pML104酵母编辑质粒

基本信息
名称：pML104酵母编辑质粒
别称:Plasmid#67638

原核抗性:	Amp
筛选标记:	URA3
克隆菌株:	Stbl3
培养条件:	37度

质粒属性

载体宿主：	酵母菌
载体用途：	基因敲除
基因种属：
基因类型：
原核抗性：	Amp
筛选标记：	URA3
荧光蛋白：

质粒简介
  pML104是一个酵母细胞CAS9和gRNA表达基因敲除质粒。

质粒图谱


质粒序列
LOCUS       Exported               11240 bp ds-DNA     circular SYN 03-DEC-2017
DEFINITION  synthetic circular DNA
FEATURES             Location/Qualifiers
     source          1..11240
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1..4113
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNL-SDAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEKEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGDEGA"
     CDS             4114..4134
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      4162..4349
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     terminator      4467..4486
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(4568..4836)
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     rep_origin      complement(5308..5896)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6067..6927)
                     /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(6928..7032)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7059..8401
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        8663..8878
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             8879..9682
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(9813..10268)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        10540..11206
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase"
ORIGIN
        1 atggacaaga agtattctat cggactggac atcgggacta atagcgtcgg gtgggccgtc
       61 atcactgacg agtacaaggt gccctctaag aagttcaagg tgctcgggaa caccgaccgg
      121 cattccatca agaaaaatct gatcggagct ctcctctttg attcagggga gaccgctgaa
      181 gcaacccgcc tcaagcggac tgctagacgg cggtacacca ggaggaagaa ccggatttgt
      241 taccttcaag agatattctc caacgaaatg gcaaaggtcg acgacagctt cttccatagg
      301 ctggaagaat cattcctcgt ggaagaggat aagaagcatg aacggcatcc catcttcggt
      361 aatatcgtcg acgaggtggc ctatcacgag aaatacccaa ccatctacca tcttcgcaaa
      421 aagctggtgg actcaaccga caaggcagac ctccggctta tctacctggc cctggcccac
      481 atgattaagt tcagaggcca cttcctgatc gagggcgacc tcaatcctga caatagcgat
      541 gtggataaac tgttcatcca gctggtgcag acttacaacc agctctttga agagaacccc
      601 atcaatgcaa gcggagtcga tgccaaggcc attctgtcag cccggctgtc aaagagccgc
      661 agacttgaga atcttatcgc tcagctgccg ggtgaaaaga aaaatggact gttcgggaac
      721 ctgattgctc tttcacttgg gctgactccc aatttcaagt ctaatttcga cctggcagag
      781 gatgccaagc tgcaactgtc caaggacacc tatgatgacg atctcgacaa cctcctggcc
      841 cagatcggtg accaatacgc cgaccttttc cttgctgcta agaatctttc tgacgccatc
      901 ctgctgtctg acattctccg cgtgaacact gaaatcacca aggcccctct ttcagcttca
      961 atgattaagc ggtatgatga gcaccaccag gacctgaccc tgcttaaggc actcgtccgg
     1021 cagcagcttc cggagaagta caaggaaatc ttctttgacc agtcaaagaa tggatacgcc
     1081 ggctacatcg acggaggtgc ctcccaagag gaattttata agtttatcaa acctatcctt
     1141 gagaagatgg acggcaccga agagctcctc gtgaaactga atcgggagga tctgctgcgg
     1201 aagcagcgca ctttcgacaa tgggagcatt ccccaccaga tccatcttgg ggagcttcac
     1261 gccatccttc ggcgccaaga ggacttctac ccctttctta aggacaacag ggagaagatt
     1321 gagaaaattc tcactttccg catcccctac tacgtgggac ccctcgccag aggaaatagc
     1381 cggtttgctt ggatgaccag aaagtcagaa gaaactatca ctccctggaa cttcgaagag
     1441 gtggtggaca agggagccag cgctcagtca ttcatcgaac ggatgactaa cttcgataag
     1501 aacctcccca atgagaaggt cctgccgaaa cattccctgc tctacgagta ctttaccgtg
     1561 tacaacgagc tgaccaaggt gaaatatgtc accgaaggga tgaggaagcc cgcattcctg
     1621 tcaggcgaac aaaagaaggc aattgtggac cttctgttca agaccaatag aaaggtgacc
     1681 gtgaagcagc tgaaggagga ctatttcaag aaaattgaat gcttcgactc tgtggagatt
     1741 agcggggtcg aagatcggtt caacgcaagc ctgggtacct accatgatct gcttaagatc
