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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pX459M
- 库存：
100
- 供应商：
上海烜雅生物科技有限公司
- 规格：
干粉/液体
基本信息
名称：pX459M哺乳编辑质粒
别称: pX459M
质粒属性
质粒简介
pX459M是一个哺乳细胞基因编辑质粒。
质粒图谱
质粒序列
LOCUS Exported 9211 bp ds-DNA circular SYN 24-SEP-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9211)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 24, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..9211
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..241
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 268..343
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_feature 428..475
/label=MCS
enhancer 476..761
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 763..1040
/label=chicken beta-actin promoter
intron 1041..1269
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 1290..1355
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1362..1382
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1407..5507
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNL-SDAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5508..5555
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 5571..5624
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 5625..6224
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
polyA_signal 6258..6465
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 6474..6614
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 6689..7144
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 7426..7530
/gene="bla"
/label=AmpR promoter
CDS 7531..8391
/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 8562..9150
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
421 aaatggctct agagtcgaca ctagtctcga gatcgataag cttcctagag gtacccgtta
481 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
541 caatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
601 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
661 atgacggtaa atggcccgcc tggcattgtg cccagtacat gaccttatgg gactttccta
721 cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
781 tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
841 tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg
901 cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga
961 gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa
1021 agcgaagcgc gcggcgggcg ggagtcgctg cgacgctgcc ttcgccccgt gccccgctcc
1081 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
1141 gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta agggtttaag
1201 ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc tgaaatcact
1261 ttttttcagg ttggaccggt gccaccatgg actataagga ccacgacgga gactacaagg
1321 atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag aagcggaagg
1381 tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg gacatcggca
1441 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
1501 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
1561 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
1621 ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg
1681 tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc
1741 acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
1801 ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc
1861 tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg
1921 acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
1981 accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt
2041 ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
2101 agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca
2161 agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg
2221 acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg
2281 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
2341 ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
2401 ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg
2461 accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct
2521 acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc
2581 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc
2641 agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
2701 tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg
2761 gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca
2821 tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg
2881 agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc
2941 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
3001 gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt
3061 tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg
3121 agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca
3181 cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg
3241 aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
3301 aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc
3361 ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca
3421 agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact
3481 tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg
3541 tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca
3601 ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc
3661 ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg
3721 gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca
3781 gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc
3841 tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc
3901 tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg
3961 acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg
4021 aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta
4081 cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata
4141 aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac
4201 agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag
4261 tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt
4321 acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg
4381 tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact
4441 acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta
4501 ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg
4561 ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg
4621 tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga
4681 atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca
4741 agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg
4801 gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca
4861 agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca
4921 gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg
4981 acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa
5041 tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg
5101 tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg
5161 agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga
5221 tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg
5281 cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt
5341 ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc
5401 ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca
5461 ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaaa aggccggcgg
5521 ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt cggcagtgga gagggcagag
5581 gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccaatgacc gagtacaagc
5641 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
5701 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
5761 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
5821 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
5881 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
5941 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
6001 ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
6061 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
6121 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
6181 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgagaattc taactagagc
6241 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
6301 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
6361 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
6421 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
6481 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
6541 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
6601 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
6661 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
6721 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
6781 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
6841 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
6901 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
6961 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
7021 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
7081 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
7141 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
7201 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
7261 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
7321 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
7381 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
7441 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
7501 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
7561 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
7621 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
7681 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
7741 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
7801 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
7861 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
7921 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
7981 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
8041 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
8101 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
8161 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
8221 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
8281 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
8341 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
8401 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
8461 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
8521 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
8581 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
8641 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
8701 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
8761 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
8821 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
8881 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
8941 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
9001 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
9061 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
9121 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
9181 ctggcctttt gctggccttt tgctcacatg t
//
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
名称：pX459M哺乳编辑质粒
别称: pX459M
| 启动子: | U6,CBH |
|---|---|
| 复制子: | pUC |
| 原核抗性: | Amp |
| 筛选标记: | Puro |
| 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： | 空载体 |
| 基因类型： | CRISPR,Cas9,sgRNA |
| 原核抗性： | Amp |
| 真核抗性： | Puro |
| 荧光蛋白： |
质粒简介
