pML107酵母编辑质粒

基本信息
名称：pML107酵母编辑质粒
别称: Plasmid#67639

原核抗性:	Amp
筛选标记:	LEU2
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：	LEU2
荧光蛋白：

质粒简介
 pML107是一个酵母CAS9和gRNA表达质粒。

质粒图谱


质粒序列
LOCUS       Exported               12367 bp ds-DNA     circular SYN 06-DEC-2018
KEYWORDS    pML107
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12367)
  AUTHORS   Triple Threat
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..12367
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        132..798
                     /gene="S. cerevisiae TDH3"
                     /note="GAP promoter"
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase"
     CDS             833..4945
                     /codon_start=1
                     /note="Cas9"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNL-SDAILLS-DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEKEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLS-DYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGDEGA"
     CDS             4946..4966
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     terminator      4994..5181
                     /gene="S. cerevisiae ADH1"
                     /note="ADH1 terminator"
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     terminator      5299..5318
                     /note="SUP4 terminator"
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(5400..5668)
                     /note="SNR52 promoter"
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     rep_origin      complement(6140..6728)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6899..7759)
                     /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(7760..7864)
                     /gene="bla"
                     /note="AmpR promoter"
     rep_origin      7891..9237
                     /note="2u ori"
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(9969..11063)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /note="LEU2"
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(11064..11471)
                     /gene="S. cerevisiae LEU2"
                     /note="LEU2 promoter"
     rep_origin      complement(11772..12227)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg
       61 ggccccccct cgaggtcgac ggtatcgata agcttgatat cgaattcagt ctactagtca
      121 gttcgagttt atcattatca atactgccat ttcaaagaat acgtaaataa ttaatagtag
      181 tgattttcct aactttattt agtcaaaaaa ttagcctttt aattctgctg taacccgtac
      241 atgcccaaaa tagggggcgg gttacacaga atatataaca tcgtaggtgt ctgggtgaac
      301 agtttattcc tggcatccac taaatataat ggagcccgct ttttaagctg gcatccagaa
      361 aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc aaccatcagt tcataggtcc
      421 attctcttag cgcaactaca gagaacaggg gcacaaacag gcaaaaaacg ggcacaacct
      481 caatggagtg atgcaacctg cctggagtaa atgatgacac aaggcaattg acccacgcat
      541 gtatctatct cattttctta caccttctat taccttctgc tctctctgat ttggaaaaag
      601 ctgaaaaaaa aggttgaaac cagttccctg aaattattcc cctacttgac taataagtat
      661 ataaagacgg taggtattga ttgtaattct gtaaatctat ttcttaaact tcttaaattc
      721 tacttttata gttagtcttt tttttagttt taaaacacca agaacttagt ttcgaataaa
      781 cacacataaa caaacaaaag atctgatatg gatcgaatta gatctcgcca ccatggacaa
      841 gaagtattct atcggactgg acatcgggac taatagcgtc gggtgggccg tcatcactga
      901 cgagtacaag gtgccctcta agaagttcaa ggtgctcggg aacaccgacc ggcattccat
      961 caagaaaaat ctgatcggag ctctcctctt tgattcaggg gagaccgctg aagcaacccg
     1021 cctcaagcgg actgctagac ggcggtacac caggaggaag aaccggattt gttaccttca
