| 质粒名称: | pEvolvR-enCas9-PolI5M |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 113078 |
| 存储实验室: | Vladislav Verkhusha |
|
载体骨架基本信息 |
|
| 载体名称: | custom |
| 空载体大小: | 12456 bp |
| 载体类型: | 细菌表达，crispr载体 |
| 载体抗性: | 卡那霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 低拷贝 |
|
插入基因信息 |
|
| 插入基因名称: |
enCas9-PolI5M
|
|
文献引用方法 |
|
| 材料和方法部分: | pEvolvR-enCas9-PolI5M was a gift from John Dueber & David Schaffer (Addgene plasmid # 113078 ; http://n2t.net/addgene:113078 ; RRID:Addgene_113078) |
| 参考文献部分: | CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Halperin SO, Tou CJ, Wong EB, Modavi C, Schaffer DV, Dueber JE. Nature. 2018 Aug 1. pii: 10.1038/s41586-018-0384-8. doi: 10.1038/s41586-018-0384-8. 10.1038/s41586-018-0384-8 PubMed 30069054 |
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载体基本信息
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载体简介
载体序列
LOCUS pEvolvR-enCas9-PolI5M 12546 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses enCas9 fused to PolI5M in E. coli. contains a
BsmbI-flanked GFP cassette for sgRNA cloning..
ACCESSION .
VERSION .
KEYWORDS pEvolvR-enCas9-PolI5M
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12546)
AUTHORS Halperin SO, Tou CJ, Wong EB, Modavi C, Schaffer DV, Dueber JE
TITLE CRISPR-guided DNA polymerases enable diversification of all
nucleotides in a tunable window.
JOURNAL Nature. 2018 Aug 1. pii: 10.1038/s41586-018-0384-8. doi:
10.1038/s41586-018-0384-8.
PUBMED 30069054
REFERENCE 2 (bases 1 to 12546)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..12546
/organism="synthetic DNA construct"
/mol_type="other DNA"
RBS 4884..4892
/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)"
CDS complement(7094..7909)
/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 7817..7836
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 7901..7920
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 8138..8726
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8627..8646
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 8880..8897
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 8912..9051
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(8977..8996)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 9137..9159
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(9153..9344)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
RBS 9474..9485
/note="strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)"
CDS 9492..10151
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKF*PAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind complement(9558..9577)
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
RBS 10451..10462
/note="strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)"
CDS 10469..11182
/codon_start=1
/product="GFP variant that folds robustly even when fused
to poorly folded proteins (Pedelacq et al., 2006)"
/label=superfolder GFP
/translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
FVTAAGITHGMDELYK"
terminator 11194..11265
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 11281..11308
/label=T7Te terminator
/note="phage T7 early transcription terminator"
misc_RNA 11322..11397
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
CDS 11418..11447
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 11463..11480
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(11536..11553)
/label=pBAD Reverse
/note="For vectors with E. coli araBAD promoter, reverse
primer"
terminator 11706..11752
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
CDS complement(11811..12434)
/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 12342..12361
/label=Tet-R
/note="Tetracycline resistance gene, reverse primer"
