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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
Plasmid#89567
- 库存：
100
- 供应商：
上海烜雅生物科技有限公司
- 规格：
干粉/液体
基本信息
名称：Lenti-dCas9-KRAB-blast哺乳编辑质粒
别称: Plasmid#89567
质粒属性
质粒简介
Lenti-dCas9-KRAB-blast质粒是一个慢病毒过表达质粒，EF-1α 启动子驱动dCas9-KRAB蛋白片段的表达，可用于哺乳细胞的基因编辑实验。本质粒的参考文献是：Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells.
质粒图谱
质粒序列
LOCUS Exported 14229 bp ds-DNA circular SYN 26-OCT-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14229)
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..14229
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 238..617
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 619..817
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2440..2557
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2687..3865
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 3881..3928
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 3929..8029
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the N863A mutation in the HNH
catalytic domain"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNL-SDAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 8084..8104
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8147..8341
/codon_start=1
/gene="ZNF10"
/gene_synonym="KOX1"
/product="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
/label=KRAB
/translation="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
QLTKPDVILRLEKGEEPWLV"
CDS 8408..8428
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8444..8497
/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 8498..8896
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=Blast
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
misc_feature 8921..9509
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 10034..10214
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 10246..10470
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 10516..10944
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 10958..11287
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 11138..11273
/label=SV40 ori
/note="SV40 origin of replication"
promoter 11335..11382
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 11401..11775
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=Zeo
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 11905..12026
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(12477..13065)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13236..14096)
/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"
promoter complement(14097..14201)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
2581 agatccagtt tggttaatta gctagctgca aagatggata aagttttaaa cagagaggaa
2641 tctttgcagc taatggacct tctaggtctt gaaaggagtg ggaattggct ccggtgcccg
2701 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
2761 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
2821 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
2881 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
2941 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct
3001 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
3061 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg
3121 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
3181 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
3241 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
3301 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
3361 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
3421 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
3481 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
3541 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
3601 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt
3661 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
3721 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
3781 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
3841 ttttcttcca tttcaggtgt cgtgacgtac ggccaccatg aaaaggccgg cggccacgaa
3901 aaaggccggc caggcaaaaa agaaaaagga caagaagtac agcatcggcc tggccatcgg
3961 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
4021 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
4081 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
4141 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
4201 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
4261 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
4321 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
4381 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
4441 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
4501 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
4561 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
4621 gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga cccccaactt
4681 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
4741 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
4801 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
4861 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
4921 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
4981 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
5041 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
5101 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
5161 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
5221 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
5281 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
5341 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
5401 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
5461 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
5521 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
5581 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
5641 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
5701 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
5761 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
5821 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
5881 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
5941 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
6001 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
6061 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
6121 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
6181 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
6241 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
6301 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
6361 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
6421 gctgtccgac tacgatgtgg accacatcgt gcctcagagc tttctgaagg acgactccat
6481 cgacaacaag gtgctgacca gaagcgacaa ggcccggggc aagagcgaca acgtgccctc
6541 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
6601 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
6661 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
6721 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
6781 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
6841 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
6901 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
6961 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
7021 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
7081 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
7141 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
7201 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
7261 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
7321 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
7381 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
7441 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
7501 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
7561 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
7621 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
7681 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
7741 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
7801 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
7861 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
7921 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
7981 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca gcgctggagg
8041 aggtggaagc ggaggaggag gaagcggagg aggaggtagc ggacctaaga aaaagaggaa
8101 ggtggcggcc gctggatccg atgctaagtc actgactgcc tggtcccgga cactggtgac
8161 cttcaaggat gtgtttgtgg acttcaccag ggaggagtgg aagctgctgg acactgctca
8221 gcagatcctg tacagaaatg tgatgctgga gaactataag aacctggttt ccttgggtta
8281 tcagcttact aagccagatg tgatcctccg gttggagaag ggagaagagc cctggctggt
8341 ggagagagaa attcaccaag agacccatcc tgattcagag actgcatttg aaatcaaatc
8401 atcagttccg aaaaagaaac gcaaagtttg tacaggcagt ggagagggca gaggaagtct
8461 gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg gccaagcctt tgtctcaaga
8521 agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga
8581 ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt
8641 atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc
8701 ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag
8761 cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt
8821 gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta
8881 tgtgtgggag ggctaagaat tcgatatcaa gcttatcggt aatcaacctc tggattacaa
8941 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
9001 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
9061 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
9121 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
9181 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
9241 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
9301 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
9361 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
9421 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
9481 tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgag acctagaaaa
9541 acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga
9601 agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat
9661 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
9721 gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg
9781 ctacttccct gattggcaga actacacacc agggccaggg atcagatatc cactgacctt
9841 tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg
