| 质粒名称: | pET-28b-T7-henAsCas12a-HF1(E174R/N282A/S542R/K548R)-NLS(nucleoplasmin)-6xHis (AAS1935) |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 114073 |
| 存储实验室: | Keith Joung, Benjamin Kleinstiver |
|
载体骨架基本信息 |
|
| 载体名称: | pET-28b-Cas9-His |
| 载体出品公司: | Schier Lab (Addgene plasmid # 47327) |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | human codon optimized enAsCas12a-HF1 (E174R/N282A/S542R/K548R) with C-terminal NLS and His-tag,AAS1935 |
| 物种来源: | Synthetic |
| 插入大小: | -- |
| 基因突变: | E174R, S542R, K548R and N282A |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | NLS（核仁蛋白酶）和6xHis（插入物上的C端） |
| 克隆方法: |
Gibson克隆
|
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | -- |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pET-28b-T7-henAsCas12a-HF1(E174R/N282A/S542R/K548R)-NLS(nucleoplasmin)-6xHis (AAS1935) was a gift from Keith Joung & Benjamin Kleinstiver (Addgene plasmid # 114073 ; http://n2t.net/addgene:114073 ; RRID:Addgene_114073) |
| 参考文献部分: | Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Kleinstiver BP, Sousa AA, Walton RT, Tak YE, Hsu JY, Clement K, Welch MM, Horng JE, Malagon-Lopez J, Scarfo I, Maus MV, Pinello L, Aryee MJ, Joung JK. Nat Biotechnol. 2019 Feb 11. pii: 10.1038/s41587-018-0011-0. doi: 10.1038/s41587-018-0011-0. 10.1038/s41587-018-0011-0 PubMed 30742127 |
pET-28b-T7-henAsCas12a-HF1(E174R/N282A/S542R/K548R)-NLS(nucl
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
-NLS(nucleoplasmin)-6xHis (AAS1935)质粒图谱.png)
载体简介
载体序列
LOCUS pET-28b-T7-henAsCas12a-HF1(E174R/N282A/S542R/K548R)-NLS(nucleoplasmin)-6xHis (AAS1935) 9213 bp ds-DNA circular SYN 29-AUG-2018
DEFINITION T7 promoter bacterial expression plasmid for human codon optimized
enAsCas12a-HF1(E174R/N282A/S542R/K548R) with C-terminal NLS and
His-tag.
ACCESSION .
VERSION .
KEYWORDS
pET-28b-T7-henAsCas12a-HF1(E174R/N282A/S542R/K548R)-NLS(nucleoplasmin)-6xHis (AAS1935)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9213)
AUTHORS Keith Joung
TITLE Joung lab Cas12a plasmids
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 9213)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 29, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..9213
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 5..144
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(70..89)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 230..252
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(246..437)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(1246..2328)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
primer_bind 2290..2309
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(2329..2406)
/gene="lacI"
/label=lacI promoter
primer_bind 2612..2631
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 2715..2734
/label=T7
/note="T7 promoter, forward primer"
promoter 2715..2733
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 2734..2758
/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)."
