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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：
ptdTomato-N1
- 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：ptdTomato-N1哺乳荧光质粒
别称: ptdTomato-N1
质粒属性
质粒简介
ptdTomato-N1 is a mammalian expression vector designed to express a protein of interest fused to the N-terminus of tdTomato. td Tomato is a member of the family of fruit fluorescent proteins (1) derived from the Discosoma sp. red fluorescent protein, DsRed (2). Because the Tomato protein has a tendency to dimerize, the vector was designed with two copies of the Tomato coding region linked together to allow intramolecular dimerization. As a result, each tdTomato RNA transcript encodes a tandem dimer of the Tomato protein (excitation and emission maxima equal 554nm and 581nm, respectively). Expression of td Tomato as a tandem dimer prevents the fused protein of interest from being forced into a dimeric complex. Fusions that retain the fluorescence properties of tdTomato can be monitored by flow cytometry and localized by fluorescence microscopy. The multiple cloning site (MCS) in ptdTomato-N1 is positioned upstream of the tdTomato coding sequence. A Kozak consensus sequence, located between the MCS and the tdTomato coding sequence, enhances translational efficiency of the unfused tdTomato protein in eukaryotic cells (3). SV40 polyadenylation signals downstream of the tdTomato coding sequence direct proper processing of the 3' end of the tdTomato (or fusion gene) mRNA.
质粒图谱
质粒序列
LOCUS Exported 5443 bp ds-DNA circular SYN 22-10-2015
DEFINITION .
ACCESSION .
VERSION .
KEYWORDS Untitled 3
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5443)
AUTHORS admin
TITLE Direct Submission
JOURNAL Exported 2015-10-22 from MLCC
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..5443
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 61..364
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 591..671
/note="MCS"
/note="multiple cloning site"
CDS 679..2109
/codon_start=1
/product="tandem dimeric (pseudo-monomeric) derivative of
DsRed (Shaner et al., 2004)"
/note="tdTomato"
/note="mammalian codon-optimized"
/translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
RSEGRHHLFLYGMDELYK"
polyA_signal 2231..2352
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(2359..2814)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2841..2945
/gene="bla"
/note="AmpR promoter"
promoter 2947..3304
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 3155..3290
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 3339..4133
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 4365..4412
/note="HSV TK poly(A) signal"
/note="herpesvirus thymidine kinase polyadenylation signal"
rep_origin 4741..5329
/direction=RIGHT
/note="ori"
/note="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 tcgccaccat ggtgagcaag ggcgaggagg tcatcaaaga gttcatgcgc
721 ttcaaggtgc gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc
781 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc
841 ctgcccttcg cctgggacat cctgtccccc cagttcatgt acggctccaa ggcgtacgtg
901 aagcaccccg ccgacatccc cgattacaag aagctgtcct tccccgaggg cttcaagtgg
961 gagcgcgtga tgaacttcga ggacggcggt ctggtgaccg tgacccagga ctcctccctg
1021 caggacggca cgctgatcta caaggtgaag atgcgcggca ccaacttccc ccccgacggc
1081 cccgtaatgc agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc
1141 gacggcgtgc tgaagggcga gatccaccag gccctgaagc tgaaggacgg cggccactac
1201 ctggtggagt tcaagaccat ctacatggcc aagaagcccg tgcaactgcc cggctactac
1261 tacgtggaca ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag
1321 tacgagcgct ccgagggccg ccaccacctg ttcctggggc atggcaccgg cagcaccggc
1381 agcggcagct ccggcaccgc ctcctccgag gacaacaaca tggccgtcat caaagagttc
1441 atgcgcttca aggtgcgcat ggagggctcc atgaacggcc acgagttcga gatcgagggc
1501 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggc
1561 ggccccctgc ccttcgcctg ggacatcctg tccccccagt tcatgtacgg ctccaaggcg
1621 tacgtgaagc accccgccga catccccgat tacaagaagc tgtccttccc cgagggcttc
1681 aagtgggagc gcgtgatgaa cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc
1741 tccctgcagg acggcacgct gatctacaag gtgaagatgc gcggcaccaa cttccccccc
1801 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga gcgcctgtac
1861 ccccgcgacg gcgtgctgaa gggcgagatc caccaggccc tgaagctgaa ggacggcggc
1921 cactacctgg tggagttcaa gaccatctac atggccaaga agcccgtgca actgcccggc
1981 tactactacg tggacaccaa gctggacatc acctcccaca acgaggacta caccatcgtg
2041 gaacagtacg agcgctccga gggccgccac cacctgttcc tgtacggcat ggacgagctg
2101 tacaagtagg cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
2161 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
2221 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
2281 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
2341 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
2401 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