9901 agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga
9961 agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca
10021 tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
10081 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
10141 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
10201 gaaaatctct agcagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
10261 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
10321 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
10381 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
10441 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
10501 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
10561 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
10621 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
10681 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
10741 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
10801 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
10861 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
10921 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
10981 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
11041 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
11101 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
11161 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
11221 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
11281 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac
11341 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc
11401 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc
11461 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt
11521 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac
11581 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag
11641 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag
11701 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc
11761 gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg
11821 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
11881 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
11941 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
12001 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
12061 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
12121 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
12181 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
12241 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
12301 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
12361 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
12421 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
12481 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
12541 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
12601 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
12661 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
12721 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
12781 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
12841 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
12901 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
12961 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
13021 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
13081 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
13141 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
13201 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
13261 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
13321 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
13381 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
13441 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
13501 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
13561 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
13621 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
13681 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
13741 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
13801 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
13861 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
13921 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
13981 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
14041 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
14101 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
14161 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
14221 ccacctgac
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
名称：Lenti-dCas9-KRAB-blast哺乳编辑质粒
别称: Plasmid#89567
| 启动子: | EF-1α promoter |
|---|---|
| 复制子: | pUC |
| 终止子: | WPRE |
| 质粒大小: | 14229bp |
| 原核抗性: | Amp |
| 筛选标记: | Blast |
| 克隆菌株: | Stbl3 |
| 培养条件: | 37度 |
质粒属性
| 载体宿主： | 哺乳细胞,慢病毒 |
|---|---|
| 载体用途： | 干扰沉默 |
| 基因种属： | |
| 基因类型： | CRISPR,Cas9 |
| 原核抗性： | Amp |
| 真核抗性： | Blast |
| 荧光蛋白： |
质粒简介
Lenti-dCas9-KRAB-blast质粒是一个慢病毒过表达质粒，EF-1α 启动子驱动dCas9-KRAB蛋白片段的表达，可用于哺乳细胞的基因编辑实验。本质粒的参考文献是：Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells.
质粒图谱
质粒序列
LOCUS Exported 14229 bp ds-DNA circular SYN 26-OCT-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14229)
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..14229
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 238..617
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 619..817
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2440..2557
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2687..3865
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 3881..3928
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 3929..8029
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the N863A mutation in the HNH
catalytic domain"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNL-SDAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 8084..8104
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8147..8341
/codon_start=1
/gene="ZNF10"
/gene_synonym="KOX1"
/product="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
/label=KRAB
/translation="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
QLTKPDVILRLEKGEEPWLV"
CDS 8408..8428
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8444..8497
/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 8498..8896
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=Blast
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
misc_feature 8921..9509
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 10034..10214
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 10246..10470
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 10516..10944
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 10958..11287
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 11138..11273
/label=SV40 ori
/note="SV40 origin of replication"
promoter 11335..11382
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 11401..11775
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=Zeo
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 11905..12026
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(12477..13065)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13236..14096)
/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"
promoter complement(14097..14201)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
2581 agatccagtt tggttaatta gctagctgca aagatggata aagttttaaa cagagaggaa
2641 tctttgcagc taatggacct tctaggtctt gaaaggagtg ggaattggct ccggtgcccg
2701 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
2761 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
2821 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
2881 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
2941 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct
3001 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
3061 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg
3121 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
3181 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
3241 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
3301 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
3361 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
3421 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
3481 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
3541 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
3601 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt
3661 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
3721 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
3781 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
3841 ttttcttcca tttcaggtgt cgtgacgtac ggccaccatg aaaaggccgg cggccacgaa
3901 aaaggccggc caggcaaaaa agaaaaagga caagaagtac agcatcggcc tggccatcgg
3961 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
4021 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
4081 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
4141 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
4201 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
4261 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
4321 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
4381 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
4441 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
4501 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
4561 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
4621 gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga cccccaactt
4681 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
4741 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
4801 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
4861 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
4921 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
4981 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
5041 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
5101 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
5161 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
5221 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
5281 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
5341 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
5401 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
5461 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