     1801 atcaaggaca aggattttct ggacaatgag gagaaagagg acatccttga ggacattgtc
     1861 ctgactctca ctctgttcga ggaccgggaa atgatcgagg agaggcttaa gacctacgcc
     1921 catctgttcg acgataaagt gatgaagcaa cttaaacgga gaagatatac cggatgggga
     1981 cgccttagcc gcaaactcat caacggaatc cgggacaaac agagcggaaa gaccattctt
     2041 gatttcctta agagcgacgg attcgctaat cgcaacttca tgcaacttat ccatgatgat
     2101 tccctgacct ttaaggagga catccagaag gcccaagtgt ctggacaagg tgactcactg
     2161 cacgagcata tcgcaaatct ggctggttca cccgctatta agaagggtat tctccagacc
     2221 gtgaaagtcg tggacgagct ggtcaaggtg atgggtcgcc ataaaccaga gaacattgtc
     2281 atcgagatgg ccagggaaaa ccagactacc cagaagggac agaagaacag cagggagcgg
     2341 atgaaaagaa ttgaggaagg gattaaggag ctcgggtcac agatccttaa agagcacccg
     2401 gtggaaaaca cccagcttca gaatgagaag ctctatctgt actaccttca aaatggacgc
     2461 gatatgtatg tggaccaaga gcttgatatc aacaggctct cagactacga cgtggaccac
     2521 atcgtccctc agagcttcct caaagacgac tcaattgaca ataaggtgct gactcgctca
     2581 gacaagaacc ggggaaagtc agataacgtg ccctcagagg aagtcgtgaa aaagatgaag
     2641 aactattggc gccagcttct gaacgcaaag ctaatcactc agcggaagtt cgacaatctc
     2701 actaaggctg agaggggcgg actgagcgaa ctggacaaag caggattcat taaacggcaa
     2761 cttgtggaga ctcggcagat tactaaacat gtagcccaaa tccttgactc acgcatgaat
     2821 accaagtacg acgaaaacga caaacttatc cgcgaggtga aggtgattac cctgaagtcc
     2881 aagctggtca gcgatttcag aaaggacttt caattctaca aagtgcggga gatcaataac
     2941 tatcatcatg ctcatgacgc atatctgaat gccgtggtgg gaaccgccct aatcaagaag
     3001 tacccaaagc tggaaagcga gttcgtgtac ggagactaca aggtctacga cgtgcgcaag
     3061 atgattgcca aatctgagca ggagatcgga aaggccaccg caaagtactt cttctacagc
     3121 aacatcatga atttcttcaa gaccgaaatc acccttgcaa acggtgagat ccggaagagg
     3181 ccgctcatcg agactaatgg ggagactggc gaaatcgtgt gggacaaggg cagagatttc
     3241 gctaccgtgc gcaaagtgct ttctatgcct caagtgaaca tcgtgaagaa aaccgaggtg
     3301 caaaccggag gcttttctaa ggaatcaatc ctccccaagc gcaactccga caagctcatt
     3361 gcaaggaaga aggattggga ccctaagaag tacggcggat tcgattcacc aactgtggct
     3421 tattctgtcc tggtcgtggc taaggtggaa aaaggaaagt ctaagaagct caagagcgtg
     3481 aaggaactgc tgggtatcac cattatggag cgcagctcct tcgagaagaa cccaattgac
     3541 tttctcgaag ccaaaggtta caaggaagtc aagaaggacc ttatcatcaa gctcccaaag
     3601 tatagcctgt tcgaactgga gaatgggcgg aagcggatgc tcgcctccgc tggcgaactt
     3661 cagaagggta atgagctggc tctcccctcc aagtacgtga atttcctcta ccttgcaagc
     3721 cattacgaga agctgaaggg gagccccgag gacaacgagc aaaagcaact gtttgtggag
     3781 cagcataagc attatctgga cgagatcatt gagcagattt ccgagttttc taaacgcgtc
     3841 attctcgctg atgccaacct cgataaagtc cttagcgcat acaataagca cagagacaaa
     3901 ccaattcggg agcaggctga gaatatcatc cacctgttca ccctcaccaa tcttggtgcc
     3961 cctgccgcat tcaagtactt cgacaccacc atcgaccgga aacgctatac ctccaccaaa
     4021 gaagtgctgg acgccaccct catccaccag agcatcaccg gactttacga aactcggatt
     4081 gacctctcac agctcggagg ggatgaggga gctcccaaga aaaagcgcaa ggtaggtagt
     4141 tccggatcct agactctcga ggcgaatttc ttatgattta tgatttttat tattaaataa
     4201 gttataaaaa aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa
     4261 ttcttattct tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagcatgag
     4321 gtcgctctta ttgaccacac ctctaccggc atgccgagca aatgcctgca aatcgctccc
     4381 catttctcta gagcggccgt ggtatcgttt agattggcaa ttacagtgtc ttagctcaca
     4441 tgcttataac taattacatg actcgaagac ataaaaaaca aaaaaagcac caccgactcg
     4501 gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat ttaaatgcat
     4561 cgattctgat catttatctt tcactgcgga gaagtttcga acgccgaaac atgcgcacca
     4621 actttcactt ctacagcgtt tgaccaaaat cttttgaaca gaacattgta gggtgtgaaa
     4681 aaatgcgcac ctttaccgct agcccaagag ggcactacaa aatctagagt tgtacttcaa