RBS 2773..2795
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 2802..6725
/codon_start=1
/product="CRISPR-associated protein from Acidaminococcus
sp. BV3L6 (Zetsche et al., 2015)"
/label=AsCpf1
/translation="MGTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARN
DHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYR
NAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSF
DKFTTYFSGFYRNRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREH
FENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEV
LALAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTL
LRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELT
GKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTT
LKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNK
ARNYATKKPYSVEKFKLNFQMPTLARGWDVNREKNNGAILFVKNGLYYLGIMPKQKGRY
KALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIE
PLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSI
DLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAK
GHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKL
KDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFF
FHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQ
RSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQA
VVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTD
QFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHY
DVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIE
NHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQ
MRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKE
SKDLKLQNGISNQDWLAYIQELRN"
CDS 6726..6773
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 6792..6809
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(6862..6880)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 6876..6923
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 6960..7415
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(7047..7066)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 7257..7278
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(7507..8322)
/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 8230..8249
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 8314..8333
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 8444..9032
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8933..8952
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 9186..9203
/label=L4440
/note="L4440 vector, forward primer"
ORIGIN
1 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
61 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
121 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
181 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
241 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
301 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
361 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
421 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
481 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
541 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
601 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
661 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
721 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
781 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
841 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
901 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
961 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
1021 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
1081 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
1141 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
1201 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
1261 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
1321 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
1381 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
1441 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
1501 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
1561 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
1621 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
1681 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
1741 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
1801 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
1861 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
1921 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
1981 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
2041 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
2101 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
2161 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
2221 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
2281 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
2341 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
2401 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
2461 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
2521 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
2581 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
2641 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
2701 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
2761 tctagaaata attttgttta actttaagaa ggagatatac catgggcaca cagttcgagg
2821 gctttaccaa cctgtatcag gtgagcaaga cactgcggtt tgagctgatc ccacagggca
2881 agaccctgaa gcacatccag gagcagggct tcatcgagga ggacaaggcc cgcaatgatc
2941 actacaagga gctgaagccc atcatcgatc ggatctacaa gacctatgcc gaccagtgcc
3001 tgcagctggt gcagctggat tgggagaacc tgagcgccgc catcgactcc tatagaaagg
3061 agaaaaccga ggagacaagg aacgccctga tcgaggagca ggccacatat cgcaatgcca
3121 tccacgacta cttcatcggc cggacagaca acctgaccga tgccatcaat aagagacacg
3181 ccgagatcta caagggcctg ttcaaggccg agctgtttaa tggcaaggtg ctgaagcagc
3241 tgggcaccgt gaccacaacc gagcacgaga acgccctgct gcggagcttc gacaagttta
3301 caacctactt ctccggcttt tatagaaaca ggaagaacgt gttcagcgcc gaggatatca
3361 gcacagccat cccacaccgc atcgtgcagg acaacttccc caagtttaag gagaattgtc
3421 acatcttcac acgcctgatc accgccgtgc ccagcctgcg ggagcacttt gagaacgtga
3481 agaaggccat cggcatcttc gtgagcacct ccatcgagga ggtgttttcc ttcccttttt
3541 ataaccagct gctgacacag acccagatcg acctgtataa ccagctgctg ggaggaatct
3601 ctcgggaggc aggcaccgag aagatcaagg gcctgaacga ggtgctggcc ctggccatcc
