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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：
pMIR-Report Luciferase
- 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：pMIR-Report Luciferase哺乳报告质粒
别称: pMIR-Report Luciferase
质粒属性
质粒简介
The pMIR-REPORT miRNA Expression Reporter Vector System consists of an experimental firefly luciferase reporter vector and an associated β-gal reporter control plasmid. By inserting predicted miRNA target sequences in the multiple cloning site, pMIR-REPORT Luciferase miRNA Expression Reporter Vector can be used to conduct accurate, quantitative evaluations of miRNA function. It can also be used to evaluate siRNA target sites and to analyze the influence of 3' UTR sequences on gene expression. pMIR-REPORT Luciferase contains a firefly luciferase reporter gene under the control of a CMV promoter/termination system. The 3' UTR of the luciferase gene contains a multiple cloning site for insertion of predicted miRNA binding targets or other nucleotide sequences. By cloning a predicted miRNA target sequence into pMIR-REPORT, the luciferase reporter is subjected to regulation that mimics the miRNA target.
质粒图谱
质粒序列
LOCUS Exported 6470 bp ds-DNA circular SYN 12-JUL-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pMIR-Reporter
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6470)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sunday, September 4, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..6470
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 379..395
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
CDS complement(551..2200)
/codon_start=1
/gene="luc"
/product="firefly luciferase"
/note="luciferase"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
RDPIFGNQIVPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
promoter complement(2225..2428)
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
enhancer 2429..2732
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter complement(2854..2872)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
promoter complement(2884..2902)
/note="SP6 promoter"
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(2922..2938)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2946..2962
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(2970..3000)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3015..3036
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3324..3912)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4083..4943)
/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"
promoter complement(4944..5048)
/gene="bla"
/note="AmpR promoter"
polyA_signal 5136..5217
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
CDS complement(5447..6046)
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
promoter complement(6143..6459)
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 6157..6292
/note="SV40 ori"
/note="SV40 origin of replication"
ORIGIN
1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
361 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctagcggcc gcatacaaaa
421 aaccaacaca cagatccaat gaaaataaaa gatcctttat taagcttaac tgtttaaacc
481 tatgatatca cgcacgcgtc tatgccggcc tatgagctcc ctcactagtg cgcagctttc
541 atcactcgag caatttggac tttccgccct tcttggcctt tatgaggatc tctctgattt
601 ttcttgcgtc gagttttccg gtaagacctt tcggtacttc gtccacaaac acaactcctc
661 cgcgcaactt tttcgcggtt gttacttgac tggcgacgta atccacgatc tctttttccg
721 tcatcgtctt tccgtgctcc aaaacaacaa cggcggcggg aagttcaccg gcgtcatcgt
781 cgggaagacc tgccacgccc gcgtcgaaga tgttggggtg ttgtaacaat atcgattcca
841 attcagcggg ggccacctga tatcctttgt atttaattaa agacttcaag cggtcaacta
901 tgaagaagtg ttcgtcttcg tcccagtaag ctatgtctcc agaatgtagc catccatcct
961 tgtcaatcaa ggcgttggtc gcttccggat tgtttacata accggacata atcataggtc
1021 ctctgacaca taattcgcct ctctgattaa cgcccagcgt tttcccggta tccagatcca
1081 caaccttcgc ttcaaaaaat ggaacaactt taccgaccgc gcccggttta tcatccccct
1141 cgggtgtaat cagaatagct gatgtagtct cagtgagccc atatccttgt cgtatccctg
1201 gaagatggaa gcgttttgca accgcttccc cgacttcttt cgaaagaggt gcgcccccag
1261 aagcaatttc gtgtaaatta gataaatcgt atttgtcaat cagagtgctt ttggcgaaga
1321 atgaaaatag ggttggtact agcaacgcac tttgaatttt gtaatcctga agggatcgta
1381 aaaacagctc ttcttcaaat ctatacatta agacgactcg aaatccacat atcaaatatc
1441 cgagtgtagt aaacattcca aaaccgtgat ggaatgggac aacacttaaa atcgcagtat
1501 ccggaacgat ttgattgcca aaaataggat ctctggcatg cgagaatctg acgcaggcag
1561 ttctatgcgg aagggccaca cccttaggta acccagtaga tccagaggaa ttcattatca
1621 gtgcaattgt tttgtcacga tcaaaggact ctggtacaaa atcgtattca ttaaaaccgg
1681 gaggtagatg agatgtgacg aacgtgtaca tcgactgaaa tccctggtaa tccgttttag
1741 aatccatgat aataattttc tggattattg gtaatttttt ttgcacgttc aaaatttttt
1801 gcaacccctt tttggaaaca aacactacgg taggctgcga aatgttcata ctgttgagca
1861 attcacgttc attataaatg tcgttcgcgg gcgcaactgc aactccgata aataacgcgc
1921 ccaacaccgg cataaagaat tgaagagagt tttcactgca tacgacgatt ctgtgatttg
1981 tattcagccc atatcgtttc atagcttctg ccaaccgaac ggacatttcg aagtattccg
2041 cgtacgtgat gttcacctcg atatgtgcat ctgtaaaagc aattgttcca ggaaccaggg
2101 cgtatctctt catagcctta tgcagttgct ctccagcggt tccatcctct agaggataga
2161 atggcgccgg gcctttcttt atgtttttgg cgtcttccat ggtggatcca cggttcacta
2221 aaccagctct gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg
2281 gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc
2341 cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc
2401 cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt
2461 actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