     1081 agagatattc tccaacgaaa tggcaaaggt cgacgacagc ttcttccata ggctggaaga
     1141 atcattcctc gtggaagagg ataagaagca tgaacggcat cccatcttcg gtaatatcgt
     1201 cgacgaggtg gcctatcacg agaaataccc aaccatctac catcttcgca aaaagctggt
     1261 ggactcaacc gacaaggcag acctccggct tatctacctg gccctggccc acatgattaa
     1321 gttcagaggc cacttcctga tcgagggcga cctcaatcct gacaatagcg atgtggataa
     1381 actgttcatc cagctggtgc agacttacaa ccagctcttt gaagagaacc ccatcaatgc
     1441 aagcggagtc gatgccaagg ccattctgtc agcccggctg tcaaagagcc gcagacttga
     1501 gaatcttatc gctcagctgc cgggtgaaaa gaaaaatgga ctgttcggga acctgattgc
     1561 tctttcactt gggctgactc ccaatttcaa gtctaatttc gacctggcag aggatgccaa
     1621 gctgcaactg tccaaggaca cctatgatga cgatctcgac aacctcctgg cccagatcgg
     1681 tgaccaatac gccgaccttt tccttgctgc taagaatctt tctgacgcca tcctgctgtc
     1741 tgacattctc cgcgtgaaca ctgaaatcac caaggcccct ctttcagctt caatgattaa
     1801 gcggtatgat gagcaccacc aggacctgac cctgcttaag gcactcgtcc ggcagcagct
     1861 tccggagaag tacaaggaaa tcttctttga ccagtcaaag aatggatacg ccggctacat
     1921 cgacggaggt gcctcccaag aggaatttta taagtttatc aaacctatcc ttgagaagat
     1981 ggacggcacc gaagagctcc tcgtgaaact gaatcgggag gatctgctgc ggaagcagcg
     2041 cactttcgac aatgggagca ttccccacca gatccatctt ggggagcttc acgccatcct
     2101 tcggcgccaa gaggacttct acccctttct taaggacaac agggagaaga ttgagaaaat
     2161 tctcactttc cgcatcccct actacgtggg acccctcgcc agaggaaata gccggtttgc
     2221 ttggatgacc agaaagtcag aagaaactat cactccctgg aacttcgaag aggtggtgga
     2281 caagggagcc agcgctcagt cattcatcga acggatgact aacttcgata agaacctccc
     2341 caatgagaag gtcctgccga aacattccct gctctacgag tactttaccg tgtacaacga
     2401 gctgaccaag gtgaaatatg tcaccgaagg gatgaggaag cccgcattcc tgtcaggcga
     2461 acaaaagaag gcaattgtgg accttctgtt caagaccaat agaaaggtga ccgtgaagca
     2521 gctgaaggag gactatttca agaaaattga atgcttcgac tctgtggaga ttagcggggt
     2581 cgaagatcgg ttcaacgcaa gcctgggtac ctaccatgat ctgcttaaga tcatcaagga
     2641 caaggatttt ctggacaatg aggagaaaga ggacatcctt gaggacattg tcctgactct
     2701 cactctgttc gaggaccggg aaatgatcga ggagaggctt aagacctacg cccatctgtt
     2761 cgacgataaa gtgatgaagc aacttaaacg gagaagatat accggatggg gacgccttag
     2821 ccgcaaactc atcaacggaa tccgggacaa acagagcgga aagaccattc ttgatttcct
     2881 taagagcgac ggattcgcta atcgcaactt catgcaactt atccatgatg attccctgac
     2941 ctttaaggag gacatccaga aggcccaagt gtctggacaa ggtgactcac tgcacgagca
     3001 tatcgcaaat ctggctggtt cacccgctat taagaagggt attctccaga ccgtgaaagt
     3061 cgtggacgag ctggtcaagg tgatgggtcg ccataaacca gagaacattg tcatcgagat
     3121 ggccagggaa aaccagacta cccagaaggg acagaagaac agcagggagc ggatgaaaag
     3181 aattgaggaa gggattaagg agctcgggtc acagatcctt aaagagcacc cggtggaaaa
     3241 cacccagctt cagaatgaga agctctatct gtactacctt caaaatggac gcgatatgta
     3301 tgtggaccaa gagcttgata tcaacaggct ctcagactac gacgtggacc acatcgtccc
     3361 tcagagcttc ctcaaagacg actcaattga caataaggtg ctgactcgct cagacaagaa
     3421 ccggggaaag tcagataacg tgccctcaga ggaagtcgtg aaaaagatga agaactattg
     3481 gcgccagctt ctgaacgcaa agctaatcac tcagcggaag ttcgacaatc tcactaaggc
     3541 tgagaggggc ggactgagcg aactggacaa agcaggattc attaaacggc aacttgtgga
     3601 gactcggcag attactaaac atgtagccca aatccttgac tcacgcatga ataccaagta
     3661 cgacgaaaac gacaaactta tccgcgaggt gaaggtgatt accctgaagt ccaagctggt
     3721 cagcgatttc agaaaggact ttcaattcta caaagtgcgg gagatcaata actatcatca
     3781 tgctcatgac gcatatctga atgccgtggt gggaaccgcc ctaatcaaga agtacccaaa
