| 质粒名称: | pFREE-RK2 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 92054 |
| 存储实验室: | Morten Norholm |
|
载体骨架基本信息 |
|
| 载体名称: | pMAZ-SK |
| 载体出品公司: | -- |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | 9524 bp |
| 载体修饰: | -- |
| 载体类型: | CRISPR |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 低拷贝 |
| 生长条件: | 30 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Cas9 |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | ptet |
| 标签/融合蛋白: | -- |
| 克隆方法: | Ligation Independent克隆 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | AAAGTCTGGTTATAACC |
| 3' 测序引物 | TACCGGTTTATTGACTACC |
|
文献引用方法 |
|
| 材料和方法部分: | pFREE-RK2 was a gift from Morten Norholm (Addgene plasmid # 92054 ; http://n2t.net/addgene:92054 ; RRID:Addgene_92054) |
| 参考文献部分: | A versatile one-step CRISPR-Cas9 based approach to plasmid-curing. Lauritsen I, Porse A, Sommer MOA, Norholm MHH. Microb Cell Fact. 2017 Aug 2;16(1):135. doi: 10.1186/s12934-017-0748-z. 10.1186/s12934-017-0748-z [pii] PubMed 28764701 |
买家导航
pFREERK2载体基本信息
pFREERK2载体图谱质粒图谱和多克隆位点信息
pFREERK2载体简介
pFREERK2载体序列
LOCUS pFREE-RK2 11507 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Allows inducible expression of gRNAs and Cas9 for plasmid curing as
well as self-curing via a temperature sensitive RK2 replicon..
ACCESSION .
VERSION .
KEYWORDS pFREE-RK2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11507)
AUTHORS Lauritsen I, Porse A, Sommer MOA, Norholm MHH
TITLE A versatile one-step CRISPR-Cas9 based approach to plasmid-curing.
JOURNAL Microb Cell Fact. 2017 Aug 2;16(1):135. doi:
10.1186/s12934-017-0748-z.
PUBMED 28764701
REFERENCE 2 (bases 1 to 11507)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..11507
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 17..35
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 42..60
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
CDS 146..4252
/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"
primer_bind complement(4287..4305)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 4301..4348
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS 4482..5105
/codon_start=1
/gene="tetR from transposon Tn10"
/product="tetracycline repressor TetR"
/label=TetR
/note="TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved by
adding tetracycline or doxycycline."
/translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
primer_bind complement(4555..4574)
/label=Tet-R
/note="Tetracycline resistance gene, reverse primer"
CDS complement(5267..6082)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind 5990..6009
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
promoter complement(6083..6174)
/gene="bla"
/label=AmpR promoter
RBS 6257..6265
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
misc_feature 6280..6298
/label=Tn5 ME
/note="hyperactive mosaic end for Tn5 transposase
recognition (Reznikoff et al., 2004)"
RBS 6802..6810
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
misc_feature 6825..6843
/label=Tn5 ME
/note="hyperactive mosaic end for Tn5 transposase
recognition (Reznikoff et al., 2004)"
rep_origin 8217..8748
/label=oriV
/note="incP origin of replication"
CDS 9477..10622
/codon_start=1
/product="trans-acting replication protein that binds to
and activates oriV"
/label=trfA
/translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDLVHCKR"
misc_feature 10662..10680
/label=Tn5 ME
/note="hyperactive mosaic end for Tn5 transposase
recognition (Reznikoff et al., 2004)"
misc_RNA complement(10901..10979)