2461 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
2521 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
2581 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
2641 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
2701 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
2761 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
2821 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
2881 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
2941 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
3001 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
3061 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
3121 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
3181 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
3241 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
3301 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
3361 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
3421 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
3481 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
3541 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
3601 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
3661 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
3721 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
3781 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
3841 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
3901 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
3961 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
4021 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
4081 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
4141 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
4201 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
4261 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
4321 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
4381 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
4441 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
4501 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
4561 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
4621 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
4681 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
4741 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
4801 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
4861 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
4921 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
4981 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
5041 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
5101 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
5161 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
5221 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
5281 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
5341 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
5401 gttatcccct gattctgtgg ataaccgtat taccgccatg cat
//
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：ptdTomato-N1哺乳荧光质粒
别称: ptdTomato-N1
| 启动子: | CMV |
|---|---|
| 复制子: | pUC |
| 终止子: | SV40 poly(A) signal |
| 质粒分类: | 哺乳细胞，荧光蛋白报告载体 |
| 质粒大小: | 5443bp |
| 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: | 哺乳细胞 |
| 诱导方式: | 无须诱导，瞬时表达 |
| 5'测序引物: | CMV-F：CGCAAATGGGCGGTAGGCGTG |
| 3'测序引物: | 根据序列设计引物 |
质粒属性
| 载体宿主： | 哺乳细胞 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | |
| 基因类型： | ORF |
| 原核抗性： | Kan |
| 真核抗性： | Neo/G418 |
| 荧光蛋白： |
质粒简介
ptdTomato-N1 is a mammalian expression vector designed to express a protein of interest fused to the N-terminus of tdTomato. td Tomato is a member of the family of fruit fluorescent proteins (1) derived from the Discosoma sp. red fluorescent protein, DsRed (2). Because the Tomato protein has a tendency to dimerize, the vector was designed with two copies of the Tomato coding region linked together to allow intramolecular dimerization. As a result, each tdTomato RNA transcript encodes a tandem dimer of the Tomato protein (excitation and emission maxima equal 554nm and 581nm, respectively). Expression of td Tomato as a tandem dimer prevents the fused protein of interest from being forced into a dimeric complex. Fusions that retain the fluorescence properties of tdTomato can be monitored by flow cytometry and localized by fluorescence microscopy. The multiple cloning site (MCS) in ptdTomato-N1 is positioned upstream of the tdTomato coding sequence. A Kozak consensus sequence, located between the MCS and the tdTomato coding sequence, enhances translational efficiency of the unfused tdTomato protein in eukaryotic cells (3). SV40 polyadenylation signals downstream of the tdTomato coding sequence direct proper processing of the 3' end of the tdTomato (or fusion gene) mRNA.
质粒图谱
质粒序列
LOCUS Exported 5443 bp ds-DNA circular SYN 22-10-2015
DEFINITION .
ACCESSION .
VERSION .