promoter 12453..12508
/gene="tetR"
/label=tetR/tetA promoters
/note="overlapping promoters for bacterial tetR and tetA"
protein_bind 12489..12507
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
RBS 12526..12537
/note="strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)"
ORIGIN
1 gacaagaagt attctatcgg actggctatc gggactaata gcgtcgggtg ggccgtgatc
61 actgacgagt acaaggtgcc ctctaagaag ttcaaggtgc tcgggaacac cgaccggcat
121 tccatcaaga aaaatctgat cggagctctc ctctttgatt caggggaaac cgctgaagca
181 acccgcctca agcggactgc tagacggcgg tacaccagga ggaagaaccg gatttgttac
241 cttcaagaga tattctccaa cgaaatggca aaggtcgacg acagcttctt ccataggctg
301 gaagaatcat tcctcgtgga agaggataag aagcatgaac ggcatcccat cttcggtaat
361 atcgtcgacg aggtggccta tcacgagaaa tacccaacca tctaccatct tcgcaaaaag
421 ctggtggact caaccgacaa ggcagacctc cggcttatct acctggccct ggcccacatg
481 atcaagttca gaggccactt cctgatcgag ggcgacctca atcctgacaa tagcgatgtg
541 gataaactgt tcatccagct ggtgcagact tacaaccagc tctttgaaga gaaccccatc
601 aatgcaagcg gagtcgatgc caaggccatt ctgtcagccc ggctgtcaaa gagccgcaga
661 cttgagaatc ttatcgctca gctgccgggt gaaaagaaaa atggactgtt cgggaacctg
721 attgctcttt cacttgggct gactcccaat ttcaagtcta atttcgacct ggcagaggat
781 gccaagctgc aactgtccaa ggacacctat gatgacgatc tcgacaacct cctggcccag
841 atcggtgacc aatacgccga ccttttcctt gctgctaaga atctttctga cgccatcctg
901 ctgtctgaca ttctccgcgt gaacactgaa atcaccaagg cccctctttc agcttcaatg
961 attaagcggt atgatgagca ccaccaggac ctgaccctgc ttaaggcact cgtccggcag
1021 cagcttccgg agaagtacaa ggaaatcttc tttgaccagt caaagaatgg atacgccggc
1081 tacatcgacg gaggtgcctc ccaagaggaa ttttataagt ttatcaaacc tatccttgag
1141 aagatggacg gcaccgaaga gctcctcgtg aaactgaatc gggaggatct gctgcggaag
1201 cagcgcactt tcgacaatgg gagcattccc caccagatcc atcttgggga gcttcacgcc
1261 atccttcggc gccaagagga cttctacccc tttcttaagg acaacaggga gaagattgag
1321 aaaattctca ctttccgcat cccctactac gtgggacccc tcgccagagg aaatagccgg
1381 tttgcttgga tgaccagaaa gtcagaagaa actatcactc cctggaactt cgaagaggtg
1441 gtggacaagg gagccagcgc tcagtcattc atcgaacgga tgactaactt cgataagaac
1501 ctccccaatg agaaggtcct gccgaaacat tccctgctct acgagtactt taccgtgtac
1561 aacgagctga ccaaggtgaa atatgtcacc gaagggatga ggaagcccgc attcctgtca
1621 ggcgaacaaa agaaggcaat tgtggacctt ctgttcaaga ccaatagaaa ggtgaccgtg
1681 aagcagctga aggaggacta tttcaagaaa attgaatgct tcgactctgt ggagattagc
1741 ggggtcgaag atcggttcaa cgcaagcctg ggtacttacc atgatctgct taagatcatc
1801 aaggacaagg attttctgga caatgaggag aacgaggaca tccttgagga cattgtcctg
1861 actctcactc tgttcgagga ccgggaaatg atcgaggaga ggcttaagac ctacgcccat
1921 ctgttcgacg ataaagtgat gaagcaactt aaacggagaa gatataccgg atggggacgc
1981 cttagccgca aactcatcaa cggaatccgg gacaaacaga gcggaaagac cattcttgat
2041 ttccttaaga gcgacggatt cgctaatcgc aacttcatgc aacttatcca tgatgattcc
2101 ctgaccttta aggaggacat ccagaaggcc caagtgtctg gacaaggtga ctcactgcac
2161 gagcatatcg caaatctggc tggttcaccc gctattaaga agggtattct ccagaccgtg
2221 aaagtcgtgg acgagctggt caaggtgatg ggtcgccata aaccagagaa cattgtcatc
2281 gagatggcca gggaaaacca gactacccag aagggacaga agaacagcag ggagcggatg
2341 aaaagaattg aggaagggat taaggagctc gggtcacaga tccttaaaga gcacccggtg
2401 gaaaacaccc agcttcagaa tgagaagctc tatctgtact accttcaaaa tggacgcgat
2461 atgtatgtgg accaagagct tgatatcaac aggctctcag actacgacgt ggaccatatc
2521 gtccctcaga gcttcctcgc agacgactca attgacaata aggtgctgac tcgctcagac
2581 aagaaccggg gaaagtcaga taacgtgccc tcagaggaag tcgtgaaaaa gatgaagaac
2641 tattggcgcc agcttctgaa cgcaaagctg atcactcagc ggaagttcga caatctcact
2701 aaggctgaga ggggcggact gagcgaactg gacaaagcag gattcattaa acggcaactt
2761 gtggagactc ggcagattac taaacatgtc gcccaaatcc ttgactcacg catgaatacc
2821 aagtacgacg aaaacgacaa acttatccgc gaggtgaagg tgattaccct gaagtccaag