pX459M是一个哺乳细胞基因编辑质粒。
质粒图谱
质粒序列
LOCUS Exported 9211 bp ds-DNA circular SYN 24-SEP-2017
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9211)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 24, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..9211
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..241
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 268..343
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_feature 428..475
/label=MCS
enhancer 476..761
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 763..1040
/label=chicken beta-actin promoter
intron 1041..1269
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 1290..1355
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1362..1382
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1407..5507
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNL-SDAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5508..5555
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 5571..5624
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 5625..6224
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
polyA_signal 6258..6465
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 6474..6614
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 6689..7144
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 7426..7530
/gene="bla"
/label=AmpR promoter
CDS 7531..8391
/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 8562..9150
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
421 aaatggctct agagtcgaca ctagtctcga gatcgataag cttcctagag gtacccgtta
481 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
541 caatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
601 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
661 atgacggtaa atggcccgcc tggcattgtg cccagtacat gaccttatgg gactttccta
721 cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
781 tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
841 tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg
901 cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga
961 gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa
1021 agcgaagcgc gcggcgggcg ggagtcgctg cgacgctgcc ttcgccccgt gccccgctcc
1081 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
1141 gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta agggtttaag
1201 ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc tgaaatcact
1261 ttttttcagg ttggaccggt gccaccatgg actataagga ccacgacgga gactacaagg
1321 atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag aagcggaagg
1381 tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg gacatcggca
1441 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
1501 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
1561 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
1621 ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg
1681 tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc
1741 acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
1801 ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc
1861 tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg
1921 acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
1981 accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt
2041 ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
2101 agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca
2161 agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg
2221 acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg
2281 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
2341 ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
2401 ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg
2461 accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct
2521 acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc
2581 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc
2641 agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
2701 tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg
2761 gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca
2821 tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg
2881 agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc
2941 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
3001 gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt
3061 tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg
3121 agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca
3181 cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg
3241 aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
3301 aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc
3361 ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca
3421 agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact
3481 tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg
3541 tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca
3601 ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc
3661 ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg
3721 gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca
3781 gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc
3841 tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc
3901 tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg
3961 acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg
4021 aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta
4081 cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata
4141 aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac
4201 agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag
4261 tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt
4321 acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg
4381 tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact
4441 acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta
4501 ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg
4561 ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg
4621 tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga
4681 atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca
4741 agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg
4801 gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca
4861 agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca
4921 gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg
4981 acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa
5041 tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg
5101 tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg
5161 agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga
5221 tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg
5281 cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt
5341 ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc
5401 ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca
5461 ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaaa aggccggcgg
5521 ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt cggcagtgga gagggcagag
5581 gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccaatgacc gagtacaagc
5641 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
5701 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
5761 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
5821 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
5881 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
5941 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
6001 ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
6061 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
6121 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
6181 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgagaattc taactagagc
6241 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
6301 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
6361 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
6421 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
6481 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
6541 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
6601 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
6661 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
6721 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
6781 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
6841 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
6901 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
6961 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
7021 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
7081 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
7141 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
7201 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
7261 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
7321 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
7381 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
7441 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
7501 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
7561 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
7621 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
7681 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
7741 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
7801 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
7861 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
7921 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
7981 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
8041 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
8101 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
8161 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
8221 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
8281 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
8341 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
8401 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
8461 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
8521 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
8581 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
8641 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
8701 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
8761 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
8821 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
8881 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
8941 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
9001 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
9061 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
9121 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
9181 ctggcctttt gctggccttt tgctcacatg t
//
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
| P2084/pENTR223-RNF141人源基因质粒 |
| P2085/pENTR223-ZBED6CL人源基因质粒 |
| P2086/pENTR223-ATP6V1E2人源基因质粒 |
| P2087/pENTR223-CDC34人源基因质粒 |
| P2088/pENTR223-ABCD3人源基因质粒 |
| P2089/pENTR223-C6orf142-A596C人源基因质粒 |
| P2091/pENTR223-CLDN12人源基因质粒 |
| P2092/pENTR223-C1QC人源基因质粒 |
| P2093/pENTR223-FAM108A1人源基因质粒 |
| P2094/pENTR223-ZNF174人源基因质粒 |
| P2095/pENTR223-EXOC7人源基因质粒 |
| P2096/pENTR223-UBE2U人源基因质粒 |
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