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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#47107
- 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：pCDNA-dCas9-VP64哺乳编辑质粒
别称: Plasmid#47107
质粒属性
质粒简介
pCDNA-dCas9-VP64质粒是CRISPR/Cas9系列质粒，复制子是f1 ori、ori和SV40 ori，质粒大小是9812bp，带有Ampicillin氨苄青霉素抗性，可转化进Stbl3中培养，培养条件是LB培养基，37℃。
质粒图谱
质粒序列
LOCUS Exported 9812 bp ds-DNA circular SYN 29-JUN-2017
DEFINITION synthetic circular DNA
KEYWORDS pcDNA-dCas9-VP64
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9812)
TITLE Direct Submission
JOURNAL Exported Thursday, June 29, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..9812
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 235..614
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 863..881
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 907..972
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 979..999
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1009..5112
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/note="dCas9"
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10 mutation in the
RuvC-like domain and the H840A mutation in the HNH domain"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNL-SDAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRL-SDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 5137..5157
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 5167..5316
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/note="VP64"
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 5323..5349
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/note="HA"
/translation="YPYDVPDYA"
polyA_signal 5412..5636
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 5682..6110
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6124..6453
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 6304..6439
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 6520..7314
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 7488..7609
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(7658..7674)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 7682..7698
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/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)."
promoter complement(7706..7736)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 7751..7772
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(8060..8645)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8816..9676)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(9677..9781)
/gene="bla"
/note="AmpR promoter"
ORIGIN
1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
901 gccatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
961 gacgatgaca agatggcccc caagaagaag aggaaggtgg gccgcggaat ggataagaaa
1021 tactcaatag gcttagctat cggcacaaat agcgtcggat gggcggtgat cactgatgaa
1081 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa
1141 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc
1201 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag
1261 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct
1321 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat
1381 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat
1441 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt
1501 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta
1561 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt
1621 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat
1681 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg
1741 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta
1801 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat
1861 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat
1921 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc
1981 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca
2041 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat
2101 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat
2161 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc
2221 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga
2281 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg
2341 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg
2401 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa
2461 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat
2521 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg
2581 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag
2641 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta
2701 aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa
2761 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa
2821 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc
2881 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat
2941 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga
3001 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa
3061 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt
3121 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt
3181 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt
3241 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca
3301 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc
3361 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact
3421 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg
3481 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatgccat tgttccacaa
3541 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt
3601 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga
3661 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa
3721 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact
3781 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat
3841 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct
3901 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc
3961 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt
4021 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag
4081 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac
4141 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa
4201 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc
4261 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga
4321 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa
4381 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta
4441 gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta
4501 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct
4561 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt
4621 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat
4681 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag
4741 ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat
4801 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat
4861 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa
4921 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt
4981 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat
5041 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag
5101 ctaggaggtg acccaattgc cggatccaag gctagcccga aaaagaaacg caaagttggg
5161 cgcgccgacg cgctggacga tttcgatctc gacatgctgg gttctgatgc cctcgatgac
5221 tttgacctgg atatgttggg aagcgacgca ttggatgact ttgatctgga catgctcggc
5281 tccgatgctc tggacgattt cgatctcgat atgttaatta actacccgta cgacgttccg
5341 gactacgctt cttgaatcgg taggaattcg cggccgtcta gacttaagtt taaaccgctg
5401 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
5461 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
5521 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
5581 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
5641 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
5701 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
5761 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
5821 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
5881 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
5941 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
6001 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
6061 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
6121 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
6181 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
6241 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
6301 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
6361 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
6421 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
6481 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
6541 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
6601 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
6661 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
6721 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
6781 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
6841 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
6901 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
6961 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
7021 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
7081 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
7141 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
7201 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
7261 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
7321 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
7381 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
7441 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
7501 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
7561 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
7621 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
7681 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
7741 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
7801 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
7861 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
7921 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
7981 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
8041 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
8101 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
8161 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
8221 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
8281 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
8341 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
8401 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
8461 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
8521 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
8581 ccaccgctgg tagcggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
8641 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
8701 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
8761 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
8821 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
8881 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
8941 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
9001 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
9061 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
9121 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
9181 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
9241 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
9301 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
9361 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
9421 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
9481 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
9541 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
9601 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
9661 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
9721 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
9781 gcacatttcc ccgaaaagtg ccacctgacg tc
//
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：pCDNA-dCas9-VP64哺乳编辑质粒
别称: Plasmid#47107
| 启动子: | CMV |
|---|---|
| 复制子: | pUC |
| 终止子: | bGH poly(A) signal |
| 质粒分类: | 哺乳细胞质粒；哺乳编辑质粒；哺乳Cas9质粒 |
| 质粒大小: | 9812bp |
| 质粒标签: | N-3×Flag,N-NLS,C-VP64,C-HA |
| 原核抗性: | Amp |
| 筛选标记: | G418 |
| 克隆菌株: | Stbl3 |
| 培养条件: | 37度 |
| 表达宿主: | 293T等哺乳细胞 |
| 培养条件: | 37℃，5%CO2 |
| 诱导方式: | 无需诱导 |
| 5'测序引物: | CMV-F(CGCAAATGGGCGGTAGGCGTG) |
| 3'测序引物: | BGH-R（TAGAAGGCACAGTCGAGG） |
质粒属性
| 载体宿主： | 哺乳细胞 |
|---|---|
| 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： | Neo/G418 |
| 荧光蛋白： |
质粒简介
pCDNA-dCas9-VP64质粒是CRISPR/Cas9系列质粒，复制子是f1 ori、ori和SV40 ori，质粒大小是9812bp，带有Ampicillin氨苄青霉素抗性，可转化进Stbl3中培养，培养条件是LB培养基，37℃。
质粒图谱
质粒序列
LOCUS Exported 9812 bp ds-DNA circular SYN 29-JUN-2017
DEFINITION synthetic circular DNA
KEYWORDS pcDNA-dCas9-VP64
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9812)
TITLE Direct Submission
JOURNAL Exported Thursday, June 29, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..9812
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 235..614
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 863..881
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 907..972
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 979..999
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1009..5112
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/note="dCas9"
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10 mutation in the
RuvC-like domain and the H840A mutation in the HNH domain"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNL-SDAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRL-SDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 5137..5157
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 5167..5316
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/note="VP64"
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 5323..5349
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/note="HA"
/translation="YPYDVPDYA"
polyA_signal 5412..5636
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 5682..6110
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6124..6453
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 6304..6439
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 6520..7314
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 7488..7609
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(7658..7674)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 7682..7698
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/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)."