2881 ctggtcagcg atttcagaaa ggactttcaa ttctacaaag tgcgggagat caataactat
2941 catcatgctc atgacgcata tctgaatgcc gtggtgggaa ccgccctgat caagaagtac
3001 ccagcactgg aaagcgagtt cgtgtacgga gactacaagg tctacgacgt gcgcaagatg
3061 attgccaaat ctgagcagga gatcggaaag gccaccgcaa agtacttctt ctacagcaac
3121 atcatgaatt tcttcaagac cgaaatcacc cttgcaaacg gtgagatccg gaaggcgccg
3181 ctcatcgaga ctaatgggga gactggcgaa atcgtgtggg acaagggcag agatttcgct
3241 accgtgcgca aagtgctttc tatgcctcaa gtgaacatcg tgaagaaaac cgaggtgcaa
3301 accggaggct tttctaagga atcaatcctc cccaagcgca actccgacaa gctcattgca
3361 aggaagaagg attgggaccc taagaagtac ggcggattcg attcaccaac tgtggcttat
3421 tctgtcctgg tcgtggctaa ggtggaaaaa ggaaagtcta agaagctcaa gagcgtgaag
3481 gaactgctgg gtatcaccat tatggagcgc agctccttcg agaagaaccc aattgacttt
3541 ctcgaagcca aaggttacaa ggaagtcaag aaggacctta tcatcaagct cccaaagtat
3601 agcctgttcg aactggagaa tgggcggaag cggatgctcg cctccgctgg cgaacttcag
3661 aagggtaatg agctggctct cccctccaag tacgtgaatt tcctctacct tgcaagccat
3721 tacgagaagc tgaaggggag ccccgaggac aacgagcaaa agcaactgtt tgtggagcag
3781 cataagcatt atctggacga gatcattgag cagatttccg agttttctaa acgcgtcatt
3841 ctcgctgatg ccaacctcga taaagtcctt agcgcataca ataagcacag agacaaacca
3901 attcgggagc aggctgagaa tatcatccac ctgttcaccc tcaccaatct tggtgcccct
3961 gccgcattca agtacttcga caccaccatc gaccggaaac gctatacctc caccaaagaa
4021 gtgctggacg ccaccctcat ccaccagagc atcaccggac tttacgaaac tcggattgac
4081 ctctcacagc tcggagggga tggttctagt gaaaccccgg gaacaagtga gtcggccacc
4141 cctgaaggtg gatcaggggg tagcggatcc gttcagatcc cgcagaaccc gctgattctg
4201 gttgacggat ctagttacct gtaccgtgct taccatgctt tcccgccttt gaccaattct
4261 gctggtgaac ctacgggagc tatgtacgga gttctgaata tgttgcgttc tttaattatg
4321 cagtacaagc ctacccacgc tgctgttgtt ttcgatgcta aaggtaagac gttccgcgac
4381 gagttattcg agcactataa gtctcaccgt cctccgatgc ctgatgactt acgcgctcag
4441 attgagccgc tgcatgctat ggtgaaggct atgggtttac ctcttttggc tgtcagcggt
4501 gttgaggctg atgatgtcat tggcacctta gctcgtgagg ctgagaaggc tggtcgccct
4561 gttttgattt ctaccggtga caaggacatg gctcaattgg ttaccccgaa catcaccctg
4621 atcaacacca tgaccaacac gattctgggt cctgaggaag ttgttaacaa atatggtgtt
4681 cctccggagt tgattattga ctttcttgct cttatgggcg attcttcaga caatatcccg
4741 ggtgttccag gtgttggaga gaagactgct caagctctgc ttcagggtct gggtggtttg
4801 gacacccttt acgctgaacc ggagaagatc gccggtctgt cttttcgcgg tgctaagacc
4861 atggctgcta aactggaaca gaataaggag gtcgcatacc tgtcttatca attggctacc
4921 atcaagacgg atgtggagtt agaacttacg tgcgagcagc ttgaggttca acagcctgct
4981 gctgaggaac tgctgggtct ttttaagaaa tacgaattta agcgttggac cgccgacgtt
5041 gaggctggta agtggctgca agctaagggt gctaagccgg ctgctaaacc gcaagaaacg
5101 agtgtcgctg atgaggctcc ggaggttacc gctaccgtta tctcttacga taattatgtt
5161 acgattctgg acgaggaaac cttaaaggct tggatcgcta aattagagaa ggctcctgtt
5221 ttcgctttcg acacggaaac ggattctctg gacaatatta gtgcgaatct tgttggtctg
5281 agtttcgcaa ttgaaccggg tgttgctgct tacatccctg tggcacacga ctacctggac
5341 gctccggacc agatttcacg tgaacgcgct ctggaactgc tgaagccttt attagaggac
5401 gagaaagctt tgaaagttgg tcagaatttg aagtatgctc gtggaatctt agctaattat
5461 ggtatcgagt tgcgcggtat cgctttcgac acgatgttgg aatcttatat cctgaactct
5521 gtcgctggtc gccatgacat ggactctctg gctgagcgct ggctgaaaca taagacgatt
5581 accttcgagg aaatcgcagg aaagggtaag aaccagctca cgttcaatca aatcgctctg
5641 gaggaagctg gtcgctatgc tgctgaggac gctgacgtta ctctgcaact gcacttgaag
5701 atgtggcctg acttgcagaa gcataagggt ccactgaatg tttttgaaaa cattgagatg
5761 cctttggttc cagttctgtc tcgtatcgag cgcaatggcg ttaaaattga cccaaaggtt
5821 ttacataacc actcagagga actgacgctg cgcttagccg aattggagaa aaaggctcac
5881 gagatcgctg gcgaagagtt caatctgtca tctacgaaac aactgcagac tatcctgttc
