pY001 (pFnCpf1_full)大肠敲除质粒

基本信息
名称：pY001 (pFnCpf1_full)大肠敲除质粒
别称: pY001(pFnCpf1_full)
启动子：CMV
大肠杆菌质粒；大肠编辑质粒；大肠Cas9质粒
质粒属性

载体宿主：
载体用途：
基因种属：
基因类型：
原核抗性：
真核抗性：
荧光蛋白：

质粒简介
 pY001 (pFnCpf1_full)大肠敲除质粒
质粒图谱


质粒序列
LOCUS       Exported               10653 bp ds-DNA     circular SYN 28-SEP-2017
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10653)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 28, 2017 from MiaoLingPlasmid
            http://www.miaolingbio.com
FEATURES             Location/Qualifiers
     source          1..10653
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        complement(220..322)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      complement(848..1393)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     CDS             1706..5608
                     /codon_start=1
                     /product="WP_003040289.1"
                     /label=FnCpf1 locus
                     /translation="MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAK
                     DYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDT
                     IKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDID
                     EALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLK
                     DKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKF
                     NTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQIL-SDTESKSFVID
                     KLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSL
                     TDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKL
                     ALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQA
                     SAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIV
                     PLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNK
                     KNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNH
                     STHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEF
                     YREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALF
                     DERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDK
                     RFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVD
                     GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVV
                     HEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKT
                     GGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQE
                     FFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTR
                     EVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTE
                     LDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNL
                     VIKNEEYFEFVQNRNN"
     CDS             7989..9179
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=Tet
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGAL-SDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     CDS             complement(10213..219)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=Chl
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
       61 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
      121 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
      181 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
      241 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt
      301 ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc
      361 ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat
      421 ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt
      481 gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg
      541 acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact
      601 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa
      661 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc
      721 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc
      781 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa
      841 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
      901 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc
      961 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc
     1021 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac
     1081 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt
     1141 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt
     1201 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg
     1261 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt
     1321 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc
     1381 aaaacgatct caagaagatc atcttattaa tcagataaaa tattttgatg caatatttgt
     1441 cttaggtcat ccaagttatt atccaaaatt tggttttaaa ccagccacag aatatcagat
     1501 aaaatgtgaa tatgatgtcc cagcggatgt ttttatggta ctagatttgt cagctaaact
     1561 agctagttta aaaggacaaa ctgtctacta tgccgatgag tttggcaaaa ttttttagat
     1621 ctacaaaatt ataaactaaa taaagattct tataataact ttatatataa tcgaaatgta
     1681 gagaatttta taaggagtct ttatcatgtc aatttatcaa gaatttgtta ataaatatag
     1741 tttaagtaaa actctaagat ttgagttaat cccacagggt aaaacacttg aaaacataaa
     1801 agcaagaggt ttgattttag atgatgagaa aagagctaaa gactacaaaa aggctaaaca
     1861 aataattgat aaatatcatc agttttttat agaggagata ttaagttcgg tttgtattag
     1921 cgaagattta ttacaaaact attctgatgt ttattttaaa cttaaaaaga gtgatgatga
     1981 taatctacaa aaagatttta aaagtgcaaa agatacgata aagaaacaaa tatctgaata
     2041 tataaaggac tcagagaaat ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa
     2101 agggcaagag tcagatttaa ttctatggct aaagcaatct aaggataatg gtatagaact
     2161 atttaaagcc aatagtgata tcacagatat agatgaggcg ttagaaataa tcaaatcttt
     2221 taaaggttgg acaacttatt ttaagggttt tcatgaaaat agaaaaaatg tttatagtag
     2281 caatgatatt cctacatcta ttatttatag gatagtagat gataatttgc ctaaatttct
     2341 agaaaataaa gctaagtatg agagtttaaa agacaaagct ccagaagcta taaactatga
     2401 acaaattaaa aaagatttgg cagaagagct aacctttgat attgactaca aaacatctga
     2461 agttaatcaa agagtttttt cacttgatga agtttttgag atagcaaact ttaataatta
     2521 tctaaatcaa agtggtatta ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg
     2581 tgaaaataca aagagaaaag gtataaatga atatataaat ctatactcac agcaaataaa
     2641 tgataaaaca ctcaaaaaat ataaaatgag tgttttattt aagcaaattt taagtgatac
     2701 agaatctaaa tcttttgtaa ttgataagtt agaagatgat agtgatgtag ttacaacgat
     2761 gcaaagtttt tatgagcaaa tagcagcttt taaaacagta gaagaaaaat ctattaaaga
     2821 aacactatct ttattatttg atgatttaaa agctcaaaaa cttgatttga gtaaaattta
     2881 ttttaaaaat gataaatctc ttactgatct atcacaacaa gtttttgatg attatagtgt
     2941 tattggtaca gcggtactag aatatataac tcaacaaata gcacctaaaa atcttgataa
     3001 ccctagtaag aaagagcaag aattaatagc caaaaaaact gaaaaagcaa aatacttatc
     3061 tctagaaact ataaagcttg ccttagaaga atttaataag catagagata tagataaaca
     3121 gtgtaggttt gaagaaatac ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat
     3181 agctcaaaac aaagacaatt tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa
     3241 agacctactt caagctagtg cggaagatga tgttaaagct atcaaggatc ttttagatca
     3301 aactaataat ctcttacata aactaaaaat atttcatatt agtcagtcag aagataaggc
     3361 aaatatttta gacaaggatg agcattttta tctagtattt gaggagtgct actttgagct
     3421 agcgaatata gtgcctcttt ataacaaaat tagaaactat ataactcaaa agccatatag
     3481 tgatgagaaa tttaagctca attttgagaa ctcgactttg gctaatggtt gggataaaaa
     3541 taaagagcct gacaatacgg caattttatt tatcaaagat gataaatatt atctgggtgt
     3601 gatgaataag aaaaataaca aaatatttga tgataaagct atcaaagaaa ataaaggcga
     3661 gggttataaa aaaattgttt ataaactttt acctggcgca aataaaatgt tacctaaggt
     3721 tttcttttct gctaaatcta taaaatttta taatcctagt gaagatatac ttagaataag
     3781 aaatcattcc acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt
     3841 taatattgaa gattgccgaa aatttataga tttttataaa cagtctataa gtaagcatcc
     3901 ggagtggaaa gattttggat ttagattttc tgatactcaa agatataatt ctatagatga
     3961 attttataga gaagttgaaa atcaaggcta caaactaact tttgaaaata tatcagagag
     4021 ctatattgat agcgtagtta atcagggtaa attgtaccta ttccaaatct ataataaaga
     4081 tttttcagct tatagcaaag ggcgaccaaa tctacatact ttatattgga aagcgctgtt
     4141 tgatgagaga aatcttcaag atgtggttta taagctaaat ggtgaggcag agctttttta
     4201 tcgtaaacaa tcaataccta aaaaaatcac tcacccagct aaagaggcaa tagctaataa
     4261 aaacaaagat aatcctaaaa aagagagtgt ttttgaatat gatttaatca aagataaacg