     3841 gctggaaagc gagttcgtgt acggagacta caaggtctac gacgtgcgca agatgattgc
     3901 caaatctgag caggagatcg gaaaggccac cgcaaagtac ttcttctaca gcaacatcat
     3961 gaatttcttc aagaccgaaa tcacccttgc aaacggtgag atccggaaga ggccgctcat
     4021 cgagactaat ggggagactg gcgaaatcgt gtgggacaag ggcagagatt tcgctaccgt
     4081 gcgcaaagtg ctttctatgc ctcaagtgaa catcgtgaag aaaaccgagg tgcaaaccgg
     4141 aggcttttct aaggaatcaa tcctccccaa gcgcaactcc gacaagctca ttgcaaggaa
     4201 gaaggattgg gaccctaaga agtacggcgg attcgattca ccaactgtgg cttattctgt
     4261 cctggtcgtg gctaaggtgg aaaaaggaaa gtctaagaag ctcaagagcg tgaaggaact
     4321 gctgggtatc accattatgg agcgcagctc cttcgagaag aacccaattg actttctcga
     4381 agccaaaggt tacaaggaag tcaagaagga ccttatcatc aagctcccaa agtatagcct
     4441 gttcgaactg gagaatgggc ggaagcggat gctcgcctcc gctggcgaac ttcagaaggg
     4501 taatgagctg gctctcccct ccaagtacgt gaatttcctc taccttgcaa gccattacga
     4561 gaagctgaag gggagccccg aggacaacga gcaaaagcaa ctgtttgtgg agcagcataa
     4621 gcattatctg gacgagatca ttgagcagat ttccgagttt tctaaacgcg tcattctcgc
     4681 tgatgccaac ctcgataaag tccttagcgc atacaataag cacagagaca aaccaattcg
     4741 ggagcaggct gagaatatca tccacctgtt caccctcacc aatcttggtg cccctgccgc
     4801 attcaagtac ttcgacacca ccatcgaccg gaaacgctat acctccacca aagaagtgct
     4861 ggacgccacc ctcatccacc agagcatcac cggactttac gaaactcgga ttgacctctc
     4921 acagctcgga ggggatgagg gagctcccaa gaaaaagcgc aaggtaggta gttccggatc
     4981 ctagactctc gaggcgaatt tcttatgatt tatgattttt attattaaat aagttataaa
     5041 aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt
     5101 cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagcatg aggtcgctct
     5161 tattgaccac acctctaccg gcatgccgag caaatgcctg caaatcgctc cccatttctc
     5221 tagagcggcc gtggtatcgt ttagattggc aattacagtg tcttagctca catgcttata
     5281 actaattaca tgactcgaag acataaaaaa caaaaaaagc accaccgact cggtgccact
     5341 ttttcaagtt gataacggac tagccttatt ttaacttgct atttaaatgc atcgattctg
     5401 atcatttatc tttcactgcg gagaagtttc gaacgccgaa acatgcgcac caactttcac
     5461 ttctacagcg tttgaccaaa atcttttgaa cagaacattg tagggtgtga aaaaatgcgc
     5521 acctttaccg ctagcccaag agggcactac aaaatctaga gttgtacttc aaacgtacat
     5581 gtaatcacct tgtatatact cgaaagaaaa catcaagttt ctgtataaat atgagtgaaa
     5641 gcataatcat acattatctt ttcaaagatt gtccgcggtg gagctccagc ttttgttccc
     5701 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5761 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5821 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5881 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5941 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     6001 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6061 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6121 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6181 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6241 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6301 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6361 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6421 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6481 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6541 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6601 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6661 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6721 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6781 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6841 