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基本信息
載體類型: | 哺乳細(xì)胞載體;啟動子增強子報告載體;海腎熒光素酶報告載體(對照載體) |
載體抗性: | Amp |
載體標(biāo)簽: | Rluc |
啟動子: | HSV TK, T7 |
復(fù)制子: | pUC ori |
終止子: | SV40 poly(A) signal |
載體大小: | 4045 bp |
克隆方法: | 多克隆位點,限制性內(nèi)切酶 |
5' 測序引物: | 5’-TAATACGACTCACTATAGGC-3’ |
3' 測序引物: | 根據(jù)序列設(shè)計引物 |
備注: | 如有侵權(quán)請產(chǎn)權(quán)持有人盡快聯(lián)系法務(wù)15927229196 |
注意:
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載體描述
pRL系列是野生型海腎螢光素酶(Rluc)報告基因?qū)φ蛰d體。pRL載體提供組成型表達的海腎螢光素酶,可與一個螢火蟲螢光素酶載體聯(lián)合共轉(zhuǎn)染哺乳動物細(xì)胞。海腎螢光素酶的表達為實驗的螢火蟲螢光素酶報告基因正態(tài)化提供內(nèi)對照值。
The pRL-TK Vector may be used in combination with any experimental reporter vector to cotransfect mammalian cells. However, it is important to realize that trans effects between promoters on cotransfected plasmids can potentially affect reporter gene expression. This is primarily of concern when either the control or experimental reporter vector, or both, contain very strong promoter/enhancer elements.The occurrence and magnitude of such effects will depend on several factors: i) the combination and activities of the genetic regulatory elements present on the cotransfected vectors, ii) the relative ratio of experimental vector to control vector introduced into the cells, and iii) the type of cell transfected.Features of the pRL-TK Vector
A. TK Promoter Region
The pRL-TK Vector contains the herpes simplex virus thymidine kinase promoter region upstream of Rluc. The HSV-TK promoter provides low-level, constitutive expression in cells of both embryonal and mature mammalian tissues.
B. Chimeric Intron
Downstream of the TK promoter region of the pRL-TK Vector is a chimeric intron comprised of the 5′-donor splice site from the first intron of the humanβ-globin gene, and the branch and 3′-acceptor splice site from an intron preceding an immunoglobulin gene heavy chain variable region (5). The sequences of the donor and acceptor splice sites, along with the branchpoint site, have been modified to match the consensus sequences for optimal splicing (6).
Transfection studies have demonstrated that the presence of an intron flanking a cDNA insert frequently increases the level of gene expression (7–10). In the pRL-SV40 Vector, the intron is positioned 5′to Rluc to minimize the utilization of cryptic 5′-donor splice sites that may reside within the reporter gene sequence.
C. T7 Promoter
A T7 promoter is located downstream of the chimeric intron, immediately preceding the Rluc reporter gene. This T7 promoter can be used to synthesize RNA transcripts in vitro using T7 RNA Polymerase (Cat.# P2075). T7 RNA Polymerase can also be used to synthesize active Renilla luciferase in a cell-free coupled eukaryotic in vitro transcription/translation reaction.
D. Renilla Luciferase Reporter Gene (Rluc)
The Renilla luciferase cDNA inserted into all of the pRL Vectors is derived from the anthozoan coelenterate Renilla reniformis but contains nucleotide changes that were engineered during the construction of the individual vectors.
The following bases were altered in the pRL-TK Vector: base 1264 (T→C), to eliminate an internal Bgl II site; base 1807 (T→C), to eliminate internal BamH I site; base 1840 (C→T), to eliminate internal Nar I, Kas K, Ban K and Acy I sites.
These nucleotide substitutions do not alter the amino acid sequence of the encoded Renilla luciferase reporter enzyme.
E. SV40 Late Polyadenylation Signal
Polyadenylation signals cause the termination of transcription by RNA polymerase II and signal the addition of approximately 200–250 adenosine residues to the 3′-end of the RNA transcript.
Polyadenylation has been shown to enhance RNA stability and translation. The late SV40 polyadenylation signal, which is extremely efficient and has been shown to increase the steadystate level of RNA approximately five-fold more than the early SV40 polyadenylation signal, has been positioned 3′to the Rluc gene in the pRL-TK Vector to increase the level of Renilla luciferase expression.