     4321 ctttactgaa gataagtttt tctttcactg tcctattaca atcaatttta aatctagtgg
     4381 agctaataag tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa atgatgttca
     4441 tatattaagt atagatagag gtgaaagaca tttagcttac tatactttgg tagatggtaa
     4501 aggcaatatc atcaaacaag atactttcaa catcattggt aatgatagaa tgaaaacaaa
     4561 ctaccatgat aagcttgctg caatagagaa agatagggat tcagctagga aagactggaa
     4621 aaagataaat aacatcaaag agatgaaaga gggctatcta tctcaggtag ttcatgaaat
     4681 agctaagcta gttatagagt ataatgctat tgtggttttt gaggatttaa attttggatt
     4741 taaaagaggg cgtttcaagg tagagaagca ggtctatcaa aagttagaaa aaatgctaat
     4801 tgagaaacta aactatctag ttttcaaaga taatgagttt gataaaactg ggggagtgct
     4861 tagagcttat cagctaacag caccttttga gacttttaaa aagatgggta aacaaacagg
     4921 tattatctac tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt
     4981 aaatcagtta tatcctaagt atgaaagtgt cagcaaatct caagagttct ttagtaagtt
     5041 tgacaagatt tgttataacc ttgataaggg ctattttgag tttagttttg attataaaaa
     5101 ctttggtgac aaggctgcca aaggcaagtg gactatagct agctttggga gtagattgat
     5161 taactttaga aattcagata aaaatcataa ttgggatact cgagaagttt atccaactaa
     5221 agagttggag aaattgctaa aagattattc tatcgaatat gggcatggcg aatgtatcaa
     5281 agcagctatt tgcggtgaga gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa
     5341 tactatctta caaatgcgta actcaaaaac aggtactgag ttagattatc taatttcacc
     5401 agtagcagat gtaaatggca atttctttga ttcgcgacag gcgccaaaaa atatgcctca
     5461 agatgctgat gccaatggtg cttatcatat tgggctaaaa ggtctgatgc tactaggtag
     5521 gatcaaaaat aatcaagagg gcaaaaaact caatttggtt atcaaaaatg aagagtattt
     5581 tgagttcgtg cagaatagga ataactaatt cattcaagaa tatattaccc tgtcagttta
     5641 gcgactatta cctctttaat aatttgcagg ggaattattt tagtaatagt aatatacaca
     5701 agagttattg attatatgga aaattatatt tagataacat ggttaaatga ttttatattc
     5761 tgtccttact cgatatattt gcataatatc tatagtaatg cctcagatac tacatactat
     5821 tcatctagcc aaacaaaagg gcgcgatgct cataaaagta tcgataaagg aatctatagt
     5881 accaaaaaag atgacctgat cggtatcgat gttattaacc ataaatatgg tttggttggt
     5941 aaaattgatg tttttcataa agataagggc ttacttgtgg agagaaaaag gcaaatcaag
     6001 actatctatg atggctataa atatcagctt tatgcgcaat atttttgtct ccaagagatg
     6061 ggctatgatg tcaaagccat taaattttat tcgatggttg ataataaatc atacccaata
     6121 gctataccaa cttcagctga gttagaaaag tttgaaaaac atattcaaac aatcaagcaa
     6181 tataatccaa tggataactc atttaggcaa aatattgaaa agtgtaaatt ttgtatatat
     6241 gcaaacttat gtgataaaac ggacttgtag attatgttta gtaaaaatga tattgaatca
     6301 aagaatatag tttttgttaa tatttttgat ggagtgaaac ttagtctatc attggggaat
     6361 atagttataa aagataaaga aactgatgag gtgaaaacta agctttctgt tcataaagtt
     6421 cttgcattgt ttatcgtagg taatatgacg atgacctcgc aacttttaga gacctgtaag
     6481 aaaaatgcta tacagctagt ttttatgaaa aatagcttta gaccatatct atgttttggt
     6541 gatattgctg aggctaattt tttagctaga tataagcaat atagtgtagt tgagcaagat
     6601 ataagtttag caaggatttt tataacatca aagatacgca atcaacataa cttagtcaaa
     6661 agcctaagag ataaaactcc agagcagcaa gagatagtca aaaagaataa acagctaata
     6721 gcagagttag aaaatacaac aagcctagcg gagctaatgg gtatagaggg caatgttgcc
     6781 aaaaatttct tcaaaggatt ctatggacat ttagatagtt ggcaagggcg caaacctaga
     6841 ataaaacagg atccatataa tgttgtttta gacttgggct atagtatgtt gtttaatttt
     6901 gtagagtgtt ttttgcgact ttttggcttt gatttataca agggcttttg tcatcagact
     6961 tggtataagc gtaaatccct agtttgtgac tttgttgagc catttagatg tatagtggat
     7021 aaccaagtta gaaaatcatg gaatctcggg caattttctg tagaggattt tggttgcaaa
     7081 aatgagcagt tttatataaa aaaagataaa acaaaagact actcaaaaat actttttgcc
     7141 gagattatca gctacaagct agagatattt gaatatgtaa gagaatttta tcgtgccttt
     7201 atgcgaggca aagaaattgc agagtatcca atattttgtt atgaaactag gagggtgtat
     7261 gttgatagtc agttatgatt ttagtaataa taaagtacgt gcaaagtttg ccaaatttct
     7321 agaaagttat ggtgtacgtt tacaatattc ggtatttgag ctcaaatata gcaagagaat
     7381 gttagacttg attttagctg agatagaaaa taactatgta ccactattta caaatgctga
     7441 tagtgtttta atctttaatg ctccagataa agatgtgata aaatatggtt atgcgattca
     7501 tagagaacaa gaggttgttt ttatagacta aaaattgcaa accttagtct ttatgttaaa