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6901 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6961 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7021 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7081 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7141 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7201 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7261 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7321 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7381 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7441 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7501 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7561 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7621 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7681 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7741 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7801 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7861 cgcgcacatt tccccgaaaa gtgccacctg aacgaagcat ctgtgcttca ttttgtagaa
     7921 caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca
     7981 gaacagaaat gcaacgcgaa agcgctattt taccaacgaa gaatctgtgc ttcatttttg
     8041 taaaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt
     8101 ttacagaaca gaaatgcaac gcgagagcgc tattttacca acaaagaatc tatacttctt
     8161 ttttgttcta caaaaatgca tcccgagagc gctatttttc taacaaagca tcttagatta
     8221 ctttttttct cctttgtgcg ctctataatg cagtctcttg ataacttttt gcactgtagg
     8281 tccgttaagg ttagaagaag gctactttgg tgtctatttt ctcttccata aaaaaagcct
     8341 gactccactt cccgcgttta ctgattacta gcgaagctgc gggtgcattt tttcaagata
     8401 aaggcatccc cgattatatt ctataccgat gtggattgcg catactttgt gaacagaaag
     8461 tgatagcgtt gatgattctt cattggtcag aaaattatga acggtttctt ctattttgtc
     8521 tctatatact acgtatagga aatgtttaca ttttcgtatt gttttcgatt cactctatga
     8581 atagttctta ctacaatttt tttgtctaaa gagtaatact agagataaac ataaaaaatg
     8641 tagaggtcga gtttagatgc aagttcaagg agcgaaaggt ggatgggtag gttatatagg
     8701 gatatagcac agagatatat agcaaagaga tacttttgag caatgtttgt ggaagcggta
     8761 ttcgcaatat tttagtagct cgttacagtc cggtgcgttt ttggtttttt gaaagtgcgt
     8821 cttcagagcg cttttggttt tcaaaagcgc tctgaagttc ctatactttc tagctagaga
     8881 ataggaactt cggaatagga acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc
     8941 aacgcgagct gcgcacatac agctcactgt tcacgtcgca cctatatctg cgtgttgcct
     9001 gtatatatat atacatgaga agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta
     9061 tatgcgtcta tttatgtagg atgaaaggta gtctagtacc tcctgtgata ttatcccatt
     9121 ccatgcgggg tatcgtatgc ttccttcagc actacccttt agctgttcta tatgctgcca
     9181 ctcctcaatt ggattagtct catccttcaa tgctatcatt tcctttgata ttggatcata
     9241 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     9301 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     9361 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     9421 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     9481 agtgcaccat atcgactacg tcgttaaggc cgtttctgac agagtaaaat tcttgaggga
     9541 actttcacca ttatgggaaa tggttcaaga aggtattgac ttaaactcca tcaaatggtc
     9601 aggtcattga gtgtttttta tttgttgtat tttttttttt ttagagaaaa tcctccaata
     9661 tataaattag gaatcatagt ttcatgattt tctgttacac ctaacttttt gtgtggtgcc
     9721 ctcctccttg tcaatattaa tgttaaagtg caattctttt tccttatcac gttgagccat
     9781 tagtatcaat ttgcttacct gtattccttt acatcctcct ttttctcctt cttgataaat
     9841 gtatgtagat tgcgtatata gtttcgtcta ccctatgaac atattccatt ttgtaatttc