/label=tracrRNA
/note="trans-activating CRISPR RNA for the Streptococcus
pyogenes CRISPR/Cas9 system"
repeat_region 11151..11186
/label=DR
/note="direct repeat for the Streptococcus pyogenes
CRISPR/Cas system"
repeat_region 11217..11252
/label=DR
/note="direct repeat for the Streptococcus pyogenes
CRISPR/Cas system"
repeat_region 11283..11318
/label=DR
/note="direct repeat for the Streptococcus pyogenes
CRISPR/Cas system"
repeat_region 11349..11384
/label=DR
/note="direct repeat for the Streptococcus pyogenes
CRISPR/Cas system"
repeat_region 11415..11450
/label=DR
/note="direct repeat for the Streptococcus pyogenes
CRISPR/Cas system"
ORIGIN
1 tcgagtctgg ttataatccc tatcagtgat agagattgac atccctatca gtgatagaga
61 tactgagcac agaaggagat atacatggat aagaaatact gagcactatc agtgatagag
121 atactgagca cagaaggaga tatacatgga taagaaatac tcaataggct tagatatcgg
181 cacaaatagc gtcggatggg cggtgatcac tgatgaatat aaggttccgt ctaaaaagtt
241 caaggttctg ggaaatacag accgccacag tatcaaaaaa aatcttatag gggctctttt
301 atttgacagt ggagagacag cggaagcgac tcgtctcaaa cggacagctc gtagaaggta
361 tacacgtcgg aagaatcgta tttgttatct acaggagatt ttttcaaatg agatggcgaa
421 agtagatgat agtttctttc atcgacttga agagtctttt ttggtggaag aagacaagaa
481 gcatgaacgt catcctattt ttggaaatat agtagatgaa gttgcttatc atgagaaata
541 tccaactatc tatcatctgc gaaaaaaatt ggtagattct actgataaag cggatttgcg
601 cttaatctat ttggccttag cgcatatgat taagtttcgt ggtcattttt tgattgaggg
661 agatttaaat cctgataata gtgatgtgga caaactattt atccagttgg tacaaaccta
721 caatcaatta tttgaagaaa accctattaa cgcaagtgga gtagatgcta aagcgattct
781 ttctgcacga ttgagtaaat caagacgatt agaaaatctc attgctcagc tccccggtga
841 gaagaaaaat ggcttatttg ggaatctcat tgctttgtca ttgggtttga cccctaattt
901 taaatcaaat tttgatttgg cagaagatgc taaattacag ctttcaaaag atacttacga
961 tgatgattta gataatttat tggcgcaaat tggagatcaa tatgctgatt tgtttttggc
1021 agctaagaat ttatcagatg ctattttact ttcagatatc ctaagagtaa atactgaaat
1081 aactaaggct cccctatcag cttcaatgat taaacgctac gatgaacatc atcaagactt
1141 gactctttta aaagctttag ttcgacaaca acttccagaa aagtataaag aaatcttttt
1201 tgatcaatca aaaaacggat atgcaggtta tattgatggg ggagctagcc aagaagaatt
1261 ttataaattt atcaaaccaa ttttagaaaa aatggatggt actgaggaat tattggtgaa
1321 actaaatcgt gaagatttgc tgcgcaagca acggaccttt gacaacggct ctattcccca
1381 tcaaattcac ttgggtgagc tgcatgctat tttgagaaga caagaagact tttatccatt
1441 tttaaaagac aatcgtgaga agattgaaaa aatcttgact tttcgaattc cttattatgt
1501 tggtccattg gcgcgtggca atagtcgttt tgcatggatg actcggaagt ctgaagaaac
1561 aattacccca tggaattttg aagaagttgt cgataaaggt gcttcagctc aatcatttat
1621 tgaacgcatg acaaactttg ataaaaatct tccaaatgaa aaagtactac caaaacatag
1681 tttgctttat gagtatttta cggtttataa cgaattgaca aaggtcaaat atgttactga
1741 aggaatgcga aaaccagcat ttctttcagg tgaacagaag aaagccattg ttgatttact
1801 cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa gaagattatt tcaaaaaaat
1861 agaatgtttt gatagtgttg aaatttcagg agttgaagat agatttaatg cttcattagg
1921 tacctaccat gatttgctaa aaattattaa agataaagat tttttggata atgaagaaaa
1981 tgaagatatc ttagaggata ttgttttaac attgacctta tttgaagata gggagatgat
2041 tgaggaaaga cttaaaacat atgctcacct ctttgatgat aaggtgatga aacagcttaa
2101 acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa ttgattaatg gtattaggga
2161 taagcaatct ggcaaaacaa tattagattt tttgaaatca gatggttttg ccaatcgcaa
2221 ttttatgcag ctgatccatg atgatagttt gacatttaaa gaagacattc aaaaagcaca
2281 agtgtctgga caaggcgata gtttacatga acatattgca aatttagctg gtagccctgc
2341 tattaaaaaa ggtattttac agactgtaaa agttgttgat gaattggtca aagtaatggg