3661 agaagaatga tgagacagcc cacatcatcg cctccctgcc acacagattc atccccctgt
3721 ttaagcagat cctgtccgat aggaacaccc tgtctttcat cctggaggag tttaagagcg
3781 acgaggaagt gatccagtcc ttctgcaagt acaagacact gctgagaaac gagaacgtgc
3841 tggagacagc cgaggccctg tttaacgagc tgaacagcat cgacctgaca cacatcttca
3901 tcagccacaa gaagctggag acaatcagca gcgccctgtg cgaccactgg gatacactga
3961 ggaatgccct gtatgagcgg agaatctccg agctgacagg caagatcacc aagtctgcca
4021 aggagaaggt gcagcgcagc ctgaagcacg aggatatcaa cctgcaggag atcatctctg
4081 ccgcaggcaa ggagctgagc gaggccttca agcagaaaac cagcgagatc ctgtcccacg
4141 cacacgccgc cctggatcag ccactgccta caaccctgaa gaagcaggag gagaaggaga
4201 tcctgaagtc tcagctggac agcctgctgg gcctgtacca cctgctggac tggtttgccg
4261 tggatgagtc caacgaggtg gaccccgagt tctctgcccg gctgaccggc atcaagctgg
4321 agatggagcc ttctctgagc ttctacaaca aggccagaaa ttatgccacc aagaagccct
4381 actccgtgga gaagttcaag ctgaactttc agatgcctac actggccaga ggctgggacg
4441 tgaatagaga gaagaacaat ggcgccatcc tgtttgtgaa gaacggcctg tactatctgg
4501 gcatcatgcc aaagcagaag ggcaggtata aggccctgag cttcgagccc acagagaaaa
4561 ccagcgaggg ctttgataag atgtactatg actacttccc tgatgccgcc aagatgatcc
4621 caaagtgcag cacccagctg aaggccgtga cagcccactt tcagacccac acaaccccca
4681 tcctgctgtc caacaatttc atcgagcctc tggagatcac aaaggagatc tacgacctga
4741 acaatcctga gaaggagcca aagaagtttc agacagccta cgccaagaaa accggcgacc
4801 agaagggcta cagagaggcc ctgtgcaagt ggatcgactt cacaagggat tttctgtcca
4861 agtataccaa gacaacctct atcgatctgt ctagcctgcg gccatcctct cagtataagg
4921 acctgggcga gtactatgcc gagctgaatc ccctgctgta ccacatcagc ttccagagaa
4981 tcgccgagaa ggagatcatg gatgccgtgg agacaggcaa gctgtacctg ttccagatct
5041 ataacaagga ctttgccaag ggccaccacg gcaagcctaa tctgcacaca ctgtattgga
5101 ccggcctgtt ttctccagag aacctggcca agacaagcat caagctgaat ggccaggccg
5161 agctgttcta ccgccctaag tccaggatga agaggatggc acaccggctg ggagagaaga
5221 tgctgaacaa gaagctgaag gatcagaaaa ccccaatccc cgacaccctg taccaggagc
5281 tgtacgacta tgtgaatcac agactgtccc acgacctgtc tgatgaggcc agggccctgc
5341 tgcccaacgt gatcaccaag gaggtgtctc acgagatcat caaggatagg cgctttacca
5401 gcgacaagtt ctttttccac gtgcctatca cactgaacta tcaggccgcc aattccccat
5461 ctaagttcaa ccagagggtg aatgcctacc tgaaggagca ccccgagaca cctatcatcg
5521 gcatcgatcg gggcgagaga aacctgatct atatcacagt gatcgactcc accggcaaga
5581 tcctggagca gcggagcctg aacaccatcc agcagtttga ttaccagaag aagctggaca
5641 acagggagaa ggagagggtg gcagcaaggc aggcctggtc tgtggtgggc acaatcaagg
5701 atctgaagca gggctatctg agccaggtca tccacgagat cgtggacctg atgatccact
5761 accaggccgt ggtggtgctg gagaacctga atttcggctt taagagcaag aggaccggca
5821 tcgccgagaa ggccgtgtac cagcagttcg agaagatgct gatcgataag ctgaattgcc
5881 tggtgctgaa ggactatcca gcagagaaag tgggaggcgt gctgaaccca taccagctga
5941 cagaccagtt cacctccttt gccaagatgg gcacccagtc tggcttcctg ttttacgtgc
6001 ctgccccata tacatctaag atcgatcccc tgaccggctt cgtggacccc ttcgtgtgga
6061 aaaccatcaa gaatcacgag agccgcaagc acttcctgga gggcttcgac tttctgcact
6121 acgacgtgaa aaccggcgac ttcatcctgc actttaagat gaacagaaat ctgtccttcc
6181 agaggggcct gcccggcttt atgcctgcat gggatatcgt gttcgagaag aacgagacac
6241 agtttgacgc caagggcacc cctttcatcg ccggcaagag aatcgtgcca gtgatcgaga
6301 atcacagatt caccggcaga taccgggacc tgtatcctgc caacgagctg atcgccctgc
6361 tggaggagaa gggcatcgtg ttcagggatg gctccaacat cctgccaaag ctgctggaga
6421 atgacgattc tcacgccatc gacacgatgg tggccctgat ccgcagcgtg ctgcagatgc
6481 ggaactccaa tgccgccaca ggcgaggact atatcaacag ccccgtgcgc gatctgaatg
6541 gcgtgtgctt cgactcccgg tttcagaacc cagagtggcc aatggacgcc gatgccaatg
6601 gcgcctacca catcgccctg aagggccagc tgctgctgaa tcacctgaag gagagcaagg
6661 atctgaagct gcagaacggc atctccaatc aggactggct ggcctacatc caggagctgc
6721 gcaacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagggagcgg
6781 ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
6841 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
6901 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
6961 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
7021 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
7081 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
7141 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
7201 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
7261 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
7321 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
7381 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
7441 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
7501 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
7561 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
7621 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
7681 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
7741 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
7801 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
7861 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
7921 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
7981 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
8041 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
8101 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
8161 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
8221 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
8281 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
8341 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
8401 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
8461 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
8521 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
8581 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
8641 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
8701 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
8761 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
8821 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
8881 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
8941 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
9001 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
9061 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
9121 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
9181 agcgcagcga gtcagtgagc gaggaagcgg aag
//
