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- 详细信息
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
Plasmid#72833
- 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：AAVS1 T2 CRIPR in pX330哺乳编辑质粒
别称: Plasmid#72833
质粒属性
质粒简介
AAVS1 T2 CRIPR in pX330哺乳编辑质粒
质粒图谱
质粒序列
LOCUS Exported 8508 bp ds-DNA circular SYN 17-JUL-2018
DEFINITION synthetic circular DNA
KEYWORDS AAVS1 T2 CRIPR in pX330
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8508)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8508
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..241
/note="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 250..269
/note="AAVS1 gRNA"
misc_RNA 270..345
/note="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 442..727
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 729..1006
/note="chicken beta-actin promoter"
intron 1007..1235
/note="hybrid intron"
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 1247..1256
/regulatory_class="other"
/note="Kozak"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1256..1321
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1328..1348
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1373..5473
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/note="Cas9"
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLS-DAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5474..5521
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
polyA_signal 5555..5762
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
repeat_region 5771..5911
/note="AAV2 ITR"
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 5986..6441
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6723..6827
/gene="bla"
/note="AmpR promoter"
CDS 6828..7688
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 7859..8447
/direction=RIGHT
/note="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 gggccactag ggacaggatg ttttagagct agaaatagca agttaaaata
301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc
481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
841 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg
901 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
961 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac
1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta
1261 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa
1321 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa
1381 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
1441 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
1501 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
1561 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
1621 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
1681 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
1741 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
1801 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
1861 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
1921 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
1981 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
2041 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
2101 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
2161 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
2221 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
2281 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
2341 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
2401 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
2461 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
2521 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
2581 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
2641 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
2701 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
2761 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
2821 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
2881 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
2941 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
3001 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
3061 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
3121 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
3181 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
3241 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
3301 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
3361 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
3421 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
3481 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
3541 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
3601 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
3661 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
3721 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
3781 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
3841 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
3901 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
3961 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
4021 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
4081 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
4141 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
4201 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
4261 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
4321 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
4381 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
4441 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
4501 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
4561 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
4621 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
4681 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
4741 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
4801 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
4861 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
4921 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
4981 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
5041 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
5101 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
5161 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
5221 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
5281 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
5341 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
5401 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
5461 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
5521 gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
5581 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
5641 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
5701 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
5761 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
5821 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
5881 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
5941 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
6001 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
6061 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
6121 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
6181 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
6241 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
6301 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
6361 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
6421 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
6481 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
6541 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
6601 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
6661 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
6721 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
6781 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
6841 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
6901 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
6961 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
7021 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
7081 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
7141 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
7201 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
7261 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
7321 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
7381 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
7441 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
7501 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
7561 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
7621 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
7681 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
7741 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
7801 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
7861 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
7921 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
7981 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
8041 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
8101 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
8161 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
8221 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
8281 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
8341 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
8401 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
8461 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
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：AAVS1 T2 CRIPR in pX330哺乳编辑质粒
别称: Plasmid#72833
| 启动子: | U6 |
|---|---|
| 复制子: | pUC |
| 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,腺相关病毒 |
|---|---|
| 载体用途： | 基因敲入 |
| 基因种属： | 空载体 |
| 基因类型： | CRISPR,Cas9,sgRNA |
| 原核抗性： | Amp |
| 真核抗性： | |
| 荧光蛋白： |
质粒简介
AAVS1 T2 CRIPR in pX330哺乳编辑质粒
质粒图谱
质粒序列
LOCUS Exported 8508 bp ds-DNA circular SYN 17-JUL-2018
DEFINITION synthetic circular DNA
KEYWORDS AAVS1 T2 CRIPR in pX330
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8508)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8508
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..241
/note="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 250..269
/note="AAVS1 gRNA"
misc_RNA 270..345
/note="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 442..727
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 729..1006
/note="chicken beta-actin promoter"
intron 1007..1235
/note="hybrid intron"
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 1247..1256
/regulatory_class="other"
/note="Kozak"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1256..1321
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1328..1348
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1373..5473
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/note="Cas9"
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLS-DAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRL-SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5474..5521
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
polyA_signal 5555..5762
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
repeat_region 5771..5911
/note="AAV2 ITR"
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 5986..6441
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6723..6827
/gene="bla"
/note="AmpR promoter"
CDS 6828..7688
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 7859..8447
/direction=RIGHT
/note="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 gggccactag ggacaggatg ttttagagct agaaatagca agttaaaata
301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc
481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
841 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg
901 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
961 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac
1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta
1261 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa
1321 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa
1381 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
1441 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
1501 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
1561 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
1621 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
1681 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
1741 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
1801 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
1861 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
1921 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
1981 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
2041 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
2101 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
2161 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
2221 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
2281 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
2341 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
2401 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
2461 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
2521 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
2581 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
2641 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
2701 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
2761 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
2821 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
2881 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
2941 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
3001 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
3061 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
3121 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
3181 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
3241 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
3301 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
3361 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
3421 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
3481 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
3541 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
3601 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
3661 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
3721 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
3781 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
3841 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
3901 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
3961 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
4021 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
4081 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
4141 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
4201 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
4261 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
4321 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
4381 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
4441 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
4501 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
4561 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
4621 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
4681 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
4741 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
4801 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
4861 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
4921 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
4981 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
5041 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
5101 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
5161 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
5221 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
5281 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
5341 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
5401 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
5461 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
5521 gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
5581 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
5641 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
5701 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
5761 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
5821 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
5881 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
5941 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
6001 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
6061 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
6121 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
6181 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
6241 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
6301 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
6361 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
6421 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
6481 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
6541 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
6601 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
6661 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
6721 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
6781 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
6841 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
6901 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
6961 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
7021 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
7081 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
7141 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
7201 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
7261 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
7321 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
7381 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
7441 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
7501 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
7561 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
7621 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
7681 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
7741 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
7801 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
7861 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
7921 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
7981 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
8041 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
8101 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
8161 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
8221 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
8281 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
8341 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
8401 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
8461 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
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
| P4101/pCDNA3.1-3×HA-TBK1人源基因质粒 |
| P4102/pCDNA3.1-3×HA-TRAF3人源基因质粒 |
| P4103/pCDNA3.1-3×HA-MAVS人源基因质粒 |
| P4104/pCDNA3.1-3×HA-MDA5人源基因质粒 |
| P4263/pCDNA3.1-HA-STAT1-Myc人源基因质粒 |
| P4105/pCDNA3.1-HA-STAT2-Myc人源基因质粒 |
| P4106/pLVX-IRES-Puro-MEN1人源基因质粒 |
| P1235/pCDH-puro-Bcl-XL人源基因质粒 |
| P1745/pActin-EGFP-beta-Actin (Homo)人源基因质粒 |
| P1246/pECMV-3×FLAG-DRD2人源基因质粒 |
| P1247/pECMV-3×FLAG-DRD3人源基因质粒 |
| P1248/pECMV-3×FLAG-PCSK9人源基因质粒 |
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AAVS1 T2 CRIPR in pX330哺乳编辑质粒
￥100 - 1000
