BE4-Gam哺乳编辑质粒

基本信息
名称：BE4-Gam哺乳编辑质粒
别称: Plasmid#100806

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
载体用途：	基因敲除
基因种属：
基因类型：
原核抗性：	Amp
真核抗性：
荧光蛋白：

质粒简介
  BE4-Gam是一个大鼠基因哺乳细胞过表达质粒。

质粒图谱


质粒序列
LOCUS       Exported                9444 bp ds-DNA     circular SYN 01-DEC-2017
DEFINITION  synthetic circular DNA
FEATURES             Location/Qualifiers
     source          1..9444
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      403..412
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     misc_feature    409..930
                     /label=Gam
     CDS             979..1662
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=rAPOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             1759..5859
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNL-SDAILLS-DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5890..6138
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNL-SDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6169..6417
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNL-SDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6430..6450
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6459..6476
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    6505..6729
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(7202..7790)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7961..8821)
                     /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(8822..8926)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        9196..131
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggctaaacca
      421 gcaaaacgta tcaagagtgc cgcagcggct tatgtgccac aaaaccgcga tgcggtgatt
      481 accgatatta aacgcatcgg ggatttacag cgcgaagcat cacgtctgga aacggaaatg
      541 aatgatgcca tcgcggaaat tacggagaaa tttgcggccc ggattgcacc gattaaaacc
      601 gatattgaaa ccctttcaaa aggcgttcag ggatggtgtg aagcgaaccg cgacgaactg
      661 acgaacggcg gcaaagtgaa gacggcgaat cttgtcaccg gtgatgtatc gtggcgggtc
      721 cgtccaccat cagtaagtat tcgtggtatg gatgcagtga tggaaacgct ggagcgtctt
      781 ggcctgcaac gctttattcg cacgaagcag gaaatcaaca aggaagcgat tttactggaa
      841 ccgaaagcgg tcgcaggcgt tgccggaatt acagttaaat caggcattga ggatttttct
      901 attattccat ttgaacagga agccggtatt agcggcagcg agactcccgg gacctcagag
      961 tccgctacac ccgaaagtag ctcagagact ggcccagtgg ctgtggaccc cacattgaga
     1021 cggcggatcg agccccatga gtttgaggta ttcttcgatc cgagagagct ccgcaaggag
     1081 acctgcctgc tttacgaaat taattggggg ggccggcact ccatttggcg acatacatca
     1141 cagaacacta acaagcacgt cgaagtcaac ttcatcgaga agttcacgac agaaagatat
     1201 ttctgtccga acacaaggtg cagcattacc tggtttctca gctggagccc atgcggcgaa
     1261 tgtagtaggg ccatcactga attcctgtca aggtatcccc acgtcactct gtttatttac
     1321 atcgcaaggc tgtaccacca cgctgacccc cgcaatcgac aaggcctgcg ggatttgatc
     1381 tcttcaggtg tgactatcca aattatgact gagcaggagt caggatactg ctggagaaac
     1441 tttgtgaatt atagcccgag taatgaagcc cactggccta ggtatcccca tctgtgggta
     1501 cgactgtacg ttcttgaact gtactgcatc atactgggcc tgcctccttg tctcaacatt
     1561 ctgagaagga agcagccaca gctgacattc tttaccatcg ctcttcagtc ttgtcattac
     1621 cagcgactgc ccccacacat tctctgggcc accgggttga aatctggtgg ttcttctggt
     1681 ggttctagcg gcagcgagac tcccgggacc tcagagtccg ccacacccga aagttctggt
     1741 ggttcttctg gtggttctga taaaaagtat tctattggtt tagccatcgg cactaattcc
     1801 gttggatggg ctgtcataac cgatgaatac aaagtacctt caaagaaatt taaggtgttg
     1861 gggaacacag accgtcattc gattaaaaag aatcttatcg gtgccctcct attcgatagt
     1921 ggcgaaacgg cagaggcgac tcgcctgaaa cgaaccgctc ggagaaggta tacacgtcgc
     1981 aagaaccgaa tatgttactt acaagaaatt tttagcaatg agatggccaa agttgacgat
     2041 tctttctttc accgtttgga agagtccttc cttgtcgaag aggacaagaa acatgaacgg
     2101 caccccatct ttggaaacat agtagatgag gtggcatatc atgaaaagta cccaacgatt
     2161 tatcacctca gaaaaaagct agttgactca actgataaag cggacctgag gttaatctac
     2221 ttggctcttg cccatatgat aaagttccgt gggcactttc tcattgaggg tgatctaaat
     2281 ccggacaact cggatgtcga caaactgttc atccagttag tacaaaccta taatcagttg
     2341 tttgaagaga accctataaa tgcaagtggc gtggatgcga aggctattct tagcgcccgc
     2401 ctctctaaat cccgacggct agaaaacctg atcgcacaat tacccggaga gaagaaaaat
     2461 gggttgttcg gtaaccttat agcgctctca ctaggcctga caccaaattt taagtcgaac