2521 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
2581 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg
2641 ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc
2701 caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta
2761 atgaccccgt aattgattac tattaataac taagatctgg taccttgaat tcatgcttct
2821 cctcccttta gtgagggtaa ttctctctct ctccctatag tgagtcgtat taattccttc
2881 tcttctatag tgtcacctaa atcgttgcaa ttcgtaatca tgtcatagct gtttcctgtg
2941 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
3001 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
3061 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
3121 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
3181 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
3241 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
3301 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
3361 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
3421 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
3481 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
3541 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
3601 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
3661 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
3721 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
3781 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
3841 aaaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
3901 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
3961 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
4021 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
4081 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
4141 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
4201 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
4261 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
4321 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
4381 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
4441 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
4501 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
4561 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
4621 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
4681 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
4741 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
4801 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
4861 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
4921 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
4981 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
5041 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
5101 acattaacct ataaaaatag gcgtatcacg agattgcagt gaaaaaaatg ctttatttgt
5161 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
5221 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
5281 agcaagtaaa acctctacaa atgtggtatg gctgattatg atcctctaga gtcggtgggc
5341 ctcgggggcg ggtgcggggt cggcggggcc gccccgggtg gcttcggtcg gagccatggg
5401 gtcgtgcgct cctttcggtc gggcgctgcg ggtcgtgggg cgggcgtcag gcaccgggct
5461 tgcgggtcat gcaccaggtg cgcggtcctt cgggcacctc gacgtcggcg gtgacggtga
5521 agccgagccg ctcgtagaag gggaggttgc ggggcgcgga ggtctccagg aaggcgggca
5581 ccccggcgcg ctcggccgcc tccactccgg ggagcacgac ggcgctgccc agacccttgc
5641 cctggtggtc gggcgagacg ccgacggtgg ccaggaacca cgcgggctcc ttgggccggt
5701 gcggcgccag gaggccttcc atctgttgct gcgcggccag ccgggaaccg ctcaactcgg
5761 ccatgcgcgg gccgatctcg gcgaacaccg cccccgcttc gacgctctcc ggcgtggtcc
5821 agaccgccac cgcggcgccg tcgtccgcga cccacacctt gccgatgtcg agcccgacgc
5881 gcgtgaggaa gagttcttgc agctcggtga cccgctcgat gtggcggtcc gggtcgacgg
5941 tgtggcgcgt ggcggggtag tcggcgaacg cggcggcgag ggtgcgtacg gcccggggga
6001 cgtcgtcgcg ggtggcgagg cgcaccgtgg gcttgtactc ggtcatggaa ggtcgtctcc
6061 ttgtgagggg tcaggggcgt gggtcagggg atggtggcgg caccggtcgt ggcggccgac
6121 ctgcaggcat gcaagctagc tttttgcaaa agcctaggcc tccaaaaaag cctcctcact
6181 acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaata aaaaaaatta
6241 gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag
6301 ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
6361 ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc atgctttgca
6421 tacttctgcc tgctggggag cctggggact ttccacacca atctttcgtc
//
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：pMIR-Report Luciferase哺乳报告质粒
别称: pMIR-Report Luciferase
| 启动子: | CMV |
|---|---|
| 复制子: | pUC |
| 终止子: | SV40 poly(A) signal |
| 质粒分类: | 哺乳细胞，信号通路报告载体 |
| 质粒大小: | 6470bp |
| 原核抗性: | Amp |
| 筛选标记: | Puro |
| 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: | M13F：TGTAAAACGACGGCCAGT |
质粒属性
| 载体宿主： | 哺乳细胞 |
|---|---|
| 载体用途： | 信号报告 |
| 基因种属： | 空载体 |
| 基因类型： | miRNA,UTR |
| 原核抗性： | Amp |
| 真核抗性： | Puro |
| 荧光蛋白： | fLuc |
质粒简介
The pMIR-REPORT miRNA Expression Reporter Vector System consists of an experimental firefly luciferase reporter vector and an associated β-gal reporter control plasmid. By inserting predicted miRNA target sequences in the multiple cloning site, pMIR-REPORT Luciferase miRNA Expression Reporter Vector can be used to conduct accurate, quantitative evaluations of miRNA function. It can also be used to evaluate siRNA target sites and to analyze the influence of 3' UTR sequences on gene expression. pMIR-REPORT Luciferase contains a firefly luciferase reporter gene under the control of a CMV promoter/termination system. The 3' UTR of the luciferase gene contains a multiple cloning site for insertion of predicted miRNA binding targets or other nucleotide sequences. By cloning a predicted miRNA target sequence into pMIR-REPORT, the luciferase reporter is subjected to regulation that mimics the miRNA target.