KEYWORDS Untitled 3
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5443)
AUTHORS admin
TITLE Direct Submission
JOURNAL Exported 2015-10-22 from MLCC
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..5443
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 61..364
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 591..671
/note="MCS"
/note="multiple cloning site"
CDS 679..2109
/codon_start=1
/product="tandem dimeric (pseudo-monomeric) derivative of
DsRed (Shaner et al., 2004)"
/note="tdTomato"
/note="mammalian codon-optimized"
/translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
RSEGRHHLFLYGMDELYK"
polyA_signal 2231..2352
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(2359..2814)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2841..2945
/gene="bla"
/note="AmpR promoter"
promoter 2947..3304
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 3155..3290
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 3339..4133
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 4365..4412
/note="HSV TK poly(A) signal"
/note="herpesvirus thymidine kinase polyadenylation signal"
rep_origin 4741..5329
/direction=RIGHT
/note="ori"
/note="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 tcgccaccat ggtgagcaag ggcgaggagg tcatcaaaga gttcatgcgc
721 ttcaaggtgc gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc
781 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc
841 ctgcccttcg cctgggacat cctgtccccc cagttcatgt acggctccaa ggcgtacgtg
901 aagcaccccg ccgacatccc cgattacaag aagctgtcct tccccgaggg cttcaagtgg
961 gagcgcgtga tgaacttcga ggacggcggt ctggtgaccg tgacccagga ctcctccctg
1021 caggacggca cgctgatcta caaggtgaag atgcgcggca ccaacttccc ccccgacggc
1081 cccgtaatgc agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc
1141 gacggcgtgc tgaagggcga gatccaccag gccctgaagc tgaaggacgg cggccactac
1201 ctggtggagt tcaagaccat ctacatggcc aagaagcccg tgcaactgcc cggctactac
1261 tacgtggaca ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag
1321 tacgagcgct ccgagggccg ccaccacctg ttcctggggc atggcaccgg cagcaccggc
1381 agcggcagct ccggcaccgc ctcctccgag gacaacaaca tggccgtcat caaagagttc
1441 atgcgcttca aggtgcgcat ggagggctcc atgaacggcc acgagttcga gatcgagggc
1501 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggc
1561 ggccccctgc ccttcgcctg ggacatcctg tccccccagt tcatgtacgg ctccaaggcg
1621 tacgtgaagc accccgccga catccccgat tacaagaagc tgtccttccc cgagggcttc
1681 aagtgggagc gcgtgatgaa cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc
1741 tccctgcagg acggcacgct gatctacaag gtgaagatgc gcggcaccaa cttccccccc
1801 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga gcgcctgtac
1861 ccccgcgacg gcgtgctgaa gggcgagatc caccaggccc tgaagctgaa ggacggcggc
1921 cactacctgg tggagttcaa gaccatctac atggccaaga agcccgtgca actgcccggc
1981 tactactacg tggacaccaa gctggacatc acctcccaca acgaggacta caccatcgtg
2041 gaacagtacg agcgctccga gggccgccac cacctgttcc tgtacggcat ggacgagctg
2101 tacaagtagg cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
2161 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
2221 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
2281 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
2341 caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
2401 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
2461 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
2521 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
2581 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
2641 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
2701 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
2761 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
2821 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
2881 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
2941 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
3001 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
3061 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
3121 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
3181 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
3241 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
3301 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
3361 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
3421 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
3481 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
3541 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
3601 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
3661 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
3721 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
3781 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
3841 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
3901 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
3961 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
4021 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
4081 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
4141 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
4201 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
4261 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
4321 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
4381 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
4441 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
4501 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
4561 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
4621 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
4681 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
4741 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
4801 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
4861 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
4921 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
4981 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
5041 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
5101 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
5161 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
5221 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
5281 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
5341 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
5401 gttatcccct gattctgtgg ataaccgtat taccgccatg cat
//
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
| P3144/pENTR223-HMGN2人源基因质粒 |
| P3145/pENTR223-GCK人源基因质粒 |
| P3146/pENTR223-DKC1（2点突变）人源基因质粒 |
| P3147/pENTR223-TUBG1（多了一碱基）人源基因质粒 |
| P3148/pENTR223-SUMO2人源基因质粒 |
| P3149/pENTR223-TCEAL7（1点突变）人源基因质粒 |
| P3150/pENTR223-VAMP3人源基因质粒 |
| P3151/pENTR223-S100A14人源基因质粒 |
| P3223/pENTR223-CRIPT人源基因质粒 |
| P3224/pENTR223-TMEM208人源基因质粒 |
| P3225/pENTR223-RAB33A人源基因质粒 |
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ptdTomato-N1哺乳荧光质粒
￥100 - 1000
