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保存条件 :-20
保质期 :2年
英文名 :Plasmid#17532
库存 :100
供应商 :上海烜雅生物科技有限公司
CAS号 :/
规格 :干粉/液体
基本信息名称:pLTR-G慢病毒包装质粒
别称: Plasmid#17532
复制子: | pUC |
---|---|
原核抗性: | Kan |
筛选标记: | G418 |
克隆菌株: | DH5a |
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: | 包装辅助 |
基因种属: | 病毒 |
基因类型: | ORF |
原核抗性: | Kan |
筛选标记: | Neo/G418 |
荧光蛋白: |
质粒简介
pLTR-G是一个慢病毒哺乳细胞VSVG包膜蛋白表达质粒。
质粒图谱
质粒序列
LOCUS Exported 5832 bp ds-DNA circular SYN 03-NOV-2017
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..5832
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 782..2317
/codon_start=1
/product="vesicular stomatitis virus G glycoprotein"
/label=VSV-G
/note="Indiana strain"
/translation="MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSD
LNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPS
VEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQ
FINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFR
SNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQT
SVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLK
YFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFP
LYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWF
SSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK"
polyA_signal 2620..2741
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(2748..3203)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3230..3334
/gene="bla"
/label=AmpR promoter
promoter 3336..3693
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 3544..3679
/label=SV40 ori
/note="SV40 origin of replication"
CDS 3728..4522
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 4754..4801
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 5130..5718
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 tagttattac cggactcaga tctcgagacc tagaaaaaca tggagcaatc acaagtagca
61 atacagcagc taacaatgct gcttgtgcct ggctagaagc acaagaggag gaagaggtgg
121 gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc
181 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caaagaagac
241 aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact
301 acacaccagg gccaggggtc agatatccac tgacctttgg atggtgctac aagctagtac
361 cagttgagcc agataaggta gaagaggcca ataaaggaga gaacaccagc ttgttacacc
421 ctgtgagcct gcatggaatg gatgaccctg agagagaagt gttagagtgg aggtttgaca
481 gccgcctagc atttcatcac gtggcccgag agctgcatcc ggagtacttc aagaactgct
541 gacatcgagc ttgctacaag ggactttccg ctggggactt tccagggagg cgtggcctgg
601 gcgggactgg ggagtggcga gccctcagat gctgcatata agcagctgct ttttgcctgt
661 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
721 ccactgctta agcctcaata aagcttctag agatccctcg acctcgagga attctgacac
781 tatgaagtgc cttttgtact tagccttttt attcattggg gtgaattgca agttcaccat
841 agtttttcca cacaaccaaa aaggaaactg gaaaaatgtt ccttctaatt accattattg
901 cccgtcaagc tcagatttaa attggcataa tgacttaata ggcacagcct tacaagtcaa
961 aatgcccaag agtcacaagg ctattcaagc agacggttgg atgtgtcatg cttccaaatg
1021 ggtcactact tgtgatttcc gctggtatgg accgaagtat ataacacatt ccatccgatc
1081 cttcactcca tctgtagaac aatgcaagga aagcattgaa caaacgaaac aaggaacttg
1141 gctgaatcca ggcttccctc ctcaaagttg tggatatgca actgtgacgg atgccgaagc
1201 agtgattgtc caggtgactc ctcaccatgt gctggttgat gaatacacag gagaatgggt
1261 tgattcacag ttcatcaacg gaaaatgcag caattacata tgccccactg tccataactc
1321 tacaacctgg cattctgact ataaggtcaa agggctatgt gattctaacc tcatttccat
1381 ggacatcacc ttcttctcag aggacggaga gctatcatcc ctgggaaagg agggcacagg
1441 gttcagaagt aactactttg cttatgaaac tggaggcaag gcctgcaaaa tgcaatactg
1501 caagcattgg ggagtcagac tcccatcagg tgtctggttc gagatggctg ataaggatct
1561 ctttgctgca gccagattcc ctgaatgccc agaagggtca agtatctctg ctccatctca
1621 gacctcagtg gatgtaagtc taattcagga cgttgagagg atcttggatt attccctctg
1681 ccaagaaacc tggagcaaaa tcagagcggg tcttccaatc tctccagtgg atctcagcta
1741 tcttgctcct aaaaacccag gaaccggtcc tgctttcacc ataatcaatg gtaccctaaa
1801 atactttgag accagataca tcagagtcga tattgctgct ccaatcctct caagaatggt
1861 cggaatgatc agtggaacta ccacagaaag ggaactgtgg gatgactggg caccatatga
1921 agacgtggaa attggaccca atggagttct gaggaccagt tcaggatata agtttccttt
1981 atacatgatt ggacatggta tgttggactc cgatcttcat cttagctcaa aggctcaggt
2041 gttcgaacat cctcacattc aagacgctgc ttcgcaactt cctgatgatg agagtttatt
2101 ttttggtgat actgggctat ccaaaaatcc aatcgagctt gtagaaggtt ggttcagtag
2161 ttggaaaagc tctattgcct cttttttctt tatcataggg ttaatcattg gactattctt
2221 ggttctccga gttggtatcc atctttgcat taaattaaag cacaccaaga aaagacagat
2281 ttatacagac atagagatga accgacttgg aaagtaactc aaatcctgca caacagattc
2341 ttcatgtttg gaccaaatca acttgtgata ccatgctcaa agaggcctca attatatttg
2401 agtttttaat ttttatgaaa aaaaaaaaaa aaaacggaat tcctcgaggg atcttccata
2461 cctaccagtt ctgcgcctgc aggtcgcggc cgatccaccg gatctagata actgatcata
2521 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
2581 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
2641 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
2701 cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt
2761 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
2821 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
2881 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
2941 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
3001 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
3061 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
3121 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
3181 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
3241 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
3301 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
3361 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
3421 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
3481 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
3541 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
3601 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
3661 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc
3721 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
3781 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
3841 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
3901 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
3961 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
4021 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
4081 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
4141 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
4201 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
4261 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
4321 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
4381 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
4441 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
4501 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
4561 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
4621 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
4681 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga
4741 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt
4801 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
4861 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
4921 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca
4981 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
5041 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
5101 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
5161 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
5221 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
5281 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
5341 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
5401 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
5461 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
5521 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
5581 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
5641 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
5701 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
5761 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
5821 accgccatgc at
//
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
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P2060/pENTR223-TWSG1人源基因质粒 |
P2061/pENTR223-TMEM86B人源基因质粒 |
P2062/pENTR223-NUDCD3人源基因质粒 |
P2063/pENTR223-TRAPPC4人源基因质粒 |
P2064/pENTR223-GJB2人源基因质粒 |
P2065/pENTR223-GSTM4人源基因质粒 |
P2066/pENTR223-FAM119A人源基因质粒 |
P2067/pENTR223-PRELID1人源基因质粒 |
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