质粒图谱
质粒序列
LOCUS Exported 6470 bp ds-DNA circular SYN 12-JUL-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pMIR-Reporter
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6470)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sunday, September 4, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..6470
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 379..395
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
CDS complement(551..2200)
/codon_start=1
/gene="luc"
/product="firefly luciferase"
/note="luciferase"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
RDPIFGNQIVPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
promoter complement(2225..2428)
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
enhancer 2429..2732
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter complement(2854..2872)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
promoter complement(2884..2902)
/note="SP6 promoter"
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(2922..2938)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2946..2962
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(2970..3000)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3015..3036
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3324..3912)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4083..4943)
/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"
promoter complement(4944..5048)
/gene="bla"
/note="AmpR promoter"
polyA_signal 5136..5217
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
CDS complement(5447..6046)
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
promoter complement(6143..6459)
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 6157..6292
/note="SV40 ori"
/note="SV40 origin of replication"
ORIGIN
1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
361 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctagcggcc gcatacaaaa
421 aaccaacaca cagatccaat gaaaataaaa gatcctttat taagcttaac tgtttaaacc
481 tatgatatca cgcacgcgtc tatgccggcc tatgagctcc ctcactagtg cgcagctttc
541 atcactcgag caatttggac tttccgccct tcttggcctt tatgaggatc tctctgattt
601 ttcttgcgtc gagttttccg gtaagacctt tcggtacttc gtccacaaac acaactcctc
661 cgcgcaactt tttcgcggtt gttacttgac tggcgacgta atccacgatc tctttttccg
721 tcatcgtctt tccgtgctcc aaaacaacaa cggcggcggg aagttcaccg gcgtcatcgt
781 cgggaagacc tgccacgccc gcgtcgaaga tgttggggtg ttgtaacaat atcgattcca
841 attcagcggg ggccacctga tatcctttgt atttaattaa agacttcaag cggtcaacta
901 tgaagaagtg ttcgtcttcg tcccagtaag ctatgtctcc agaatgtagc catccatcct
961 tgtcaatcaa ggcgttggtc gcttccggat tgtttacata accggacata atcataggtc
1021 ctctgacaca taattcgcct ctctgattaa cgcccagcgt tttcccggta tccagatcca
1081 caaccttcgc ttcaaaaaat ggaacaactt taccgaccgc gcccggttta tcatccccct
1141 cgggtgtaat cagaatagct gatgtagtct cagtgagccc atatccttgt cgtatccctg
1201 gaagatggaa gcgttttgca accgcttccc cgacttcttt cgaaagaggt gcgcccccag
1261 aagcaatttc gtgtaaatta gataaatcgt atttgtcaat cagagtgctt ttggcgaaga
1321 atgaaaatag ggttggtact agcaacgcac tttgaatttt gtaatcctga agggatcgta
1381 aaaacagctc ttcttcaaat ctatacatta agacgactcg aaatccacat atcaaatatc
1441 cgagtgtagt aaacattcca aaaccgtgat ggaatgggac aacacttaaa atcgcagtat
1501 ccggaacgat ttgattgcca aaaataggat ctctggcatg cgagaatctg acgcaggcag
1561 ttctatgcgg aagggccaca cccttaggta acccagtaga tccagaggaa ttcattatca
1621 gtgcaattgt tttgtcacga tcaaaggact ctggtacaaa atcgtattca ttaaaaccgg
1681 gaggtagatg agatgtgacg aacgtgtaca tcgactgaaa tccctggtaa tccgttttag
1741 aatccatgat aataattttc tggattattg gtaatttttt ttgcacgttc aaaatttttt
1801 gcaacccctt tttggaaaca aacactacgg taggctgcga aatgttcata ctgttgagca
1861 attcacgttc attataaatg tcgttcgcgg gcgcaactgc aactccgata aataacgcgc
1921 ccaacaccgg cataaagaat tgaagagagt tttcactgca tacgacgatt ctgtgatttg
1981 tattcagccc atatcgtttc atagcttctg ccaaccgaac ggacatttcg aagtattccg
2041 cgtacgtgat gttcacctcg atatgtgcat ctgtaaaagc aattgttcca ggaaccaggg
2101 cgtatctctt catagcctta tgcagttgct ctccagcggt tccatcctct agaggataga
2161 atggcgccgg gcctttcttt atgtttttgg cgtcttccat ggtggatcca cggttcacta
2221 aaccagctct gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg
2281 gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc
2341 cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc
2401 cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt
2461 actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
2521 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
2581 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg
2641 ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc
