| 质粒名称: | TLCV2 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 87360 |
| 存储实验室: | Adam Karpf |
|
载体骨架基本信息 |
|
| 载体名称: | lentiCRISPR v2 |
| 载体出品公司: | Feng Zhang lab |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 16733 bp |
| 载体修饰: | 将LentiCRISPR v2修改为多合一诱导系统，强力霉素诱导Cas9-P2A-GFP表达。sgRNA和嘌呤霉素的表达由组成型启动子驱动。对lentiCRISPR v2进行了以下修改：移除EF1启动子并替换为紧密TRE启动子；去除P2A-嘌呤霉素，并用T2A-eGFP代替；并添加EF1启动子-嘌呤霉素-P2A-rtTA盒。 |
| 载体类型: | 哺乳动物表达，慢病毒，CRISPR；强力霉素诱导；eGFP报告程序 |
| 筛选标记: | 嘌呤霉素 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Cas9-2A-eGFP |
| 启动子 | Tight TRE promoter |
| 5' 测序引物 | LNCX [5'-AGCTCGTTTAGTGAACCGTCAGATC-3'] |
| 3' 测序引物 | EF1a-R [5'CACGGCGACTACTGCACTTA-3'] |
|
文献引用方法 |
|
| 材料和方法部分: | TLCV2 was a gift from Adam Karpf (Addgene plasmid # 87360 ; http://n2t.net/addgene:87360 ; RRID:Addgene_87360) |
| 参考文献部分: | Pan-Cancer Analyses Reveal Genomic Features of FOXM1 Overexpression in Cancer. Barger CJ, Branick C, Chee L, Karpf AR. Cancers (Basel). 2019 Feb 21;11(2). pii: cancers11020251. doi: 10.3390/cancers11020251. 10.3390/cancers11020251 PubMed 30795624 |
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 TLCV2 16733 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION LentiCRISPR v2 was modified into an all-in-one dox inducible system.
The addition of doxycycline induces Cas9-2A-eGFP. The U6 promoter
drives constitutive sgRNA expression..
ACCESSION .
VERSION .
KEYWORDS TLCV2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 16733)
TITLE Inducible LentiCRISPR v2
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 16733)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..16733
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 14..32
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 49..67
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 85..103
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 121..139
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 156..174
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 192..210
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 257..281
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 310..319
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 316..4419
/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 4420..4467
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 4468..4491
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 4501..4554
/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 4555..5271
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(4597..4618)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(4858..4877)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 5205..5226
/label=EGFP-C
/note="EGFP, forward primer"
promoter 5282..5493
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 5515..6111
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(5515..5534)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 6011..6031
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
CDS 6121..6177
/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 6178..6924
/codon_start=1
/product="improved tetracycline-controlled transactivator"
/label=rtTA-Advanced
/note="In the Tet-On(R) system, rtTA-Advanced binds to the
Tet-responsive element and stimulates transcription only in
the presence of tetracycline or doxycycline."
/translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
misc_feature 6945..7533
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(6998..7018)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(7416..7427)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 7605..7838
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(7864..7881)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 7870..8094
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 8140..8568
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(8227..8246)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 8437..8458
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(8577..8597)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 8582..8911
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 8762..8897
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 8824..8843
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 8959..9006
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 9025..9399
/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 9529..9650
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(9566..9585)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 9620..9639
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(9699..9715)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9699..9715)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(9712..9734)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 9723..9739
/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(9747..9777)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9792..9813
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(9930..9947)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(10101..10689)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(10181..10200)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(10860..11720)
/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 11483..11502
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(11721..11825)
/gene="bla"
/label=AmpR promoter
primer_bind complement(11900..11919)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 12091..12470
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 12472..12670
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 12625..12645
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 12688..12868
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 12915..13040
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 13533..13766
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 14293..14410
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 14461..14701
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 14461..14481
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 14632..14651
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 16591..16666
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 16703..284
/label=tight TRE promoter
/note="Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter"
protein_bind 16711..16729
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
1 atgtcgagtt tactccctat cagtgataga gaacgatgtc gagtttactc cctatcagtg
61 atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac
121 tccctatcag tgatagagaa cgtatgtcga gtttatccct atcagtgata gagaacgtat
181 gtcgagttta ctccctatca gtgatagaga acgtatgtcg aggtaggcgt gtacggtggg
241 aggcctatat aagcagagct cgtttagtga accgtcagat cgcctggaga ataccggttc
301 tagagcgctg ccaccatgga caagaagtac agcatcggcc tggacatcgg caccaactct
361 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
421 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc
481 ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg
541 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
601 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
661 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
721 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
781 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
841 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
901 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
961 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
1021 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
1081 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
1141 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
1201 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
1261 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
1321 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
1381 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc
1441 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
1501 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