5941 gagaagcaag gtatcaagcc attaaaaaag acccctggcg gtgctccgtc tacctctgag
6001 gaagttttgg aggagttagc tttggattac cctctgccga aggttatctt ggaataccgc
6061 ggtttggcta aattgaagtc tacttatacg gataaacttc ctttgatgat taatccaaag
6121 acgggtcgcg ttcacacgtc gtaccatcaa gctgttaccg ctaccggtcg cctgtcttct
6181 acggatccga atttacagaa tattcctgtg cgcaatgagg agggccgccg cattcgtcaa
6241 gcttttatcg ctccggaaga ctacgttatc gtttctgctg attattctca aaatgaatta
6301 cgtatcatgg ctcacctgtc tcgcgataag ggtctgttga cggcctttgc tgagggtaag
6361 gacattcatc gtgctaccgc tgctgaggtt tacggcctgc cgttggaaac ggttacgtct
6421 gaacagcgtc gctctgctaa gcgtattaat ttcggcttaa tctacggtat gtctgcgttt
6481 ggcttagctc gtcagctgaa tatcccgcgc aaggaagctc aaaaatatat ggatctgtat
6541 tttgagcgtt accacggtgt tttggaatac atggagcgta cgcgcgcgca agctaaggaa
6601 caaggttatg tggaaacctt ggatggtcgt cgcttgtact tgcctgacat taagtcttct
6661 aacggcgccc gccgcgctgc tgccgagcgc gctgctatca atgctccgat gcaaggtact
6721 gctgctgata ttattaagcg tgctatgatc gctgtggacg cttggctgca agctgaacag
6781 cctcgcgttc gcatgattat gcaagttcat gacgagttgg ttttcgaggt gcataaggac
6841 gacgtggacg ctgttgctaa acaaatccac cagttgatgg agaattgcac gcgcttagac
6901 gttccgctgc tggttgaagt tggttctggt gaaaactggg accaggctca ctaataatgg
6961 cctcggtacc aaagacgaac aataagacgc tgaaaagcgt cttttttcgt tttggtccgc
7021 tgagcagtta cagagatgtt acgaaccact agtgcactgc agtacagtgt tacaaccaat
7081 taaccaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
7141 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
7201 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
7261 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
7321 catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt
7381 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
7441 tcattcgtga ttgcgcctga gcgaggcgaa atacgcgatc gctgttaaaa ggacaattac
7501 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
7561 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
7621 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
7681 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
7741 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
7801 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
7861 aatttaatcg cggcctggag caagacgttt cccgttgaat atggctcata acaccccttg
7921 tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg
7981 caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat cgaacttttg
8041 ctgagttgaa ggatcaggag ttcatgacca aaatccctta acgtgagttt tcgttccact
8101 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
8161 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
8221 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
8281 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
8341 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
8401 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
8461 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
8521 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
8581 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
8641 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
8701 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
8761 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
8821 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
8881 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
8941 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
9001 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
9061 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
9121 caagctgtga ccttctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
9181 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
9241 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
9301 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