     2521 ttcgacttag ctgaagatgc caaattgcag cttagtaagg acacgtacga tgacgatctc
     2581 gacaatctac tggcacaaat tggagatcag tatgcggact tatttttggc tgccaaaaac
     2641 cttagcgatg caatcctcct atctgacata ctgagagtta atactgagat taccaaggcg
     2701 ccgttatccg cttcaatgat caaaaggtac gatgaacatc accaagactt gacacttctc
     2761 aaggccctag tccgtcagca actgcctgag aaatataagg aaatattctt tgatcagtcg
     2821 aaaaacgggt acgcaggtta tattgacggc ggagcgagtc aagaggaatt ctacaagttt
     2881 atcaaaccca tattagagaa gatggatggg acggaagagt tgcttgtaaa actcaatcgc
     2941 gaagatctac tgcgaaagca gcggactttc gacaacggta gcattccaca tcaaatccac
     3001 ttaggcgaat tgcatgctat acttagaagg caggaggatt tttatccgtt cctcaaagac
     3061 aatcgtgaaa agattgagaa aatcctaacc tttcgcatac cttactatgt gggacccctg
     3121 gcccgaggga actctcggtt cgcatggatg acaagaaagt ccgaagaaac gattactcca
     3181 tggaattttg aggaagttgt cgataaaggt gcgtcagctc aatcgttcat cgagaggatg
     3241 accaactttg acaagaattt accgaacgaa aaagtattgc ctaagcacag tttactttac
     3301 gagtatttca cagtgtacaa tgaactcacg aaagttaagt atgtcactga gggcatgcgt
     3361 aaacccgcct ttctaagcgg agaacagaag aaagcaatag tagatctgtt attcaagacc
     3421 aaccgcaaag tgacagttaa gcaattgaaa gaggactact ttaagaaaat tgaatgcttc
     3481 gattctgtcg agatctccgg ggtagaagat cgatttaatg cgtcacttgg tacgtatcat
     3541 gacctcctaa agataattaa agataaggac ttcctggata acgaagagaa tgaagatatc
     3601 ttagaagata tagtgttgac tcttaccctc tttgaagatc gggaaatgat tgaggaaaga
     3661 ctaaaaacat acgctcacct gttcgacgat aaggttatga aacagttaaa gaggcgtcgc
     3721 tatacgggct ggggacgatt gtcgcggaaa cttatcaacg ggataagaga caagcaaagt
     3781 ggtaaaacta ttctcgattt tctaaagagc gacggcttcg ccaataggaa ctttatgcag
     3841 ctgatccatg atgactcttt aaccttcaaa gaggatatac aaaaggcaca ggtttccgga
     3901 caaggggact cattgcacga acatattgcg aatcttgctg gttcgccagc catcaaaaag
     3961 ggcatactcc agacagtcaa agtagtggat gagctagtta aggtcatggg acgtcacaaa
     4021 ccggaaaaca ttgtaatcga gatggcacgc gaaaatcaaa cgactcagaa ggggcaaaaa
     4081 aacagtcgag agcggatgaa gagaatagaa gagggtatta aagaactggg cagccagatc
     4141 ttaaaggagc atcctgtgga aaatacccaa ttgcagaacg agaaacttta cctctattac
     4201 ctacaaaatg gaagggacat gtatgttgat caggaactgg acataaaccg tttatctgat
     4261 tacgacgtcg atcacattgt accccaatcc tttttgaagg acgattcaat cgacaataaa
     4321 gtgcttacac gctcggataa gaaccgaggg aaaagtgaca atgttccaag cgaggaagtc
     4381 gtaaagaaaa tgaagaacta ttggcggcag ctcctaaatg cgaaactgat aacgcaaaga
     4441 aagttcgata acttaactaa agctgagagg ggtggcttgt ctgaacttga caaggccgga
     4501 tttattaaac gtcagctcgt ggaaacccgc caaatcacaa agcatgttgc acagatacta
     4561 gattcccgaa tgaatacgaa atacgacgag aacgataagc tgattcggga agtcaaagta
     4621 atcactttaa agtcaaaatt ggtgtcggac ttcagaaagg attttcaatt ctataaagtt
     4681 agggagataa ataactacca ccatgcgcac gacgcttatc ttaatgccgt cgtagggacc
     4741 gcactcatta agaaataccc gaagctagaa agtgagtttg tgtatggtga ttacaaagtt
     4801 tatgacgtcc gtaagatgat cgcgaaaagc gaacaggaga taggcaaggc tacagccaaa
     4861 tacttctttt attctaacat tatgaatttc tttaagacgg aaatcactct ggcaaacgga
     4921 gagatacgca aacgaccttt aattgaaacc aatggggaga caggtgaaat cgtatgggat
     4981 aagggccggg acttcgcgac ggtgagaaaa gttttgtcca tgccccaagt caacatagta
     5041 aagaaaactg aggtgcagac cggagggttt tcaaaggaat cgattcttcc aaaaaggaat
     5101 agtgataagc tcatcgctcg taaaaaggac tgggacccga aaaagtacgg tggcttcgat
     5161 agccctacag ttgcctattc tgtcctagta gtggcaaaag ttgagaaggg aaaatccaag
     5221 aaactgaagt cagtcaaaga attattgggg ataacgatta tggagcgctc gtcttttgaa
     5281 aagaacccca tcgacttcct tgaggcgaaa ggttacaagg aagtaaaaaa ggatctcata
     5341 attaaactac caaagtatag tctgtttgag ttagaaaatg gccgaaaacg gatgttggct
     5401 agcgccggag agcttcaaaa ggggaacgaa ctcgcactac cgtctaaata cgtgaatttc
     5461 ctgtatttag cgtcccatta cgagaagttg aaaggttcac ctgaagataa cgaacagaag
     5521 caactttttg ttgagcagca caaacattat ctcgacgaaa tcatagagca aatttcggaa
     5581 ttcagtaaga gagtcatcct agctgatgcc aatctggaca aagtattaag cgcatacaac
     5641 aagcacaggg ataaacccat acgtgagcag gcggaaaata ttatccattt gtttactctt
     5701 accaacctcg gcgctccagc cgcattcaag tattttgaca caacgataga tcgcaaacga
     5761 tacacttcta ccaaggaggt gctagacgcg acactgattc accaatccat cacgggatta
     5821 tatgaaactc ggatagattt gtcacagctt gggggtgact ctggtggttc tggaggatct