     4741 acgtacatgt aatcaccttg tatatactcg aaagaaaaca tcaagtttct gtataaatat
     4801 gagtgaaagc ataatcatac attatctttt caaagattgt ccgcggtgga gctccagctt
     4861 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     4921 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4981 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5041 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5101 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5161 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5221 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5281 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5341 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5401 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5461 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5521 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5581 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5641 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5701 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5761 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5821 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5881 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5941 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6001 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6061 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6121 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6181 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6241 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6301 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6361 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6421 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6481 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6541 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6601 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6661 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6721 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6781 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6841 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6901 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6961 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7021 aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt
     7081 ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca
     7141 tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt
     7201 catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag
     7261 ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta
     7321 tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc
     7381 ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc
     7441 actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa
     7501 aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt
     7561 tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga
     7621 acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct
     7681 attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca
     7741 ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat
     7801 aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt
     7861 tatataggga tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg
     7921 aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga
     7981 aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta
     8041 gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa
     8101 atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt
     8161 gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta
     8221 cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc
     8281 cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct
     8341 gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt gatattggat
     8401 catactaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     8461 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     8521 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     8581 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     8641 tgagagtgca ccataccacc ttttcaattc atcatttttt ttttattctt ttttttgatt
     8701 tcggtttcct tgaaattttt ttgattcggt aatctccgaa cagaaggaag aacgaaggaa
     8761 ggagcacaga cttagattgg tatatatacg catatgtagt gttgaagaaa catgaaattg
     8821 cccagtattc ttaacccaac tgcacagaac aaaaacctgc aggaaacgaa gataaatcat
     8881 gtcgaaagct acatataagg aacgtgctgc tactcatcct agtcctgttg ctgccaagct
     8941 atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg ttcgtaccac
     9001 caaggaatta ctggagttag ttgaagcatt aggtcccaaa atttgtttac taaaaacaca
     9061 tgtggatatc ttgactgatt tttccatgga gggcacagtt aagccgctaa aggcattatc
     9121 cgccaagtac aattttttac tcttcgaaga cagaaaattt gctgacattg gtaatacagt
     9181 caaattgcag tactctgcgg gtgtatacag aatagcagaa tgggcagaca ttacgaatgc
     9241 acacggtgtg gtgggcccag gtattgttag cggtttgaag caggcggcag aagaagtaac
     9301 aaaggaacct agaggccttt tgatgttagc agaattgtca tgcaagggct ccctatctac
     9361 tggagaatat actaagggta ctgttgacat tgcgaagagc gacaaagatt ttgttatcgg
     9421 ctttattgct caaagagaca tgggtggaag agatgaaggt tacgattggt tgattatgac
     9481 acccggtgtg ggtttagatg acaagggaga cgcattgggt caacagtata gaaccgtgga
     9541 tgatgtggtc tctacaggat ctgacattat tattgttgga agaggactat ttgcaaaggg
     9601 aagggatgct aaggtagagg gtgaacgtta cagaaaagca ggctgggaag catatttgag
     9661 aagatgcggc cagcaaaact aaaaaactgt attataagta aatgcatgta tactaaactc
     9721 acaaattaga gcttcaattt aattatatca gttattaccc tatgcggtgt gaaataccgc
     9781 acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa
     9841 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     9901 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     9961 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
    10021 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
    10081 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
    10141 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
    10201 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
    10261 gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
    10321 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
    10381 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac
    10441 gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcgacgg tatcgataag
    10501 cttgatatcg aattcagtct actagtcagt tcgagtttat cattatcaat actgccattt
    10561 caaagaatac gtaaataatt aatagtagtg attttcctaa ctttatttag tcaaaaaatt
    10621 agccttttaa ttctgctgta acccgtacat gcccaaaata gggggcgggt tacacagaat
    10681 atataacatc gtaggtgtct gggtgaacag tttattcctg gcatccacta aatataatgg
    10741 agcccgcttt ttaagctggc atccagaaaa aaaaagaatc ccagcaccaa aatattgttt
    10801 tcttcaccaa ccatcagttc ataggtccat tctcttagcg caactacaga gaacaggggc
    10861 acaaacaggc aaaaaacggg cacaacctca atggagtgat gcaacctgcc tggagtaaat
    10921 gatgacacaa ggcaattgac ccacgcatgt atctatctca ttttcttaca ccttctatta
    10981 ccttctgctc tctctgattt ggaaaaagct gaaaaaaaag gttgaaacca gttccctgaa
    11041 attattcccc tacttgacta ataagtatat aaagacggta ggtattgatt gtaattctgt
    11101 aaatctattt cttaaacttc ttaaattcta cttttatagt tagtcttttt tttagtttta
    11161 aaacaccaag aacttagttt cgaataaaca cacataaaca aacaaaagat ctgatatgga
    11221 tcgaattaga tctcgccacc
//

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

P3053/pENTR223-CMIP（476-2322bp）人源基因质粒

P3054/pENTR223-ACSF2（1同义突变）人源基因质粒

P3073/pENTR223-P3H2人源基因质粒

P3074/pENTR223-DBR1（1点突变1同义突变）人源基因质粒

P3078/pENTR223-IP6K2人源基因质粒

P3079/pENTR223-ENY2人源基因质粒

P3080/pENTR223-HIST2H4A人源基因质粒

P3081/pENTR223-MANBAL人源基因质粒

P3082/pENTR223-C16orf57人源基因质粒

P3083/pENTR223-ACBD7(一同义突变）人源基因质粒

P3084/pENTR223-GNG4人源基因质粒