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- 详细信息
- 询价记录
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pEGFPN2
- 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：pEGFP-N2哺乳荧光质粒
别称:pEGFPN2
质粒属性
质粒简介
pEGFP-N2 encodes a red-shifted variant of wild-type GFP which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) pEGFP-N1 encodes the GFPmut1 variant which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences. Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-N1 is between the immediate early promoter of CMV and the EGFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of EGFP if they are in the same reading frame as EGFP and there are no intervening stop codons. SV40 polyadenylation signals downstream of the EGFP gene direct proper processing of the 3' end of the EGFP mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette, consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pEGFP-N1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
Fusions to the N terminus of EGFP retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pEGFP-N1 so that it is in frame with the EGFP coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 . pEGFP-N1 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).
质粒图谱
质粒序列
LOCUS Exported 4737 bp ds-DNA circular SYN 30-AUG-2016
DEFINITION synthetic circular DNA
KEYWORDS Untitled 8
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4737)
AUTHORS admin
TITLE Direct Submission
JOURNAL Exported 2015-10-22 from MLCC http://www.miaolingbio.com
REFERENCE 2 (bases 1 to 4737)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Tuesday, August 30, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..4737
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 61..364
/note="CMV enhancer"
/note="CMV enhancer;human cytomegalovirus immediate early
enhancer"
promoter 365..568
/note="CMV promoter"
/note="CMV promoter;human cytomegalovirus (CMV) immediate
early promoter"
misc_feature 609..665
/note="MCS"
/note="MCS;multiple cloning site"
CDS 683..1402
/codon_start=1
/product="enhanced GFP"
/note="enhanced GFP"
/note="EGFP;mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 1525..1646
/note="SV40 poly(A) signal"
/note="SV40 poly(A) signal;SV40 polyadenylation signal"
rep_origin complement(1653..2108)
/direction=LEFT
/note="f1 ori"
/note="f1 ori;f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2135..2239
/gene="bla"
/note="bla AmpR promoter"
/note="AmpR promoter"
promoter 2241..2598
/note="SV40 promoter"
/note="SV40 promoter;SV40 enhancer and early promoter"
rep_origin 2449..2584
/note="SV40 ori"
/note="SV40 ori;SV40 origin of replication"
CDS 2633..3427
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="aph(3')-II (or nptII)"
/note="NeoR/KanR;confers resistance to neomycin, kanamycin,
and G418 (Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATC-PFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3659..3706
/note="HSV TK poly(A) signal"
/note="HSV TK poly(A) signal;herpesvirus thymidine kinase
polyadenylation signal"
rep_origin 4035..4623
/direction=RIGHT
/note="ori"
/note="ori;high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
661 gatccaccgg ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
721 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
781 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
841 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
901 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
961 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
1021 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
1081 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
1141 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
1201 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
1261 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
1321 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
1381 catggacgag ctgtacaagt aaagcggccg cgactctaga tcataatcag ccataccaca
1441 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
1501 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
1561 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
1621 ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa
1681 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
1741 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
1801 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
1861 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
1921 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
1981 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
2041 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
2101 gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
2161 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
2221 atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
2281 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
2341 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
2401 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
2461 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
2521 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
2581 ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga
2641 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
2701 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
2761 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
2821 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
2881 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
2941 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
3001 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
3061 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
3121 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
3181 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
3241 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
3301 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
3361 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
3421 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
3481 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
3541 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct
3601 agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
3661 gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt
3721 cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc
3781 cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca
3841 gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt
3901 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
3961 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
4021 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
4081 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
4141 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
4201 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
4261 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
4321 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
4381 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
4441 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
4501 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
4561 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
4621 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
4681 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcat
//
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：pEGFP-N2哺乳荧光质粒
别称:pEGFPN2
| 启动子: | CMV |
|---|---|
| 复制子: | pUC |
| 终止子: | SV40 poly(A) signal |
| 质粒分类: | 哺乳系列质粒；哺乳荧光质粒；哺乳绿色质粒 |
| 质粒大小: | 4737bp |
| 质粒标签: | C-EGFP |
| 原核抗性: | Kan |
| 筛选标记: | G418 |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 293T等哺乳细胞 |
| 培养条件: | 37℃，有氧，5%CO2 |
| 诱导方式: | 无须诱导，瞬时表达 |
| 5'测序引物: | pEGFP-N-5：TGGGAGGTCTATATAAGCAGAG |
| 3'测序引物: | pEGFP-N-3: CGTCGCCGTCCAGCTCGACCAG |
质粒属性
| 载体宿主： | 哺乳细胞 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | 空载体 |
| 基因类型： | ORF |
| 原核抗性： | Kan |
| 真核抗性： | Neo/G418 |
| 荧光蛋白： |
质粒简介
pEGFP-N2 encodes a red-shifted variant of wild-type GFP which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) pEGFP-N1 encodes the GFPmut1 variant which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences. Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-N1 is between the immediate early promoter of CMV and the EGFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of EGFP if they are in the same reading frame as EGFP and there are no intervening stop codons. SV40 polyadenylation signals downstream of the EGFP gene direct proper processing of the 3' end of the EGFP mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette, consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pEGFP-N1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
Fusions to the N terminus of EGFP retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pEGFP-N1 so that it is in frame with the EGFP coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 . pEGFP-N1 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).