     7561 ataactacta agttcttaga gatatttaaa aatatgactg ttgttatata tcaaaatgct
     7621 aaaaaaatca tagattttag gtcttttttt gctgatttag gcaaaaacgg gtctaagaac
     7681 tttaaataat ttctactgtt gtagatgaga agtcatttaa taaggccact gttaaaagtc
     7741 taagaacttt aaataatttc tactgttgta gatgctacta ttcctgtgcc ttcagataat
     7801 tcagtctaag aactttaaat aatttctact gttgtagatg tctagagcct tttgtattag
     7861 tagccggtct aagaacttta aataatttct actgttgtag attagcgatt tatgaaggtc
     7921 atttttttgt ctagctttaa tgcggtagtt tatcacagtt aaattgctaa cgcagtcagg
     7981 caccgtgtat gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc accctggatg
     8041 ctgtaggcat aggcttggtt atgccggtac tgccgggcct cttgcgggat atcgtccatt
     8101 ccgacagcat cgccagtcac tatggcgtgc tgctagcgct atatgcgttg atgcaatttc
     8161 tatgcgcacc cgttctcgga gcactgtccg accgctttgg ccgccgccca gtcctgctcg
     8221 cttcgctact tggagccact atcgactacg cgatcatggc gaccacaccc gtcctgtgga
     8281 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     8341 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     8401 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     8461 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     8521 gcttcctaat gcaggagtcg cataagggag agcgtcgacc gatgcccttg agagccttca
     8581 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg
     8641 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg
     8701 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct
     8761 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc
     8821 aggccattat cgccggcatg gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga
     8881 cgcgaggctg gatggccttc cccattatga ttcttctcgc ttccggcggc atcgggatgc
     8941 ccgcgttgca ggccatgctg tccaggcagg tagatgacga ccatcaggga cagcttcaag
     9001 gatcgctcgc ggctcttacc agcctaactt cgatcattgg accgctgatc gtcacggcga
     9061 tttatgccgc ctcggcgagc acatggaacg ggttggcatg gattgtaggc gccgccctat
     9121 accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa
     9181 tggaagccgg cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt
     9241 cttgcggaga actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc
     9301 catctccagc agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg
     9361 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
     9421 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc
     9481 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg
     9541 gaagtcccct acgtgctgct gaagttgccc gcaacagaga gtggaaccaa ccggtgatac
     9601 cacgatacta tgactgagag tcaacgccat gagcggcctc atttcttatt ctgagttaca
     9661 acagtccgca ccgctgtccg gtagctcctt ccggtgggcg cggggcatga ctatcgtcgc
     9721 cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgcccaa
     9781 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc cctgcaccat
     9841 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt
     9901 attaacgaag cgctaaccgt ttttatcagg ctctgggagg cagaataaat gatcatatcg
     9961 tcaattatta cctccacggg gagagcctga gcaaactggc ctcaggcatt tgagaagcac
    10021 acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct
    10081 atttaacgac cctgccctga accgacgacc gggtcgaatt tgctttcgaa tttctgccat
    10141 tcatccgctt attatcactt attcaggcgt agcaccaggc gtttaagggc accaataact
    10201 gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag
    10261 cattctgccg acatggaagc catcacagac ggcatgatga acctgaatcg ccagcggcat
    10321 cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt
    10381 gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac
    10441 gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc
    10501 cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag
    10561 cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca
    10621 tatcaccagc tcaccgtctt tcattgccat acg
//

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

P1074/pLV-mCherry慢病毒表达质粒

P1092/FUGW慢病毒表达质粒质粒

P1216/pLenti CMV Puro LUC (w168-1) 慢病毒表达质粒

P1387/pLenti CMV V5-LUC Blast (w567-1)慢病毒表达质粒

P1589/Clover-Geminin(1-110) 慢病毒表达质粒

P1598/pLenti CMV GFP Blast (659-1)慢病毒表达质粒

P2883/pSLIK-GFP四环素诱导绿色荧光质粒

P2953/pLNCX2慢病毒表达质粒

P1686/pLVX-TetOne-Puro慢病毒四环素激活表达质粒

P4364/pLVX-TetOne-Puro-Luc慢病毒四环素激活表达质粒

P4208/pLIX_403慢病毒四环素诱导表达质粒