1561 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
1621 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
1681 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
1741 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg
1801 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
1861 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
1921 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc
1981 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
2041 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
2101 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
2161 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
2221 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
2281 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
2341 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
2401 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
2461 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
2521 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
2581 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
2641 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
2701 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
2761 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
2821 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
2881 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
2941 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
3001 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
3061 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
3121 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
3181 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
3241 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
3301 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
3361 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
3421 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
3481 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
3541 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
3601 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
3661 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac
3721 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
3781 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
3841 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
3901 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
3961 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
4021 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
4081 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
4141 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
4201 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
4261 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg
4321 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
4381 tacgagacac ggatcgacct gtctcagctg ggaggcgaca agcgacctgc cgccacaaag
4441 aaggctggac aggctaagaa gaagaaagat tacaaagacg atgacgataa gggatccgga
4501 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccagtgagc
4561 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
4621 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
4681 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
4741 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
4801 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
4861 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
4921 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
4981 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
5041 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
5101 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
5161 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
5221 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta agctagcatc
5281 cgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
5341 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
5401 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
5461 ctttttcgca acgggtttgc cgccagaaca cagactgcga tcgcaatgta cagtatgacc
5521 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
5581 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
5641 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
5701 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
5761 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
5821 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
5881 tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc
5941 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
6001 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
6061 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc cggttccggc
6121 gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccgatg
6181 tctagactgg acaagagcaa agtcataaac ggagctctgg aattactcaa tggtgtcggt
6241 atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg
6301 tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tgccaatcga gatgctggac
6361 aggcatcata cccacttctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac
6421 aacgccaagt cataccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc
6481 ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt
6541 cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca
6601 ctgggctgcg tattggagga acaggagcat caagtagcaa aagaggaaag agagacacct
6661 accaccgatt ctatgccccc acttctgaga caagcaattg agctgttcga ccggcaggga
6721 gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta
6781 aagtgcgaaa gcggcgggcc gaccgacgcc cttgacgatt ttgacttaga catgctccca
6841 gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt
6901 gaccttgaca tgctccccgg gtaactcgag cgcgttaagt cgacaatcaa cctctggatt
6961 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
7021 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
7081 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
7141 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
7201 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
7261 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
7321 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
7381 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
7441 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
7501 cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa
7561 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
7621 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
7681 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
7741 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
7801 cctcagaccc ttttagtcag tgtggaaaat ctctagcagg gcccgtttaa acccgctgat
7861 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
7921 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
7981 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
8041 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
8101 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
8161 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
8221 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
8281 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
8341 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
8401 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
8461 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
8521 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
8581 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
8641 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
8701 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
8761 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
8821 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
8881 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
8941 cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
9001 tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc ggtgctcacc
9061 gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac
9121 ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg
9181 gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac
9241 gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg
9301 gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc
