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保存条件 :-20
保质期 :2年
英文名 :p2Luc
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:p2Luc哺乳报告质粒
别称: NM_001308090.1;HD7;HD7A;HDAC7A
启动子: | SV40 |
---|---|
复制子: | pUC |
终止子: | SV40 poly(A) signal |
原核抗性: | Amp |
筛选标记: | 荧光素酶Luc |
克隆菌株: | DH5a |
培养条件: | 37度 |
质粒属性
载体宿主: | 哺乳细胞 |
---|---|
载体用途: | 信号报告 |
基因种属: | |
基因类型: | |
原核抗性: | Amp |
真核抗性: | |
荧光蛋白: | fLuc |
质粒简介
p2Luc是一个哺乳细胞双荧光素酶报告质粒。
质粒图谱
质粒序列
LOCUS Exported 5397 bp ds-DNA circular SYN 17-JAN-2018
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..5397
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 66..423
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 274..409
/note="SV40 ori"
/note="SV40 origin of replication"
intron 493..625
/note="chimeric intron"
/note="chimera between introns from human beta-globin and
immunoglobulin heavy chain genes"
promoter 670..688
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 698..1630
/codon_start=1
/product="luciferase from the anthozoan coelenterate
Renilla reniformis (sea pansy)"
/note="Rluc"
/translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
misc_feature 1633..1662
/note="MCS"
CDS 1663..3312
/codon_start=1
/gene="luc"
/product="firefly luciferase"
/note="luciferase"
/translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH
IEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
NDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI
QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
VDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
polyA_signal 3352..3473
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
promoter 3606..3710
/gene="bla"
/note="AmpR promoter"
CDS 3711..4571
/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 4742..5330
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 agatcgatct gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt
61 accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
121 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
181 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
241 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
301 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
361 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
421 aaaaagcttg attcttctga cacaacagtc tcgaacttaa gctgcagaag ttggtcgtga
481 ggcactgggc aggtaagtat caaggttaca agacaggttt aaggagacca atagaaactg
541 ggcttgtcga gacagagaag actcttgcgt ttctgatagg cacctattgg tcttactgac
601 atccactttg cctttctctc cacaggtgtc cactcccagt tcaattacag ctcttaaggc
661 tagagtactt aatacgactc actataggct agccaccatg acttcgaaag tttatgatcc
721 agaacaaagg aaacggatga taactggtcc gcagtggtgg gccagatgta aacaaatgaa
781 tgttcttgat tcatttatta attattatga ttcagaaaaa catgcagaaa atgctgttat
841 ttttttacat ggtaacgcgg cctcttctta tttatggcga catgttgtgc cacatattga
901 gccagtagcg cggtgtatta taccagacct tattggtatg ggcaaatcag gcaaatctgg
961 taatggttct tataggttac ttgatcatta caaatatctt actgcatggt ttgaacttct
1021 taatttacca aagaagatca tttttgtcgg ccatgattgg ggtgcttgtt tggcatttca
1081 ttatagctat gagcatcaag ataagatcaa agcaatagtt cacgctgaaa gtgtagtaga
1141 tgtgattgaa tcatgggatg aatggcctga tattgaagaa gatattgcgt tgatcaaatc
1201 tgaagaagga gaaaaaatgg ttttggagaa taacttcttc gtggaaacca tgttgccatc
1261 aaaaatcatg agaaagttag aaccagaaga atttgcagca tatcttgaac cattcaaaga
1321 gaaaggtgaa gttcgtcgtc caacattatc atggcctcgt gaaatcccgt tagtaaaagg
1381 tggtaaacct gacgttgtac aaattgttag gaattataat gcttatctac gtgcaagtga
1441 tgatttacca aaaatgttta ttgaatcgga cccaggattc ttttccaatg ctattgttga
1501 aggtgccaag aagtttccta atactgaatt tgtcaaagta aaaggtcttc atttttcgca
1561 agaagatgca cctgatgaaa tgggaaaata tatcaaatcg ttcgttgagc gagttctcaa
1621 aaatgaacaa atgtcgacgg atccttcaac ttccctgagc tcgaagacgc caaaaacata
1681 aagaaaggcc cggcgccatt ctatcctcta gaggatggaa ccgctggaga gcaactgcat
1741 aaggctatga agagatacgc cctggttcct ggaacaattg cttttacaga tgcacatatc
1801 gaggtgaaca tcacgtacgc ggaatacttc gaaatgtccg ttcggttggc agaagctatg
