| 质粒名称: | pLenti-PE2-BSD |
|---|---|
| 出品公司: | addgene |
| 目录编号: | 161514 |
| 存储实验室: | Hyongbum Kim |
|
载体骨架基本信息 |
|
| 载体名称: | lentiCas9-Blast |
| 载体出品公司: | Feng Zhang |
| 完整质粒大小: | 15008bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | Blasticidin |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | PE2-P2A-BSD/Prime editor 2-P2A-Blast/Cas9(H840A)-M-MLV reversetranscriptase (D200N, T306K, W313F, T330P, L603W)-P2A-BSD |
| 物种来源: | Synthetic |
| 插入大小: | 6816 bp |
| 标签/融合蛋白: |
SV40 NLS (N terminal on insert)
SV40 NLS (C terminal on insert)
|
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | EFS_forward (5'-GGTCGGCAATTGATCCGGTG-3') |
| 3’测序引物 | WPRE_reverse (5'-GGTTGCGTCAGCAAACACAG-3') |
|
文献引用方法 |
|
| 材料和方法部分: | pLenti-PE2-BSD was a gift from Hyongbum Kim (Addgene plasmid # 161514 ; http://n2t.net/addgene:161514 ; RRID:Addgene_161514) |
| 参考文献部分: | Predicting the efficiency of prime editing guide RNAs in human cells. Kim HK, Yu G, Park J, Min S, Lee S, Yoon S, Kim HH. Nat Biotechnol. 2020 Sep 21. pii: 10.1038/s41587-020-0677-y. doi: 10.1038/s41587-020-0677-y. 10.1038/s41587-020-0677-y PubMed 32958957 |
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 pLenti-PE2-BSD 15008 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expression of PE2 in mammalian cells. Can be used to generate stable
cell lines by Blasticidin selection.
ACCESSION .
VERSION .
KEYWORDS pLenti-PE2-BSD
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15008)
AUTHORS Kim HK, Yu G, Park J, Min S, Lee S, Yoon S, Kim HH
TITLE Predicting the efficiency of prime editing guide RNAs in human
cells.
JOURNAL Nat Biotechnol. 2020 Sep 21. pii: 10.1038/s41587-020-0677-y. doi:
10.1038/s41587-020-0677-y.
PUBMED 32958957
REFERENCE 2 (bases 1 to 15008)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..15008
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 382..580
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 535..555
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 598..778
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 825..950
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1443..1676
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1861..1905
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
misc_feature 2203..2320
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2413..2624
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 2680..2700
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2701..6801
/codon_start=1
/product="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
/label=Cas9(H840A)
/note="generates RNA-guided single strand nicks in DNA due
to the H840A mutation in the HNH catalytic domain"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 8974..8994
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 9004..9060
/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 9061..9459
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=BSD
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
misc_feature 9484..10072
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(9537..9557)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(9955..9966)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(10075..10091)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10076..10092)
/label=pBluescriptKS
/note="For pBluescript vector"
LTR 10597..10777
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(10803..10820)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 10809..11033
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 11079..11507
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(11166..11185)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 11376..11397
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(11516..11536)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 11681..11815
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 11742..11761
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 11877..11924
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 11943..12317
/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 12447..12568
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(12484..12503)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 12538..12557
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(12617..12633)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12617..12633)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12630..12652)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 12641..12657
/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(12665..12695)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 12710..12731
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12848..12865)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(13019..13607)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(13099..13118)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(13778..14638)
/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 14401..14420
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(14639..14743)
/gene="bla"
/label=AmpR promoter
primer_bind complement(14818..14837)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
2341 cagagatcca gtttggttaa ttagctagct aggtcttgaa aggagtggga attggctccg
2401 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
2461 tcggcaattg atccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
2521 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
2581 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggaccgg ttctagagcc
2641 accatgaaac ggacagccga cggaagcgag ttcgagtcac caaagaagaa gcggaaagtc
2701 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
2761 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
2821 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
2881 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
2941 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
3001 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
3061 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
3121 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
3181 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
3241 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
3301 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
3361 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
3421 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
3481 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
3541 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
3601 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
3661 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
3721 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
3781 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
3841 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
3901 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
3961 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
4021 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
4081 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
4141 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
4201 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
4261 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
4321 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
4381 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
4441 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
4501 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
4561 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
4621 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
4681 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
4741 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
4801 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
4861 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
4921 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
4981 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
5041 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
5101 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
5161 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggacgctatc
5221 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
5281 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
5341 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
5401 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
5461 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
5521 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
5581 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
5641 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
5701 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
5761 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
5821 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
5881 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
5941 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
6001 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
6061 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
6121 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
6181 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
6241 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
6301 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
6361 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
6421 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
6481 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
6541 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
6601 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
6661 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
6721 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
6781 ctgtctcagc tgggaggtga ctctggagga tctagcggag gatcctctgg cagcgagaca
6841 ccaggaacaa gcgagtcagc aacaccagag agcagtggcg gcagcagcgg cggcagcagc
6901 accctaaata tagaagatga gtatcggcta catgagacct caaaagagcc agatgtttct
6961 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga
7021 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc
7081 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga
7141 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc
7201 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag
7261 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca
7321 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc
7381 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca
7441 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttaat
7501 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta
7561 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact
7621 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa
7681 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg
7741 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta
7801 agggagttcc tagggaaggc aggcttctgt cgcctcttca tccctgggtt tgcagaaatg
7861 gcagcccccc tgtaccctct caccaaaccg gggactctgt ttaattgggg cccagaccaa
7921 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca
7981 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc
8041 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac
8101 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca
8161 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta
8221 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat
8281 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg
8341 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc
8401 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc
8461 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg
8521 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg
8581 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt
8641 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg
8701 cgtgggtggc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta
8761 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag
8821 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc
8881 atcacagaga ctccagacac ctctaccctc ctcatagaaa attcatcacc ctctggcggc
8941 tcaaaaagaa ccgccgacgg cagcgaattc gagcccaaga agaagaggaa agtcggatcc
9001 ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg
9061 atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc
9121 aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc
9181 cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg
9241 gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga
9301 aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg
9361 catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt
9421 cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag aattcgatat caagcttatc
9481 ggtaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
9541 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
9601 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
9661 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
9721 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
9781 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
9841 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
9901 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
9961 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
10021 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
10081 cgtcgacctc gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc
10141 aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca
10201 cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
10261 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat
10321 ctgtggatct accacacaca aggctacttc cctgattggc agaactacac accagggcca
10381 gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt tgagcaagag
10441 aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt gagcctgcat
10501 gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg cctagcattt
10561 catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt agaccagatc
10621 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
10681 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
10741 ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa cccgctgatc
10801 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
10861 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
10921 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
10981 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
11041 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
11101 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
11161 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
11221 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
11281 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
11341 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
11401 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
11461 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
11521 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
11581 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
11641 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
11701 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
11761 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
11821 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg
11881 acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa
11941 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
12001 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
12061 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
12121 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg
12181 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc
12241 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg
12301 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa
12361 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
12421 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
12481 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
12541 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
12601 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
12661 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
12721 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
12781 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
12841 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
12901 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
12961 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
13021 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
13081 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
13141 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
13201 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
13261 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
13321 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
13381 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
13441 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
13501 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
13561 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
13621 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
13681 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
13741 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
13801 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
13861 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
13921 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
13981 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
14041 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
14101 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
14161 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
14221 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
14281 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
14341 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
14401 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
14461 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
14521 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
14581 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
14641 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
14701 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
14761 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
14821 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
14881 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
14941 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
15001 tacgcgtt
//
