| 质粒名称: | pU6-sgRosa26-1_CBh-Cas9-T2A-BFP |
|---|---|
| 出品公司: | addgene |
| 目录编号: | 64216 |
| 存储实验室: | Ralf Kuehn |
|
载体骨架基本信息 |
|
| 载体名称: | pU6-(BbsI)_CBh-Cas9-T2A-BFP |
| 载体出品公司: | Kuhn lab (Addgene plasmid# 64323) |
| 空载体大小: | 4038 bp |
| 完整质粒大小: | 4989 bp |
| 载体修饰: | sgRNA targeting mouse Rosa26 locus was cloned into the BbsI sites |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称1: | Cas9/3xFLAG-NLS-Cas9-NLS-T2A-BFP |
| 物种来源: | Streptococcus pyogenes |
| 标签/融合蛋白: |
3xFLAG (N terminal on insert)
NLS (N terminal on insert)
NLS (C terminal on insert)
T2A-BFP (C terminal on insert)
|
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | AgeI(未破坏) |
| 3’克隆位点: | NotI (未破坏) |
| 5’测序引物 | pCBhPro-F (5'-agggatggttggttggtggg-3') for Cas9 |
| 3’测序引物 | BGH-rev |
| 插入基因名称2: | sgRNA targeting ROSA26-1/5'-GACTCCAGTCTTTCTAGAAGA-3' |
| 启动子: | U6 |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BbsI (destroyed during cloning) |
| 3’克隆位点: | BbsI (destroyed during cloning) |
| 5’测序引物 | hU6-F (5'-GAGGGCCTATTTCCCATGATT-3') |
|
文献引用方法 |
|
| 材料和方法部分: | pU6-sgRosa26-1_CBh-Cas9-T2A-BFP was a gift from Ralf Kuehn (Addgene plasmid # 64216 ; http://n2t.net/addgene:64216 ; RRID:Addgene_64216) |
| 参考文献部分: | Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R. Nat Biotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198. 10.1038/nbt.3198 PubMed 25803306 |
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 pU6-sgRosa26-1_CBh-Cas9-T2A-BFP 9288 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expression vector for a sgRNA against the mouse Rosa26 locus and
Cas9 linked to BFP via a T2A peptide.
ACCESSION .
VERSION .
KEYWORDS pU6-sgRosa26-1_CBh-Cas9-T2A-BFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9288)
AUTHORS Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R
TITLE Increasing the efficiency of homology-directed repair for
CRISPR-Cas9-induced precise gene editing in mammalian cells.
JOURNAL Nat Biotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198.
PUBMED 25803306
REFERENCE 2 (bases 1 to 9288)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..9288
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 6..246
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 6..26
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 177..196
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 276..351
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 448..733
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 735..1012
/label=chicken beta-actin promoter
intron 1013..1240
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 1252..1261
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1261..1326
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1333..1353
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1378..5478
/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 5479..5526
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 5530..5583
/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 5590..6309
/codon_start=1
/product="enhanced blue variant of GFP (Ai et al., 2007)"
/label=EBFP2
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
EFRTAAGITLGMDELYK"
primer_bind complement(5635..5656)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(6334..6351)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 6340..6547
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 6556..6696
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 6556..6685
/label=AAV2 ITR
rep_origin 6771..7226
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(6858..6877)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 7068..7089
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(7243..7262)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 7362..7384
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(7422..7440)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 7508..7612
/gene="bla"
/label=AmpR promoter
CDS 7613..8473
/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(7831..7850)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 8644..9232
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 9133..9152
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
61 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
121 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
181 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
241 aaggacgaaa caccgactcc agtctttcta gaagagtttt agagctagaa atagcaagtt
301 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt
361 ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt
421 ctgcagacaa atggctctag aggtacccgt tacataactt acggtaaatg gcccgcctgg
481 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca
541 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
601 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg
661 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
721 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
781 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
841 gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc
901 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
961 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
1021 tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
1081 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
1141 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt
1201 taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg tgccaccatg
1261 gactataagg accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac
1321 gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccgac
1381 aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc
1441 gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc
1501 atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
1561 cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg
1621 caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa
1681 gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc
1741 gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg
1801 gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc
1861 aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac
1921 aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
1981 gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg
2041 gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt
2101 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc
2161 aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc
2221 ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg
2281 agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc
2341 aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag
2401 ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac
2461 attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag
2521 atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag
2581 cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt
2641 ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag
2701 atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc
2761 gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg
2821 gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg
2881 cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac
2941 gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
3001 gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag
3061 cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc
3121 gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag
3181 gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc
3241 ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg
3301 ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg
3361 agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc
3421 ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg
3481 acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag
3541 cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag
3601 gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa
3661 atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag
3721 cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa
3781 aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg
3841 tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg
3901 cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag
3961 aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac
4021 tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag
4081 gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg
4141 gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag
4201 tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg
4261 gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac
4321 cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct
4381 aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
4441 gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc
4501 atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg
4561 atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc
4621 gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca
4681 ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga
4741 aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct
4801 gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
4861 ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg
4921 gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc
4981 ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag
5041 ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat
5101 gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac
5161 aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg
5221 gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc
5281 agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc
5341 gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg
5401 ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg
5461 tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag
5521 aaaaagcttg agggcagagg aagtctgcta acatgcggtg acgtggagga gaatcccggc
5581 cctgctagca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
5641 gagctggacg gcgacgtaaa cggccacaag ttcagcgtga ggggcgaggg cgagggcgat
5701 gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
5761 tggcccaccc tcgtgaccac cctgagccac ggcgtgcagt gcttcgcccg ctaccccgac
5821 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
5881 accatcttct tcaaggacga cggcacctac aagacccgcg ccgaggtgaa gttcgagggc
5941 gacaccctgg tgaaccgcat cgagctgaag ggcgtcgact tcaaggagga cggcaacatc
6001 ctggggcaca agctggagta caacttcaac agccacaaca tctatatcat ggccgtcaag
6061 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acgtggagga cggcagcgtg
6121 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
6181 gacagccact acctgagcac ccagtccgtg ctgagcaaag accccaacga gaagcgcgat
6241 cacatggtcc tgctggagtt ccgcaccgcc gccgggatca ctctcggcat ggacgagctg
6301 tacaagtgag aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
6361 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
6421 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
6481 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg
6541 ctggggagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
6601 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
6661 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
6721 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
6781 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
6841 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
6901 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
6961 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
7021 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
7081 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
7141 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
7201 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
7261 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
7321 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
7381 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
7441 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
7501 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
7561 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
7621 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
7681 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
7741 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
7801 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
7861 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
7921 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
7981 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
8041 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
8101 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
8161 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
8221 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
8281 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat
8341 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
8401 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
8461 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
8521 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
8581 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
8641 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
8701 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
8761 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
8821 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
8881 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
8941 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
9001 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
9061 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
9121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
9181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
9241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctca
//