9361 gggggatttc tgttcatccc tgaattcgca tctagactga actggccgat aattgcagac
9421 gaacggattt actttacagc tagctcagtc ctaggtatta tgctagcgct agcaaagagg
9481 agaaaagatc tatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
9541 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
9601 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttaac
9661 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattt cgtatggcaa
9721 tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg
9781 agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc
9841 tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag
9901 ggtttattga gaatatgttt ttcgtttcag ccaatccctg ggtgagtttc accagttttg
9961 atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt
10021 atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg
10081 atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg
10141 gcggggcgta aggatcctaa ctcgagctcg gtaccaaatt ccagaaaaga ggcctcccga
10201 aaggggggcc ttttttcgtt ttggtccgct gccaaaccag atgtcaacac agctacaacg
10261 tttatagcta gctcagccct tggtacaatg ctagctgaga cggaaagtga aacgtgattt
10321 catgcgtcat tttgaacatt ttgtaaatct tatttaataa tgtgtgcggc aattcacatt
10381 taatttatga atgttttctt aacatcgcgg caactcaaga aacggcaggt tcggatctta
10441 gctactagag aaagaggaga aatactagat gcgtaaaggc gaagagctgt tcactggtgt
10501 cgtccctatt ctggtggaac tggatggtga tgtcaacggt cataagtttt ccgtgcgtgg
10561 cgagggtgaa ggtgacgcaa ctaatggtaa actgacgctg aagttcatct gtactactgg
10621 taaactgccg gttccttggc cgactctggt aacgacgctg acttatggtg ttcagtgctt
10681 tgctcgttat ccggaccata tgaagcagca tgacttcttc aagtccgcca tgccggaagg
10741 ctatgtgcag gaacgcacga tttcctttaa ggatgacggc acgtacaaaa cgcgtgcgga
10801 agtgaaattt gaaggcgata ccctggtaaa ccgcattgag ctgaaaggca ttgactttaa
10861 agaggacggc aatatcctgg gccataagct ggaatacaat tttaacagcc acaatgttta
10921 catcaccgcc gataaacaaa aaaatggcat taaagcgaat tttaaaattc gccacaacgt
10981 ggaggatggc agcgtgcagc tggctgatca ctaccagcaa aacactccaa tcggtgatgg
11041 tcctgttctg ctgccagaca atcactatct gagcacgcaa agcgttctgt ctaaagatcc
11101 gaacgagaaa cgcgatcata tggttctgct ggagttcgta accgcagcgg gcatcacgca
11161 tggtatggat gaactgtaca aatgaccagg catcaaataa aacgaaaggc tcagtcgaaa
11221 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact
11281 ggctcacctt cgggtgggcc tttctgcgtt tatacgtctc agttttagag ctagaaatag
11341 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
11401 ttttgaagct tgggcccgaa caaaaactca tctcagaaga ggatctgaat agcgccgtcg
11461 accatcatca tcatcatcat tgagtttaaa cggactccag cttggctgtt ttggcggatg
11521 agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca
11581 gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt
11641 gaaacgccgt agcgccgatg gtagtgtggg gactccccat gcgagagtag ggaactgcca
11701 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
11761 tgtcggtgaa cgacagggct gccaaaccag atgtcaacac agctacaacg ttaagaccca
11821 ctttcacatt taagttgttt ttctaatccg catatgatca attcaaggcc gaataagaag
11881 gctggctctg caccttggtg atcaaataat tcgatagctt gtcgtaataa tggcggcata
11941 ctatcagtag taggtgtttc cctttcttct ttagcgactt gatgctcttg atcttccaat
12001 acgcaaccta aagtaaaatg ccccacagcg ctgagtgcat ataatgcatt ctctagtgaa
12061 aaaccttgtt ggcataaaaa ggctaattga ttttcgagag tttcatactg tttttctgta
12121 ggccgtgtac ctaaatgtac ttttgctcca tcgcgatgac ttagtaaagc acatctaaaa
12181 cttttagcgt tattacgtaa aaaatcttgc cagctttccc cttctaaagg gcaaaagtga
12241 gtatggtgcc tatctaacat ctcaatggct aaggcgtcga gcaaagcccg cttatttttt
12301 acatgccaat acaatgtagg ctgctctaca cctagcttct gggcgagttt acgggttgtt
12361 aaaccttcga ttccgacctc attaagcagc tctaatgcgc tgttaatcac tttactttta
12421 tctaatctag acatcattaa ttcctaattt ttgttgacac tctatcgttg atagagttat
12481 tttaccactc cctatcagtg atagagaaaa gaattcaaaa gatctaaaga ggagaaaaga
12541 tctatg
//