9361 ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat
9421 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
9481 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
9541 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
9601 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
9661 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
9721 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
9781 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
9841 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
9901 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
9961 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
10021 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
10081 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
10141 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
10201 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
10261 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
10321 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
10381 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
10441 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
10501 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
10561 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
10621 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
10681 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
10741 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
10801 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
10861 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
10921 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
10981 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
11041 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
11101 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
11161 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
11221 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
11281 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
11341 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
11401 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
11461 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
11521 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
11581 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
11641 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
11701 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
11761 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
11821 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
11881 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
11941 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
12001 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
12061 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
12121 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
12181 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
12241 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
12301 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
12361 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
12421 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
12481 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
12541 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
12601 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
12661 atataagcag cgcgttttgc ctgtactggg tctctctggt tagaccagat ctgagcctgg
12721 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
12781 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
12841 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag
12901 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag
12961 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga
13021 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa
13081 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca
13141 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac
13201 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat
13261 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg
13321 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg
13381 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
13441 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
13501 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
13561 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
13621 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
13681 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
13741 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
13801 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
13861 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
13921 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
13981 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
14041 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
14101 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
14161 aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga
14221 tccattcgat tagtgaacgg atcggcactg cgtgcgccaa ttctgcagac aaatggcagt
14281 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
14341 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
14401 tcaaaatttt cgggtttatt acagggacag cagagatcca gtttggttaa ttaaggtacc
14461 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
14521 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
14581 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
14641 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
14701 cgaaacaccg gagacggttg taaatgagca cacaaaatac acatgctaaa atattatatt
14761 ctatgacctt tataaaatca accaaaatct tctttttaat aactttagta tcaataatta
14821 gaatttttat gttccttttt gcaaactttt aataaaaatg agcaaaataa aaaaacgcta
14881 gttttagtaa ctcgcgttgt tttcttcacc tttaataata gctactccac cacttgttcc
14941 taagcggtca gctcctgctt caatcatttt ttgagcatct tcaaatgttc taactccacc
15001 agctgcttta actaaagcat tgtctttaac aactgacttc attagtttaa catcttcaaa
15061 tgttgcacct gattttgaaa atcctgttga tgttttaaca aattctaatc cagcttcaac
15121 agctatttca caagctttca tgatttcttc ttttgttaat aaacaatttt ccataataca
15181 tttaacaaca tgtgatccag ctgctttttt tacagctttc atgtcttcta aaactaattc
15241 ataatttttg tcttttaatg caccaatatt taataccata tcaatttctg ttgcaccatc
15301 tttaattgct tcagaaactt cgaatgcttt tgtagctgtt gtgcatgcac ctagaggaaa
15361 acctacaaca tttgttattc ctacatttgt gccttttaat aattctttac aatagcttgt
15421 tcaatatgaa ttaacacaaa ctgttgcaaa atcaaattca attgcttcat cacataattg
15481 tttaatttca gctttcgtag catcttgttt taataatgtg tgatctatat atttgtttag
15541 tttcattttt tctcctatat attcattttt aattttaatt ctttaataat ttcgtctact
15601 ttaactttag cgttttgaac agattcacca acacctataa aataaatttt tagtttaggt
15661 tcagttccac ttgggcgaac agcaaatcat gacttatctt ctaaataaaa ttttagtaag
15721 tcttgtcctg gcatattata cattccatcg atgtagtctt caacattaac aactttaagt
15781 ccagcaattt gagttaaggg tgttgctctc aatgatttca ttaatggttc aatttttaat
15841 ttcttttctt ctggtttaaa attcaagttt aaagtgaaag tgtaatatgc acccatttct
15901 ttaaataaat cttctaaata gtctactaat gttttatttt gttttttata aaatcaagca
15961 gcctctgcta ttaatataga agcttgtatt ccatctttat ctctagctga gtcatcaatt
16021 acatatccat aactttcttc ataagcaaaa acaaaattta atccgttatc ttcttcttta
16081 gcaatttctc tacccattca tttaaatcca gttaaagttt ttacaatatt aactccatat
16141 ttttcatgag cgattctatc acccaaatca cttgttacaa aacttgaata tagagccgga
16201 ttttttggaa tgctatttaa gcgttttaga tttgataatt ttcaatcaat taaaattggt
16261 cctgtttgat ttccatctaa tcttacaaaa tgaccatcat gttttattgc cattccaaat
16321 ctgtcagcat ctgggtcatt cataataata atatctgcat catgtttaat accatattca
16381 agcggtattt ttcatgcagg atcaaattct ggatttggat ttacaacatt tttaaatgtt
16441 tcatcttcaa atgcatgctc ttcaacctca ataacgttat atcctgattc acgtaatatt
16501 tttggggtaa atttagttcc tgttccatta actgcgctaa aaataatttt taaatctttt
16561 ttagcttctt gctctttttt gtacgtctct gttttagagc tagaaatagc aagttaaaat
16621 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgaattcac
16681 tttggccgcg aatcgatatg tcgagtttac tccctatcag tgatagagaa cgt
//
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