2401 gcggcataag ccagaaaata tcgttattga aatggcacgt gaaaatcaga caactcaaaa
2461 gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca aagaattagg
2521 aagtcagatt cttaaagagc atcctgttga aaatactcaa ttgcaaaatg aaaagctcta
2581 tctctattat ctccaaaatg gaagagacat gtatgtggac caagaattag atattaatcg
2641 tttaagtgat tatgatgtcg atcacattgt tccacaaagt ttccttaaag acgattcaat
2701 agacaataag gtcttaacgc gttctgataa aaatcgtggt aaatcggata acgttccaag
2761 tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg ccaagttaat
2821 cactcaacgt aagtttgata atttaacgaa agctgaacgt ggaggtttga gtgaacttga
2881 taaagctggt tttatcaaac gccaattggt tgaaactcgc caaatcacta agcatgtggc
2941 acaaattttg gatagtcgca tgaatactaa atacgatgaa aatgataaac ttattcgaga
3001 ggttaaagtg attaccttaa aatctaaatt agtttctgac ttccgaaaag atttccaatt
3061 ctataaagta cgtgagatta acaattacca tcatgcccat gatgcgtatc taaatgccgt
3121 cgttggaact gctttgatta agaaatatcc aaaacttgaa tcggagtttg tctatggtga
3181 ttataaagtt tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa taggcaaagc
3241 aaccgcaaaa tatttctttt actctaatat catgaacttc ttcaaaacag aaattacact
3301 tgcaaatgga gagattcgca aacgccctct aatcgaaact aatggggaaa ctggagaaat
3361 tgtctgggat aaagggcgag attttgccac agtgcgcaaa gtattgtcca tgccccaagt
3421 caatattgtc aagaaaacag aagtacagac aggcggattc tccaaggagt caattttacc
3481 aaaaagaaat tcggacaagc ttattgctcg taaaaaagac tgggatccaa aaaaatatgg
3541 tggttttgat agtccaacgg tagcttattc agtcctagtg gttgctaagg tggaaaaagg
3601 gaaatcgaag aagttaaaat ccgttaaaga gttactaggg atcacaatta tggaaagaag
3661 ttcctttgaa aaaaatccga ttgacttttt agaagctaaa ggatataagg aagttaaaaa
3721 agacttaatc attaaactac ctaaatatag tctttttgag ttagaaaacg gtcgtaaacg
3781 gatgctggct agtgccggag aattacaaaa aggaaatgag ctggctctgc caagcaaata
3841 tgtgaatttt ttatatttag ctagtcatta tgaaaagttg aagggtagtc cagaagataa
3901 cgaacaaaaa caattgtttg tggagcagca taagcattat ttagatgaga ttattgagca
3961 aatcagtgaa ttttctaagc gtgttatttt agcagatgcc aatttagata aagttcttag
4021 tgcatataac aaacatagag acaaaccaat acgtgaacaa gcagaaaata ttattcattt
4081 atttacgttg acgaatcttg gagctcccgc tgcttttaaa tattttgata caacaattga
4141 tcgtaaacga tatacgtcta caaaagaagt tttagatgcc actcttatcc atcaatccat
4201 cactggtctt tatgaaacac gcattgattt gagtcagcta ggaggtgact gagatcccat
4261 gttaccggta tcctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
4321 ctctaaacgg gtcttgaggg gttttttgct gaaacctcag gcatttgaga agcacacggt
4381 cacactgctt ccggtagtca ataaaccggt attgacgcca acgggtcaca cgggtacagt
4441 gctagcagaa acatatccat gaaatcccgc cccgaattca tatgtctaga ttagataaaa
4501 gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa
4561 cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa
4621 ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact
4681 tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta
4741 gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag
4801 aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac
4861 tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg
4921 aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc
4981 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct
5041 tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt
5101 cttaagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac gacaagctga
5161 cgaccgggtc tccgcaagtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
5221 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
5281 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
5341 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
5401 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
