pLVCT-tTR-KRAB慢病毒干扰质粒

基本信息
名称：pLVCT-tTR-KRAB慢病毒干扰质粒
别称:Plasmid#11643

别称:	Plasmid#11643
原核抗性:	Amp
克隆菌株:	Stbl3
培养条件:	37度

质粒属性

载体宿主：	哺乳细胞,慢病毒
载体用途：	干扰沉默
基因种属：
基因类型：	shRNA
原核抗性：	Amp
筛选标记：
荧光蛋白：	绿色

质粒简介
   Tet-regulated (Tet-on) lentiviral vector for transgene (CAG promoter) - OR - shRNA (H1 promoter when subcloned from pLVTHM (Addgene#12247)) - 2nd generation.
质粒图谱


质粒序列
LOCUS       Exported               12877 bp ds-DNA     circular SYN 27-SEP-2017
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12877)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 27, 2017 from MiaoLingPlasmid
            http://www.miaolingbio.com
FEATURES             Location/Qualifiers
     source          1..12877
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             6..639
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    686..811
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1304..1537
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2061..3728
                     /label=CAG promoter
     misc_feature    3814..3932
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             4015..4731
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    4827..5378
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             5381..5998
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MARLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     CDS             6002..6022
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6125..6319
                     /codon_start=1
                     /gene="ZNF10"
                     /gene_synonym="KOX1"
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     misc_feature    6461..7049
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     protein_bind    7187..7457
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     LTR             7496..7676
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        8507..8611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             8612..9472
                     /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"
     rep_origin      9643..10231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        10477..10806
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10657..10792
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          11940..12005
                     /label=small t intron
                     /note="simian virus 40 (SV40) small t antigen intron"
     CDS             12135..12155
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    12580..12714
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 gtagttggaa gggctaattc actcccaaag aagacaagat atccttgatc tgtggatcta
       61 ccacacacaa ggctacttcc ctgattagca gaactacaca ccagggccag gggtcagata
      121 tccactgacc tttggatggt gctacaagct agtaccagtt gagccagata aggtagaaga
      181 ggccaataaa ggagagaaca ccagcttgtt acaccctgtg agcctgcatg ggatggatga
      241 cccggagaga gaagtgttag agtggaggtt tgacagccgc ctagcatttc atcacgtggc
      301 ccgagagctg catccggagt acttcaagaa ctgctgatat cgagcttgct acaagggact
      361 ttccgctggg gactttccag ggaggcgtgg cctgggcggg actggggagt ggcgagccct
      421 cagatcctgc atataagcag ctgctttttg cctgtactgg gtctctctgg ttagaccaga
      481 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
      541 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
      601 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
      661 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
      721 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
      781 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
      841 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
      901 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
      961 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     1021 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     1081 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     1141 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     1201 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     1261 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     1321 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     1381 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     1441 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     1501 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     1561 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     1621 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     1681 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     1741 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     1801 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     1861 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     1921 tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag
     1981 acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgatca cgagactagc
     2041 ctcgaccatc gatgtcgatc gacattgatt attgactagt tattaatagt aatcaattac
     2101 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
     2161 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     2221 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     2281 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     2341 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     2401 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt
     2461 ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt
     2521 ttaattattt tgtgcagcga tgggggcggg gggggggggg gcgcgcgcca ggcggggcgg
     2581 ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
     2641 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     2701 gaagcgcgcg gcgggcggga gtcgctgcgt tgccttcgcc ccgtgccccg ctccgcgccg
     2761 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
     2821 acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctc gtttcttttc
     2881 tgtggctgcg tgaaagcctt aaagggctcc gggagggccc tttgtgcggg ggggagcggc
     2941 tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggccc gcgctgcccg