1861 aaacgatatg ggctgaatac aaatcacaga atcgtcgtat gcagtgaaaa ctctcttcaa
1921 ttctttatgc cggtgttggg cgcgttattt atcggagttg cagttgcgcc cgcgaacgac
1981 atttataatg aacgtgaatt gctcaacagt atgaacattt cgcagcctac cgtagtgttt
2041 gtttccaaaa aggggttgca aaaaattttg aacgtgcaaa aaaaattacc aataatccag
2101 aaaattatta tcatggattc taaaacggat taccagggat ttcagtcgat gtacacgttc
2161 gtcacatctc atctacctcc cggttttaat gaatacgatt ttgtaccaga gtcctttgat
2221 cgtgacaaaa caattgcact gataatgaat tcctctggat ctactgggtt acctaagggt
2281 gtggcccttc cgcatagaac tgcctgcgtc agattctcgc atgccagaga tcctattttt
2341 ggcaatcaaa tcattccgga tactgcgatt ttaagtgttg ttccattcca tcacggtttt
2401 ggaatgttta ctacactcgg atatttgata tgtggatttc gagtcgtctt aatgtataga
2461 tttgaagaag agctgttttt acgatccctt caggattaca aaattcaaag tgcgttgcta
2521 gtaccaaccc tattttcatt cttcgccaaa agcactctga ttgacaaata cgatttatct
2581 aatttacacg aaattgcttc tgggggcgca cctctttcga aagaagtcgg ggaagcggtt
2641 gcaaaacgct tccatcttcc agggatacga caaggatatg ggctcactga gactacatca
2701 gctattctga ttacacccga gggggatgat aaaccgggcg cggtcggtaa agttgttcca
2761 ttttttgaag cgaaggttgt ggatctggat accgggaaaa cgctgggcgt taatcagaga
2821 ggcgaattat gtgtcagagg acctatgatt atgtccggtt atgtaaacaa tccggaagcg
2881 accaacgcct tgattgacaa ggatggatgg ctacattctg gagacatagc ttactgggac
2941 gaagacgaac acttcttcat agttgaccgc ttgaagtctt taattaaata caaaggatat
3001 caggtggccc ccgctgaatt ggaatcgata ttgttacaac accccaacat cttcgacgcg
3061 ggcgtggcag gtcttcccga cgatgacgcc ggtgaacttc ccgccgccgt tgttgttttg
3121 gagcacggaa agacgatgac ggaaaaagag atcgtggatt acgtcgccag tcaagtaaca
3181 accgcgaaaa agttgcgcgg aggagttgtg tttgtggacg aagtaccgaa aggtcttacc
3241 ggaaaactcg acgcaagaaa aatcagagag atcctcataa aggccaagaa gggcggaaag
3301 tccaaattgt aacacgtgta attctagagc ggccgcttcg agcagacatg ataagataca
3361 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
3421 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
3481 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
3541 agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc aggtggcact tttcggggaa
3601 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
3661 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
3721 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
3781 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
3841 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
3901 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
3961 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
4021 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
4081 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
4141 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
4201 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
4261 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
4321 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
4381 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
4441 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
4501 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
4561 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
4621 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
4681 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
4741 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
4801 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
4861 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
4921 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
4981 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
5041 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
5101 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
5161 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
5221 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
5281 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
5341 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg gctcgac
//
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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