttctgcg ccgttacaga tccaagctgt gaccggcgcc tacaagctgc tagccaccat
5821 gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
5881 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
5941 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
6001 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
6061 ggagttcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
6121 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
6181 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
6241 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
6301 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
6361 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
6421 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
6481 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
6541 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
6601 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
6661 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
6721 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
6781 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
6841 atagttttac gtattgagta tttaactaac taaacttgtt tattgcagct tataatggtt
6901 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
6961 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggaattga ctcaaatgat
7021 gtcaattagt ctatcagaag ctcatctggt ctcccttccg ggggacaaga catccctgtt
7081 taatatttaa acagcagtgt tcccaaactg ggttcttata tcccttgctc tggtcaacca
7141 ggttgcaggg tttcctgtcc tcacaggaac gaagtcccta aagaaacagt ggcagccagg
7201 tttagccccg gaattgactg gattcctttt ttagggccca ttggtatggc tttttccccg
7261 tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc
7321 ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg
7381 ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg
7441 acgtaattac atccctgggg gctttggggg ggggctgtcc ctgatatcta taacaagaaa
7501 atatatatat aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat
7561 gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg
7621 ttatagttca aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa
7681 gcggcgactg agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag
7741 caatatttca agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaatctag
7801 ctgcatcagg atcatatcgt cgggtctttt ttccggctca gtcatcgccc aagctggcgc
7861 tatctgggca tcggggagga agaagcccgt gccttttccc gcgaggttga agcggcatgg
7921 aaagagtttg ccgaggatga ctgctgctgc attgacgttg agcgaaaacg cacgtttacc
7981 atgatgattc gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt cgttcaggcc
8041 acctgggata ccagttcgtc gcggcttttc cggacacagt tccggatggt cagcccgaag
8101 cgcatcagca acccgaacaa taccggcgac agccggaact gccgtgccgg tgtgcagatt
8161 aatgacagcg gtgcggcgct gggatattac gtcagcgagg acgggtatcc tggctggatg
8221 ccgcagaaat ggacatggat accccgtgag ttacccggcg ggcgcgcttg gcgtaatcat
8281 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
8341 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
8401 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
8461 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
8521 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
8581 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
8641 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
8701 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
8761 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
8821 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
8881 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
8941 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
9001 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
9061 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
9121 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
9181 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
9241 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
9301 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
9361 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
9421 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
9481 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
9541 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
9601 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
9661 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
9721 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
9781 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
9841 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
9901 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
9961 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
10021 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
10081 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
10141 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
10201 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
10261 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
10321 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
10381 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
10441 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
10501 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
10561 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
10621 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
10681 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
10741 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
10801 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
10861 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
10921 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
10981 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
11041 ggccagtgag cgcgcctcgt tcattcacgt ttttgaaccc gtggaggacg ggcagactcg
11101 cggtgcaaat gtgttttaca gcgtgatgga gcagatgaag atgctcgaca cgctgcagaa
11161 cacgcagcta gattaaccct agaaagataa tcatattgtg acgtacgtta aagataatca
11221 tgtgtaaaat tgacgcatgt gttttatcgg tctgtatatc gaggtttatt tattaatttg
11281 aatagatatt aagttttatt atatttacac ttacatacta ataataaatt caacaaacaa
11341 tttatttatg tttatttatt tattaaaaaa aacaaaaact caaaatttct tctataaagt
11401 aacaaaactt ttatgaggga cagccccccc ccaaagcccc cagggatgta attacgtccc
11461 tcccccgcta gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc
11521 cgcatccccg agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat
11581 cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg
11641 ggaaaaggcc tccacggcca gactagttc
//
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