     3001 gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcgtg tgcgcgaggg
     3061 gagcgcggcc gggggcggtg ccccgcggtg cgggggggct gcgaggggaa caaaggctgc
     3121 gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcggcggt cgggctgtaa
     3181 cccccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct
     3241 ccgtgcgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt
     3301 gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc
     3361 cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg
     3421 tgcgagaggg cgcagggact tcctttgtcc caaatctggc ggagccgaaa tctgggaggc
     3481 gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa
     3541 tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc
     3601 ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct
     3661 tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt
     3721 tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat
     3781 taaatttaat taatctcgac ggtatcggtt aacttttaaa agaaaagggg ggattggggg
     3841 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa tttaaagaat
     3901 tacaaaaaca aattacaaaa attcaaaatt ttatcgatca cgagactagc ctcgaggttt
     3961 aaactacggg atccaggcct aagcttacgc gtcctagcgc taccggtcgc caccatggtg
     4021 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     4081 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     4141 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     4201 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     4261 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     4321 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     4381 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     4441 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     4501 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     4561 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     4621 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     4681 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggactc
     4741 agatctcgac tagctagtag ctagctagct agtcgagctc aagcttcgaa ttcccgggct
     4801 gcaggaattc cgcccccccc cccctaacgt tactggccga agccgcttgg aataaggccg
     4861 gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc
     4921 ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa
     4981 aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag
     5041 acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg
     5101 cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg
     5161 ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa
     5221 caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg
     5281 gtgcacatgc tttacatgtg tttagtcgag gttaaaaaac gtctaggccc cccgaaccac
     5341 ggggacgtgg ttttcctttg aaaaacacga tgataatacc atggctagat tagataaaag
     5401 taaagtgatt aacagcgcat tagagctgct taatgaggtc ggaatcgaag gtttaacaac
     5461 ccgtaaactc gcccagaagc taggtgtaga gcagcctaca ttgtattggc atgtaaaaaa
     5521 taagcgggct ttgctcgacg ccttagccat tgagatgtta gataggcacc atactcactt
     5581 ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta aaagttttag
     5641 atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac ggcctacaga
     5701 aaaacagtat gaaactctcg aaaatcaatt agccttttta tgccaacaag gtttttcact
     5761 agagaatgca ttatatgcac tcagcgctgt ggggcatttt actttaggtt gcgtattgga
     5821 agatcaagag catcaagtcg ctaaagaaga aagggaaaca cctactactg atagtatgcc
     5881 gccattatta cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc cagccttctt
     5941 attcggcctt gaattgatca tatgcggatt agaaaaacaa cttaaatgtg aaagtgggtc
     6001 gccaaaaaag aagagaaagg tcgacggcgg tggtgctttg tctcctcagc actctgctgt
     6061 cactcaagga agtatcatca agaacaagga gggcatggat gctaagtcac taactgcctg
     6121 gtcccggaca ctggtgacct tcaaggatgt atttgtggac ttcaccaggg aggagtggaa
     6181 gctgctggac actgctcagc agatcgtgta cagaaatgtg atgctggaga actataagaa
     6241 cctggtttcc ttgggttatc agcttactaa gccagatgtg atcctccggt tggagaaggg
     6301 agaagagccc tggctggtgg agagagaaat tcaccaagag acccatcctg attcagagac
     6361 tgcatttgaa atcaaatcat cagtttaagg atctcgacta gctagtagct agctagctag
     6421 tcgagctcaa cttcgaattc gatgggacta gtcatatgat aatcaacctc tggattacaa
     6481 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     6541 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     6601 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     6661 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     6721 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     6781 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     6841 ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat
     6901 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     6961 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     7021 tcggatctcc ctttgggccg cctccccgca tcggtacgta tggccaggta cctttaagac
     7081 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     7141 aagggctaat tcactcccaa cgaagacaag atctcgagtt taccactccc tatcagtgat
     7201 agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa gtgaaagtcg
     7261 agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc
     7321 agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga
     7381 aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc
     7441 cctatcagtg atagagaaaa gtgaaagtcg gatcgatctg ctttttgctt gtactgggtc
     7501 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     7561 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     7621 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagcat
     7681 ctagaattaa ttccgtgtat tctatagtgt cacctaaatc gtatgtgtat gatacataag
     7741 gttatgtatt aattgtagcc gcgttctaac gacaatatgt acaagcctaa ttgtgtagca
     7801 tctggcttac tgaagcagac cctatcatct ctctcgtaaa ctgccgtcag agtcggtttg
     7861 gttggacgaa ccttctgagt ttctggtaac gccgtcccgc acccggaaat ggtcagcgaa
     7921 ccaatcagca gggtcatcgc tagccagatc ctctacgccg gacgcatcgt ggccggcatc
     7981 accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat
     8041 cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc
     8101 gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg
     8161 ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag
     8221 cgtcgaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     8281 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     8341 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     8401 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     8461 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     8521 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     8581 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     8641 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     8701 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     8761 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     8821 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     8881 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     8941 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     9001 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     9061 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     9121 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     9181 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     9241 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     9301 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     9361 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     9421 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     9481 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     9541 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     9601 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     9661 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     9721 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     9781 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     9841 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     9901 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     9961 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
    10021 cctacagcgt gagcattgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
    10081 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
    10141 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
    10201 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
    10261 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
    10321 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
    10381 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
    10441 cgcgcgttgg ccgattcatt aatgcagctg tggaatgtgt gtcagttagg gtgtggaaag
    10501 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
    10561 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
    10621 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
    10681 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
    10741 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
    10801 tgcaaaaagc ttggacacaa gacaggcttg cgagatatgt ttgagaatac cactttatcc
    10861 cgcgtcaggg agaggcagtg cgtaaaaaga cgcggactca tgtgaaatac tggtttttag
    10921 tgcgccagat ctctataatc tcgcgcaacc tattttcccc tcgaacactt tttaagccgt
    10981 agataaacag gctgggacac ttcacatgag cgaaaaatac atcgtcacct gggacatgtt
    11041 gcagatccat gcacgtaaac tcgcaagccg actgatgcct tctgaacaat ggaaaggcat
    11101 tattgccgta agccgtggcg gtctgtaccg ggtgcgttac tggcgcgtga actgggtatt
    11161 cgtcatgtcg ataccgtttg tatttccagc tacgatcacg acaaccagcg cgagcttaaa
    11221 gtgctgaaac gcgcagaagg cgatggcgaa ggcttcatcg ttattgatga cctggtggat
    11281 accggtggta ctgcggttgc gattcgtgaa atgtatccaa aagcgcactt tgtcaccatc
    11341 ttcgcaaaac cggctggtcg tccgctggtt gatgactatg ttgttgatat cccgcaagat
    11401 acctggattg aacagccgtg ggatatgggc gtcgtattcg tcccgccaat ctccggtcgc
    11461 taatcttttc aacgcctggc actgccgggc gttgttcttt ttaacttcag gcgggttaca
    11521 atagtttcca gtaagtattc tggaggctgc atccatgaca caggcaaacc tgagcgaaac
    11581 cctgttcaaa ccccgcttta aacatcctga aacctcgacg ctagtccgcc gctttaatca
    11641 cggcgcacaa ccgcctgtgc agtcggccct tgatggtaaa accatccctc actggtatcg
    11701 catgattaac cgtctgatgt ggatctggcg cggcattgac ccacgcgaaa tcctcgacgt
    11761 ccaggcacgt attgtgatga gcgatgccga acgtaccgac gatgatttat acgatacggt
    11821 gattggctac cgtggcggca actggattta tgagtgggcc ccggatcttt gtgaaggaac
    11881 cttacttctg tggtgtgaca taattggaca aactacctac agagatttaa agctctaagg
    11941 taaatataaa atttttaagt gtataatgtg ttaaactact gattctaatt gtttgtgtat
    12001 tttagattcc aacctatgga actgatgaat gggagcagtg gtggaatgcc tttaatgagg
    12061 aaaacctgtt ttgctcagaa gaaatgccat ctagtgatga tgaggctact gctgactctc
    12121 aacattctac tcctccaaaa aagaagagaa aggtagaaga ccccaaggac tttccttcag
    12181 aattgctaag ttttttgagt catgctgtgt ttagtaatag aactcttgct tgctttgcta
    12241 tttacaccac aaaggaaaaa gctgcactgc tatacaagaa aattatggaa aaatattctg
    12301 taacctttat aagtaggcat aacagttata atcataacat actgtttttt cttactccac
    12361 acaggcatag agtgtctgct attaataact atgctcaaaa attgtgtacc tttagctttt
    12421 taatttgtaa aggggttaat aaggaatatt tgatgtatag tgccttgact agagatcata
    12481 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
    12541 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
    12601 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
    12661 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcaactgg
    12721 ataactcaag ctaaccaaaa tcatcccaaa cttcccaccc cataccctat taccactgcc
    12781 aattacctag tggtttcatt tactctaaac ctgtgattcc tctgaattat tttcatttta
    12841 aagaaattgt atttgttaaa tatgtactac aaactta
//

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

P3220/pMIND质粒

P0155/pCDNA3.0哺乳表达质粒

P0942/pCDNA3-HA-N哺乳表达质粒

P0156/pCDNA3.1(-)哺乳表达质粒

P0157/pCDNA3.1(+)哺乳表达质粒

P0417/pCDNA3.1-Zeo哺乳表达质粒

P3066/pCDNA3.1-HA-Hygro哺乳表达质粒

P1266/pCDNA3.1-Hygro哺乳表达质粒

P1828/pCDNA3.1-GST哺乳表达质粒

P0794/pCDNA3.1-FLAG-N哺乳表达质粒

P0159/pECMV-3×FLAG-N哺乳表达质粒

P1303/pECMV-3×FLAG-C哺乳表达质粒