5461 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
5521 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
5581 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
5641 gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
5701 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
5761 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
5821 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
5881 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
5941 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
6001 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
6061 tttcccgttg aatatggctc atactcttcc tttttcaata ttattgaagc atttatcagg
6121 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggca
6181 tgctagcgca gaataaatat ctaacaccgt gcgtgttgac tattttacct ctggcggtga
6241 taatggttgc atgtactaag gaggttgtat ggaacaacga gatgtgtata agagacagct
6301 gaaacgccgt cgaagccgtg tgcgagacac cgcggccgcc ggcgttgtgg atacctcgcg
6361 gaaaacttgg ccctcactga cagatgaggg gcggacgttg acacttgagg ggccgactca
6421 cccggcgcgg cgttgacaga tgaggggcag gctcgatttc ggccggcgac gtggagctgg
6481 ccagcctcgc aaatcggcga aaacgcctga ttttacgcga gtttcccaca gatgatgtgg
6541 acaagcctgg ggataagtgc cctgcggtat tgacacttga ggggcgcgac tactgacaga
6601 tgaggggcgc gatccttgac acttgagggg cagagtgctg acagatgagg ggcgcaccta
6661 ttgacatttg aggggctgtc cacaggcaga aaatccagca tttgcaaggg tttccgcccg
6721 tttttcggcc accgctaacc tgtcttttaa cctgctttta aaccaatatt tataaacctt
6781 gtttttaatg gttgcatgta ctaaggaggt tgtatggaac aacgagatgt gtataagaga
6841 cagctgaaac gccgtcgaag ccgtgtgctg agagatcccc tcataatttc cccaaagcgt
6901 aaccatgtgt gaataaattt tgagctagta gggttgcagc cacgagtaag tcttcccttg
6961 ttattgtgta gccagaatgc cgcaaaactt ccatgcctaa gcgaactgtt gagagtacgt
7021 ttcgatttct gactgtgtta gcctggaagt gcttgtccca accttgtttc tgagcatgaa
7081 cgcccgcaag ccaacatgtt agttgaagca tcagggcgat tagcagcatg atatcaaaac
7141 gctctgagct gctcgttcgg ctatggcgta ggcctagtcc gtaggcagga cttttcaagt
7201 ctcggaaggt ttcttcaatc tgcattcgct tcgaatagat attaacaagt tgtttgggtg
7261 ttcgaatttc aacaggtaag ttagttgcta gaacccatgg ctcctttgcc gacgctgagt
7321 agattttagg tgacgggtgg tgacaatgag tccgtgtcga gcgctgattt tttcggcctt
7381 tagagcgaga tttatacaat agaatttggc atgagattgg attgctttta gtcagcctct
7441 tatagcctaa agtctttgag tgactagatg acatatcatg taagttgctg ataggtttcc
7501 agttttccgc tcctaggtct gcatattgta cttttcctct tactcgactt aaccagtacc
7561 aacccagctt ctcaacggat ttataccatg gcactttaaa gccagcatca ctgacaatga
7621 gcggtgtggt gttactcggt agaatgctcg caaggtcggc tagaaattgg tcatgagctt
7681 tctttgaaca ttgctctgaa agcgggaacg ctttctcata aagagtaaca gaacgaccgt
7741 gtagtgcgac tgaagctcgc aataccataa gtcgtttttg ctcacgaata tcagaccagt
7801 caacaagtac aatgggcatc gtattgcccg aacagataaa gctagcatgc caacggtata
7861 cagcgagtcg ctctttgtgg aggtgacgat tacctaacaa tcggtcgatt cgtttgatgt
7921 tatgttttgt tctcgctttg gttggcaggt tacggccaag ttcggtaaga gtgagagttt
7981 tacagtcaag taatgcgtgg caagccaacg ttaagctgtt gagtcgtttt aagtgtaatt
8041 cggggcagaa ttggtaaaga gagtcgtgta aaatatcgag ttcgcacatc ttgttgtctg
8101 attattgatt tttcgcgaaa ccatttgatc atatgacaag atgtgtatcc accttaactt
8161 aatgattttt accaaaatca ttaggggatt catcagagcc gtgtgcgaga caccgcggcc
8221 gccggcgttg tggatacctc gcggaaaact tggccctcac tgacagatga ggggcggacg
8281 ttgacacttg aggggccgac tcacccggcg cggcgttgac agatgagggg caggctcgat
8341 ttcggccggc gacgtggagc tggccagcct cgcaaatcgg cgaaaacgcc tgattttacg
8401 cgagtttccc acagatgatg tggacaagcc tggggataag tgccctgcgg tattgacact
8461 tgaggggcgc gactactgac agatgagggg cgcgatcctt gacacttgag gggcagagtg