載體序列
ORIGIN
1 agatctaaat gagtcttcgg acctcgcggg ggccgcttaa gcggtggtta gggtttgtct
61 gacgcggggg gagggggaag gaacgaaaca ctctcattcg gaggcggctc ggggtttggt
121 cttggtggcc acgggcacgc agaagagcgc cgcgatcctc ttaagcaccc ccccgccctc
181 cgtggaggcg ggggtttggt cggcgggtgg taactggcgg gccgctgact cgggcgggtc
241 gcgcgcccca gagtgtgacc ttttcggtct gctcgcagac ccccgggcgg cgccgccgcg
301 gcggcgacgg gctcgctggg tcctaggctc catggggacc gtatacgtgg acaggctctg
361 gagcatccgc acgactgcgg tgatattacc ggagaccttc tgcgggacga gccgggtcac
421 gcggctgacg cggagcgtcc gttgggcgac aaacaccagg acggggcaca ggtacactat
481 cttgtcaccc ggaggcgcga gggactgcag gagcttcagg gagtggcgca gctgcttcat
541 ccccgtggcc cgttgctcgc gtttgctggc ggtgtccccg gaagaaatat atttgcatgt
601 ctttagttct atgatgacac aaaccccgcc cagcgtcttg tcattggcga attcgaacac
661 gcagatgcag tcggggcggc gcggtcccag gtccacttcg catattaagg tgacgcgtgt
721 ggcctcgaac accgagcgac cctgcagcga cccgcttaaa agcttgattc ttctgacaca
781 acagtctcga acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag
841 gttacaagac aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc
901 ttgcgtttct gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca
961 ggtgtccact cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta
1021 taggctagcc accatgactt cgaaagttta tgatccagaa caaaggaaac ggatgataac
1081 tggtccgcag tggtgggcca gatgtaaaca aatgaatgtt cttgattcat ttattaatta
1141 ttatgattca gaaaaacatg cagaaaatgc tgttattttt ttacatggta acgcggcctc
1201 ttcttattta tggcgacatg ttgtgccaca tattgagcca gtagcgcggt gtattatacc
1261 agaccttatt ggtatgggca aatcaggcaa atctggtaat ggttcttata ggttacttga
1321 tcattacaaa tatcttactg catggtttga acttcttaat ttaccaaaga agatcatttt
1381 tgtcggccat gattggggtg cttgtttggc atttcattat agctatgagc atcaagataa
1441 gatcaaagca atagttcacg ctgaaagtgt agtagatgtg attgaatcat gggatgaatg
1501 gcctgatatt gaagaagata ttgcgttgat caaatctgaa gaaggagaaa aaatggtttt
1561 ggagaataac ttcttcgtgg aaaccatgtt gccatcaaaa atcatgagaa agttagaacc
1621 agaagaattt gcagcatatc ttgaaccatt caaagagaaa ggtgaagttc gtcgtccaac
1681 attatcatgg cctcgtgaaa tcccgttagt aaaaggtggt aaacctgacg ttgtacaaat
1741 tgttaggaat tataatgctt atctacgtgc aagtgatgat ttaccaaaaa tgtttattga
1801 atcggaccca ggattctttt ccaatgctat tgttgaaggt gccaagaagt ttcctaatac
1861 tgaatttgtc aaagtaaaag gtcttcattt ttcgcaagaa gatgcacctg atgaaatggg
1921 aaaatatatc aaatcgttcg ttgagcgagt tctcaaaaat gaacaataat tctagagcgg
1981 ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
2041 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
2101 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
2161 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat
2221 aaggatccag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
2281 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
2341 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
2401 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
2461 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
2521 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
2581 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
2641 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
2701 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
2761 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
2821 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
2881 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
2941 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
3001 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
3061 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
3121 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
3181 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
3241 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
3301 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
3361 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
3421 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
3481 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
3541 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
3601 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
3661 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
3721 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
3781 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
3841 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
3901 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
3961 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
4021 tggccttttg ctcacatggc tcgac
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