2701 caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta
2761 atgaccccgt aattgattac tattaataac taagatctgg taccttgaat tcatgcttct
2821 cctcccttta gtgagggtaa ttctctctct ctccctatag tgagtcgtat taattccttc
2881 tcttctatag tgtcacctaa atcgttgcaa ttcgtaatca tgtcatagct gtttcctgtg
2941 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
3001 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
3061 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
3121 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
3181 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
3241 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
3301 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
3361 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
3421 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
3481 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
3541 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
3601 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
3661 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
3721 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
3781 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
3841 aaaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
3901 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
3961 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
4021 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
4081 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
4141 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
4201 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
4261 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
4321 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
4381 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
4441 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
4501 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
4561 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
4621 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
4681 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
4741 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
4801 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
4861 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
4921 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
4981 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
5041 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
5101 acattaacct ataaaaatag gcgtatcacg agattgcagt gaaaaaaatg ctttatttgt
5161 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
5221 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
5281 agcaagtaaa acctctacaa atgtggtatg gctgattatg atcctctaga gtcggtgggc
5341 ctcgggggcg ggtgcggggt cggcggggcc gccccgggtg gcttcggtcg gagccatggg
5401 gtcgtgcgct cctttcggtc gggcgctgcg ggtcgtgggg cgggcgtcag gcaccgggct
5461 tgcgggtcat gcaccaggtg cgcggtcctt cgggcacctc gacgtcggcg gtgacggtga
5521 agccgagccg ctcgtagaag gggaggttgc ggggcgcgga ggtctccagg aaggcgggca
5581 ccccggcgcg ctcggccgcc tccactccgg ggagcacgac ggcgctgccc agacccttgc
5641 cctggtggtc gggcgagacg ccgacggtgg ccaggaacca cgcgggctcc ttgggccggt
5701 gcggcgccag gaggccttcc atctgttgct gcgcggccag ccgggaaccg ctcaactcgg
5761 ccatgcgcgg gccgatctcg gcgaacaccg cccccgcttc gacgctctcc ggcgtggtcc
5821 agaccgccac cgcggcgccg tcgtccgcga cccacacctt gccgatgtcg agcccgacgc
5881 gcgtgaggaa gagttcttgc agctcggtga cccgctcgat gtggcggtcc gggtcgacgg
5941 tgtggcgcgt ggcggggtag tcggcgaacg cggcggcgag ggtgcgtacg gcccggggga
6001 cgtcgtcgcg ggtggcgagg cgcaccgtgg gcttgtactc ggtcatggaa ggtcgtctcc
6061 ttgtgagggg tcaggggcgt gggtcagggg atggtggcgg caccggtcgt ggcggccgac
6121 ctgcaggcat gcaagctagc tttttgcaaa agcctaggcc tccaaaaaag cctcctcact
6181 acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaata aaaaaaatta
6241 gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag
6301 ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
6361 ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc atgctttgca
6421 tacttctgcc tgctggggag cctggggact ttccacacca atctttcgtc
//
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
| P3364/pCMV-SPORT6-KDELC1(1同义突变1点突变）人源基因质粒 |
| P3365/pCMV-SPORT6-CDK9（1同义突变）人源基因质粒 |
| P3366/pCMV-SPORT6-NMI（2同义突变）人源基因质粒 |
| P3367/pCMV-SPORT6-SH3GL1（1同义突变）人源基因质粒 |
| P3368/pCMV-SPORT6-ANXA1人源基因质粒 |
| P3369/pCMV-SPORT6-NCAPG人源基因质粒 |
| P3370/pCMV-SPORT6-IDH3G人源基因质粒 |
| P3371/pCMV-SPORT6-THOC3（1同义突变）人源基因质粒 |
| P3372/pCMV-SPORT6-OXSM人源基因质粒 |
| P3373/pCMV-SPORT6-KAT5人源基因质粒 |
| P3374/pCMV-SPORT6-MLH1人源基因质粒 |
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pMIR-Report Luciferase哺乳报告质粒
￥100 - 1000