     9901 gtgtcgtttc tattatgaat ttcatttata aagtttatgt acaaatatca taaaaaaaga
     9961 gaatcttttt aagcaaggat tttcttaact tcttcggcga cagcatcacc gacttcggtg
    10021 gtactgttgg aaccacctaa atcaccagtt ctgatacctg catccaaaac ctttttaact
    10081 gcatcttcaa tggccttacc ttcttcaggc aagttcaatg acaatttcaa catcattgca
    10141 gcagacaaga tagtggcgat agggttgacc ttattctttg gcaaatctgg agcagaaccg
    10201 tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc caaggacgca
    10261 gatggcaaca aacccaagga acctgggata acggaggctt catcggagat gatatcacca
    10321 aacatgttgc tggtgattat aataccattt aggtgggttg ggttcttaac taggatcatg
    10381 gcggcagaat caatcaattg atgttgaacc ttcaatgtag gaaattcgtt cttgatggtt
    10441 tcctccacag tttttctcca taatcttgaa gaggccaaaa cattagcttt atccaaggac
    10501 caaataggca atggtggctc atgttgtagg gccatgaaag cggccattct tgtgattctt
    10561 tgcacttctg gaacggtgta ttgttcacta tcccaagcga caccatcacc atcgtcttcc
    10621 tttctcttac caaagtaaat acctcccact aattctctga caacaacgaa gtcagtacct
    10681 ttagcaaatt gtggcttgat tggagataag tctaaaagag agtcggatgc aaagttacat
    10741 ggtcttaagt tggcgtacaa ttgaagttct ttacggattt ttagtaaacc ttgttcaggt
    10801 ctaacactac ctgtacccca tttaggacca cccacagcac ctaacaaaac ggcatcagcc
    10861 ttcttggagg cttccagcgc ctcatctgga agtggaacac ctgtagcatc tatagcagca
    10921 ccaccaatta aatgattttc gaaatcgaac ttgacattgg aacgaacatc agaaatagct
    10981 ttaagaacct taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc tggcaaaacg
    11041 acgatcttct taggggcaga cattagaatg gtatatcctt gaaatatata tatatatatt
    11101 gctgaaatgt aaaaggtaag aaaagttaga aagtaagacg attgctaacc acctattgga
    11161 aaaaacaata ggtccttaaa taatattgtc aacttcaagt attgtgatgc aagcatttag
    11221 tcatgaacgc ttctctattc tatatgaaaa gccggttccg gcgctctcac ctttcctttt
    11281 tctcccaatt tttcagttga aaaaggtata tgcgtcaggc gacctctgaa attaacaaaa
    11341 aatttccagt catcgaattt gattctgtgc gatagcgccc ctgtgtgttc tcgttatgtt
    11401 gaggaaaaaa ataatggttg ctaagagatt cgaactcttg catcttacga tacctgagta
    11461 ttcccacagt taactgcggt caagatattt cttgaatcag gcgccttaga ccgctcggcc
    11521 aaacaaccaa ttacttgttg agaaatagag tataattatc ctataaatat aacgtttttg
    11581 aacacacatg aacaaggaag tacaggacaa ttgattttga agagaatgtg gattttgatg
    11641 taattgttgg gattccattt ttaataaggc aataatatta ggtatgtaga tatactagaa
    11701 gttctcctcg accggtcgat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
    11761 accgcatcag gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
    11821 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
    11881 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
    11941 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
    12001 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
    12061 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
    12121 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
    12181 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc
    12241 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
    12301 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
    12361 cgacgtt
//

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

P3086/pENTR223-CTNNBIP1人源基因质粒

P3087/pENTR223-RPL39L人源基因质粒

P3088/pENTR223-NRGN人源基因质粒

P3089/pENTR223-ERC1人源基因质粒

P3090/pENTR223-CEP72（568-1944bp）人源基因质粒

P3091/pENTR223-INPP5K人源基因质粒

P3092/pENTR223-CCKBR（1同义突变）人源基因质粒

P3093/pENTR223-DACH1(580-2121bp）人源基因质粒

P3094/pENTR223-THOC1（1点突变1同义突变）人源基因质粒

P3095/pENTR223-SLCO6A1（有一同义突变）人源基因质粒

P3096/pENTR223-GPKOW人源基因质粒