5521 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
5581 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
5641 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
5701 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
5761 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
5821 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
5881 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
5941 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
6001 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
6061 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
6121 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
6181 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
6241 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
6301 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
6361 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
6421 gctgtccgac tacgatgtgg accacatcgt gcctcagagc tttctgaagg acgactccat
6481 cgacaacaag gtgctgacca gaagcgacaa ggcccggggc aagagcgaca acgtgccctc
6541 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
6601 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
6661 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
6721 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
6781 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
6841 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
6901 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
6961 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
7021 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
7081 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
7141 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
7201 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
7261 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
7321 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
7381 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
7441 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
7501 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
7561 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
7621 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
7681 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
7741 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
7801 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
7861 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
7921 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
7981 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca gcgctggagg
8041 aggtggaagc ggaggaggag gaagcggagg aggaggtagc ggacctaaga aaaagaggaa
8101 ggtggcggcc gctggatccg atgctaagtc actgactgcc tggtcccgga cactggtgac
8161 cttcaaggat gtgtttgtgg acttcaccag ggaggagtgg aagctgctgg acactgctca
8221 gcagatcctg tacagaaatg tgatgctgga gaactataag aacctggttt ccttgggtta
8281 tcagcttact aagccagatg tgatcctccg gttggagaag ggagaagagc cctggctggt
8341 ggagagagaa attcaccaag agacccatcc tgattcagag actgcatttg aaatcaaatc
8401 atcagttccg aaaaagaaac gcaaagtttg tacaggcagt ggagagggca gaggaagtct
8461 gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg gccaagcctt tgtctcaaga
8521 agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga
8581 ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt
8641 atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc
8701 ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag
8761 cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt
8821 gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta
8881 tgtgtgggag ggctaagaat tcgatatcaa gcttatcggt aatcaacctc tggattacaa
8941 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
9001 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
9061 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
9121 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
9181 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
9241 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
9301 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
9361 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
9421 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
9481 tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgag acctagaaaa
9541 acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga
9601 agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat
9661 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
9721 gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg
9781 ctacttccct gattggcaga actacacacc agggccaggg atcagatatc cactgacctt
9841 tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg
9901 agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga
9961 agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca
10021 tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
10081 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
10141 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
10201 gaaaatctct agcagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
10261 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
10321 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
10381 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
10441 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
10501 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
10561 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
10621 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
10681 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
10741 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
10801 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
10861 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
10921 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
10981 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
11041 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
11101 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
11161 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
11221 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
11281 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac
11341 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc
11401 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc
11461 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt
11521 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac
11581 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag
11641 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag
11701 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc
11761 gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg
11821 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
11881 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
11941 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
12001 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
12061 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
12121 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
12181 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
12241 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
12301 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
12361 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
12421 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
12481 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
12541 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
12601 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
12661 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
12721 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
12781 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
12841 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
12901 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
12961 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
13021 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
13081 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
13141 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
13201 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
13261 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
13321 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
13381 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
13441 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
13501 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
13561 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
13621 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
13681 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
13741 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
13801 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
13861 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
13921 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
13981 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
14041 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
14101 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
14161 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
14221 ccacctgac
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
| P1377/pEGFP-DIS3人源基因质粒 |
| P1378/pEGFP-SAMD4A人源基因质粒 |
| P1379/pEGFP-DNMT3A人源基因质粒 |
| P1380/pEGFP-ARID5A人源基因质粒 |
| P1381/pEGFP-RC3H1人源基因质粒 |
| P1382/pEGFP-SMG6人源基因质粒 |
| P1389/pEGFP-RC3H2人源基因质粒 |
| P1392/pEGFP-SMG7人源基因质粒 |
| P1571/pEGFP-ADRB2人源基因质粒 |
| P1606/pEGFP-ADRA2A人源基因质粒 |
| P1425/pEGFP-ABCD1-T332G人源基因质粒 |
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器 CRISPRoff，其瞬时表达便可实现持久可遗传且可逆的 DNA 甲基化修饰和基因转录调控，有效地弥补了现有的转录调控 / 表观遗传编辑工具如 CRISPRi（dCas9-KRAB）及 dCas9-Dnmt3a（D3A）-Dnmt3L（D3L）的转录抑制效果缺乏持久性和可遗传性的功能短板！ CRISPR 系统的开发可谓是具有划时代意义的研究，望后续研究再接再厉为人类生命健康保驾护航！ 图 10. 来源 Cell本周推荐：三句话读懂一篇 CNS：宝玉拒绝黛玉的生物学原因；压力导致脱发的机制...参考
RNA 靶向酶。Cas13b 具有靶向和编辑 RNA 的能力，仅需一个指导 RNA 即可找到靶标，并可靶向多个 RNA 转录本。该系统将使研究人员可以特异性地操纵 RNA，并以高通量的方式使用 CRISPR 研究广泛的生物学过程。 目前，已发现于 2018 年发现的Cas13d 家族可干扰哺乳动物细胞系中的 RNA 以实现基因沉默。与 RNA 干扰技术相比，Cas13d 介导的基因沉默具有更高的特异性和敲除效率。与 Cas9 介导的基因敲除技术相比，Cas13d 介导的基因沉默不会改变基因组 DNA
提交可以由Sequin程序完成。Sequin程序能方便的编辑和处理复杂注释，并包含一系列内建的检查函数来提高序列的质量保证。它还被设计用于提交来自系统进化、种群和突变研究的序列，可以加入比对的数据。Sequin除了用于编辑和修改序列数据记录，还可以用于序列的分析，任何以FASTA或ASN.1格式序列为输入数据的序列分析程序都可以整合到Sequin程序下。在不同操作系统下运行的Sequin程序都可以在ftp://ncbi.nlm.nih.gov/sequin/ 下找到，Sequin的使用说明可详见其网页。
Lenti-dCas9-KRAB-blast哺乳编辑质粒
￥100 - 1000