质粒图谱
质粒序列
LOCUS Exported 4737 bp ds-DNA circular SYN 30-AUG-2016
DEFINITION synthetic circular DNA
KEYWORDS Untitled 8
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4737)
AUTHORS admin
TITLE Direct Submission
JOURNAL Exported 2015-10-22 from MLCC http://www.miaolingbio.com
REFERENCE 2 (bases 1 to 4737)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Tuesday, August 30, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..4737
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 61..364
/note="CMV enhancer"
/note="CMV enhancer;human cytomegalovirus immediate early
enhancer"
promoter 365..568
/note="CMV promoter"
/note="CMV promoter;human cytomegalovirus (CMV) immediate
early promoter"
misc_feature 609..665
/note="MCS"
/note="MCS;multiple cloning site"
CDS 683..1402
/codon_start=1
/product="enhanced GFP"
/note="enhanced GFP"
/note="EGFP;mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 1525..1646
/note="SV40 poly(A) signal"
/note="SV40 poly(A) signal;SV40 polyadenylation signal"
rep_origin complement(1653..2108)
/direction=LEFT
/note="f1 ori"
/note="f1 ori;f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2135..2239
/gene="bla"
/note="bla AmpR promoter"
/note="AmpR promoter"
promoter 2241..2598
/note="SV40 promoter"
/note="SV40 promoter;SV40 enhancer and early promoter"
rep_origin 2449..2584
/note="SV40 ori"
/note="SV40 ori;SV40 origin of replication"
CDS 2633..3427
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="aph(3')-II (or nptII)"
/note="NeoR/KanR;confers resistance to neomycin, kanamycin,
and G418 (Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATC-PFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3659..3706
/note="HSV TK poly(A) signal"
/note="HSV TK poly(A) signal;herpesvirus thymidine kinase
polyadenylation signal"
rep_origin 4035..4623
/direction=RIGHT
/note="ori"
/note="ori;high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
661 gatccaccgg ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
721 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
781 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
841 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
901 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
961 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
1021 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
1081 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
1141 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
1201 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
1261 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
1321 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
1381 catggacgag ctgtacaagt aaagcggccg cgactctaga tcataatcag ccataccaca
1441 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
1501 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
1561 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
1621 ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa
1681 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
1741 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
1801 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
1861 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
1921 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
1981 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
2041 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
2101 gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
2161 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
2221 atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
2281 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
2341 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
2401 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
2461 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
2521 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
2581 ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga
2641 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
2701 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
2761 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
2821 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
2881 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
2941 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
3001 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
3061 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
3121 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
3181 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
3241 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
3301 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
3361 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
3421 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
3481 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
3541 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct
3601 agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
3661 gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt
3721 cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc
3781 cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca
3841 gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt
3901 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
3961 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
4021 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
4081 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
4141 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
4201 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
4261 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
4321 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
4381 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
4441 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
4501 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
4561 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
4621 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
4681 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcat
//
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
| P4780/pCMV-SPORT6-RNF11人源基因质粒 |
| P4781/pCMV-SPORT6-MEOX2人源基因质粒 |
| P4782/pCMV-SPORT6-VPS33B（点突变）人源基因质粒 |
| P4783/pCMV-SPORT6-SDF4（1同义突变）人源基因质粒 |
| P4784/pCMV-SPORT6-SEMA3B人源基因质粒 |
| P4785/pCMV-SPORT6-STAR人源基因质粒 |
| P4786/pCMV-SPORT6-SCAPER人源基因质粒 |
| P4787/pCMV-SPORT6-MGME1人源基因质粒 |
| P4788/pCMV-SPORT6-ETFA人源基因质粒 |
| P4789/pCMV-SPORT6-CD40人源基因质粒 |
| P4790/pCMV-SPORT6-FA2H人源基因质粒 |
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文献和实验相关实验
Restriction Map and Multiple Cloning Site of pEGFP-N2. (Unique restriction sites are in color or bold.) The Not I site follows the EGFP stop codon. The Nhe I site cannot be used for fusions since it contains an in-frame stop codon
出来，可能两个原因，第一个是抗体不好，第二个可能是质粒没做好，发生移码了，因为你用的是pEGFP-N2载体，gfp是在n端，就算发生了移码一样可以检测到绿色萤光，仔细检查下你做的质粒看看是否正确。 liuruya 谢谢大家的回复 1.WB应该是没有技术问题，用的是GFP或flag的标签抗体，而且设置了之前做过的基因做阳性对照 2.GFP质粒测序过是完整的且没有移码，flag的是自己构建的也反复check过没有问题 觉得很妖怪 问了当时给我质粒
doctorchihlee 大家好，请问一下空白载体（含GFP,但不含靶基因）成功转染哺乳动物细胞后，荧光显微镜下观察GFP，观察到细胞全部呈绿色还是仅仅外周发光或是核发光？谢谢！ doctorchihlee GFP在细胞质，是全部发光？！？ real_madrid_146 这个是我用pEGFP-C2转HEK293的图。 shylook 就是这样的
pEGFP-N2哺乳荧光质粒
￥100 - 1000