     5881 ggtggttcta ctaatctgtc agatattatt gaaaaggaga ccggtaagca actggttatc
     5941 caggaatcca tcctcatgct cccagaggag gtggaagaag tcattgggaa caagccggaa
     6001 agcgatatac tcgtgcacac cgcctacgac gagagcaccg acgagaatgt catgcttctg
     6061 actagcgacg cccctgaata caagccttgg gctctggtca tacaggatag caacggtgag
     6121 aacaagatta agatgctctc tggtggttct ggaggatctg gtggttctac taatctgtca
     6181 gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat cctcatgctc
     6241 ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact cgtgcacacc
     6301 gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc ccctgaatac
     6361 aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa gatgctctct
     6421 ggtggttctc ccaagaagaa gaggaaagtc taaccggtca tcatcaccat caccattgag
     6481 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     6541 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     6601 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     6661 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     6721 gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg acctctagct
     6781 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     6841 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc taatgagtga
     6901 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6961 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     7021 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     7081 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     7141 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     7201 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     7261 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     7321 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     7381 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     7441 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     7501 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     7561 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     7621 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     7681 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     7741 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     7801 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     7861 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     7921 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     7981 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     8041 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     8101 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     8161 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     8221 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     8281 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     8341 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     8401 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     8461 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     8521 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     8581 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     8641 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     8701 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     8761 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     8821 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     8881 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     8941 aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc
     9001 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     9061 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     9121 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     9181 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     9241 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     9301 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     9361 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     9421 acttggcagt acatcaagtg tatc
//

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

P1709/pUB6-V5-HisB哺乳表达质粒

P1710/pUB6-V5-His-LacZ哺乳表达质粒

P0699/pENTR5-UbCp哺乳表达质粒

P1133/pDEST27-CMV-GST哺乳表达质粒

P2190/pCMV-SPORT6哺乳表达质粒

P3016/pcDNA3.1-ccdB-3xFLAG-V5哺乳表达质粒

P2055/pAP3neo哺乳表达质粒

P2275/pSG5哺乳表达质粒

P0164/pVAX1哺乳表达质粒

P0375/pVAX1-FLAG-N哺乳表达质粒

P0342/pTT5哺乳表达质粒