promoter complement(7706..7736)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 7751..7772
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(8060..8645)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8816..9676)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(9677..9781)
/gene="bla"
/note="AmpR promoter"
ORIGIN
1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
901 gccatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
961 gacgatgaca agatggcccc caagaagaag aggaaggtgg gccgcggaat ggataagaaa
1021 tactcaatag gcttagctat cggcacaaat agcgtcggat gggcggtgat cactgatgaa
1081 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa
1141 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc
1201 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag
1261 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct
1321 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat
1381 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat
1441 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt
1501 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta
1561 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt
1621 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat
1681 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg
1741 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta
1801 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat
1861 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat
1921 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc
1981 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca
2041 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat
2101 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat
2161 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc
2221 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga
2281 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg
2341 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg
2401 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa
2461 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat
2521 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg
2581 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag
2641 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta
2701 aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa
2761 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa
2821 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc
2881 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat
2941 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga
3001 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa
3061 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt
3121 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt
3181 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt
3241 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca
3301 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc
3361 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact
3421 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg
3481 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatgccat tgttccacaa
3541 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt
3601 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga
3661 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa
3721 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact
3781 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat
3841 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct
3901 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc
3961 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt
4021 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag
4081 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac
4141 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa
4201 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc
4261 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga
4321 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa
4381 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta
4441 gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta
4501 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct
4561 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt
4621 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat
4681 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag
4741 ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat
4801 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat
4861 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa
4921 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt
4981 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat
5041 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag
5101 ctaggaggtg acccaattgc cggatccaag gctagcccga aaaagaaacg caaagttggg
5161 cgcgccgacg cgctggacga tttcgatctc gacatgctgg gttctgatgc cctcgatgac
5221 tttgacctgg atatgttggg aagcgacgca ttggatgact ttgatctgga catgctcggc
5281 tccgatgctc tggacgattt cgatctcgat atgttaatta actacccgta cgacgttccg
5341 gactacgctt cttgaatcgg taggaattcg cggccgtcta gacttaagtt taaaccgctg
5401 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
5461 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
5521 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
5581 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
5641 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
5701 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
5761 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
5821 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
5881 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
5941 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
6001 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
6061 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
6121 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
6181 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
6241 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
6301 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
6361 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
6421 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
6481 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
6541 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
6601 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
6661 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
6721 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
6781 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
6841 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
6901 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
6961 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
7021 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
7081 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
7141 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
7201 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
7261 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
7321 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
7381 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
7441 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
7501 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
7561 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
7621 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
7681 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
7741 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
7801 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
7861 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
7921 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
7981 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
8041 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
8101 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
8161 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
8221 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
8281 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
8341 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
8401 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
8461 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
8521 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
8581 ccaccgctgg tagcggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
8641 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
8701 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
8761 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
8821 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
8881 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
8941 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
9001 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
9061 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
9121 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
9181 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
9241 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
9301 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
9361 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
9421 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
9481 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
9541 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
9601 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
9661 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
9721 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
9781 gcacatttcc ccgaaaagtg ccacctgacg tc
//
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
| P1264/pLVX-TRE3G-IDH1-Zsgreen1人源基因质粒 |
| P1297/pLVX-TRE3G-IDH2-ZsGreen1人源基因质粒 |
| P1298/pLVX-TRE3G-IDH2-R172K-Zsgreen1人源基因质粒 |
| P1305/pLVX-IRE3G-IDH2-R140Q-ZsGreen1人源基因质粒 |
| P1271/pCDNA3.1-PPP3cb-EGFP人源基因质粒 |
| P0122/pEGFP-TYR人源基因质粒 |
| P1311/ptyrEGFP-TYR人源基因质粒 |
| P1312/pTriEx-mCherry-PA-Rac1人源基因质粒 |
| P1313/pTriEx-mCherry-PA-Rac1-T17N人源基因质粒 |
| P1314/FU-tet-o-hOct4人源基因质粒 |
| P1338/pCI-neo.mCherry-NEDD4人源基因质粒 |
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pCDNA-dCas9-VP64哺乳编辑质粒
￥100 - 1000