8521 ctgacagatg aggggcgcac ctattgacat ttgaggggct gtccacaggc agaaaatcca
8581 gcatttgcaa gggtttccgc ccgtttttcg gccaccgcta acctgtcttt taacctgctt
8641 ttaaaccaat atttataaac cttgttttta accagggctg cgccctgtgc gcgtgaccgc
8701 gcacgccgaa ggggggtgcc cccccttctc gaaccctccc ggcccgctaa cgcgggcctc
8761 ccatcccccc aggggctgcg cccctcggcc gcgaacggcc tcaccccaaa aatggcagcg
8821 ctggcagtcc ttgccattgc cgggatcggg gcagtaacgg gatgggcgat cagcccgagc
8881 gcgacgcccg gaagcattga cgtgccgcag gtgctggcat cgacattcag cgaccaggtg
8941 ccgggcagtg agggcggcgg cctgggtggc ggcctgccct tcacttcggc cgtcggggca
9001 ttcacggact tcatggcggg gccggcaatt tttaccttgg gcattcttgg catagtggtc
9061 gcgggtgccg tgctcgtgtt cgggggtgaa ttaattcccc ggatccgtca gcttcacgct
9121 gccgcaagca ctcagggcgc aagggctgct aaaggaagcg gaacacgtag aaagccagtc
9181 cgcagaaacg gtgctgaccc cggatgaatg tcagctactg ggctatctgg acaagggaaa
9241 acgcaagcgc aaagagaaag caggtagctt gcagtgggct tacatggcga tagctagact
9301 gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg
9361 ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca
9421 ggggatcaag atcgacggat ccggggaatt ccggggcaat cccgcaagga gggtgaatga
9481 atcggacgtt tgaccggaag gcatacaggc aagaactgat cgacgcgggg ttttccgccg
9541 aggatgccga aaccatcgca agccgcaccg tcatgcgtgc gccccgcgaa accttccagt
9601 ccgtcggctc gatggtccag caagctacgg ccaagatcga gcgcgacagc gtgcaactgg
9661 ctccccctgc cctgcccgcg ccatcggccg ccgtggagcg ttcgcgtcgt ctcgaacagg
9721 aggcggcagg tttggcgaag tcgatgacca tcgacacgcg aggaactatg acgaccaaga
9781 agcgaaaaac cgccggcgag gacctggcaa aacaggtcag cgaggccaag caggccgcgt
9841 tgctgaaaca cacgaagcag cagatcaagg aaatgcagct ttccttgttc gatattgcgc
9901 cgtggccgga cacgatgcga gcgatgccaa acgacacggc ccgctctgcc ctgttcacca
9961 cgcgcaacaa gaaaatcccg cgcgaggcgc tgcaaaacaa ggtcattttc cacgtcaaca
10021 aggacgtgaa gatcacctac accggcgtcg agctgcgggc cgacgatgac gaactggtgt
10081 ggcagcaggt gttggagtac gcgaagcgca cccctatcgg cgagccgatc accttcacgt
10141 tctacgagct ttgccaggac ctgggctggt cgatcaatgg ccggtattac acgaaggccg
10201 aggaatgcct gtcgcgccta caggcgacgg cgatgggctt cacgtccgac cgcgttgggc
10261 acctggaatc ggtgtcgctg ctgcaccgct tccgcgtcct ggaccgtggc aagaaaacgt
10321 cccgttgcca ggtcctgatc gacgaggaaa tcgtcgtgct gtttgctggc gaccactaca
10381 cgaaattcat atgggagaag taccgcaagc tgtcgccgac ggcccgacgg atgttcgact
10441 atttcagctc gcaccgggag ccgtacccgc tcaagctgga aaccttccgc ctcatgtgcg
10501 gatcggattc cacccgcgtg aagaagtggc gcgagcaggt cggcgaagcc tgcgaagagt
10561 tgcgaggcag cggcctggtg gaacacgcct gggtcaatga cctggtgcat tgcaaacgct
10621 agggccttgt ggggtcagtt ccggctgggg gttcagcaca gctgtctctt atacacatct
10681 cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt
10741 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
10801 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgca caccgactag
10861 cgaaaaaacc ccgccgaagc ggggtttttt gcgaaaaaaa gcaccgactc ggtgccactt
10921 tttcaagttg ataacggact agccttattt taacttgcta tgctgttttg aatggttccg
10981 ctagcactgt acctaggact gagctagccg tcaaaagcag cgtatatacc atggccacaa
11041 ttcagcaaat tgtgaacatc atcacgttca tctttccctg gttgccaatg gcccattttc
11101 ctgtcagtaa cgagaaggtc gcgaattcag gcgcttttta gactggtcgt gttttagagc
11161 tatgctgttt tgaatggtcc caaaacatga actagcgatt agtcgctatg acttaagttt
11221 tagagctatg ctgttttgaa tggtcccaaa acaaccacac tagagaacat actggctaaa
11281 tagttttaga gctatgctgt tttgaatggt cccaaaacgg ttggactcaa gacgatagtt
11341 accggatagt tttagagcta tgctgttttg aatggtccca aaacggcgaa acccgacagg
11401 actataaaga taccgtttta gagctatgct gttttgaatg gtcccaaaac aaaaaaaaac
11461 cccgcccctg acagggcggg gttttttttt taattaaagt ctggtcc
//
