pMetLuc2-Control

pMetLuc2-Control

pMetLuc2-Control载体基本信息：

pMetLuc2-Control载体质粒图谱和多克隆位点信息

pMetLuc2-Control载体描述：

The pMetLuc2-Control vector encodes a sequence-optimized, secreted luciferase from the marine copepod Metridia longa. The 24 kDa Metridia luciferase (MetLuc) contains a 17 amino acid, N-terminal signal peptide that allows the protein to be efficiently secreted into the cell culture medium (1), making it easy to detect without cell lysis.Expression of MetLuc is driven by the constitutively active cytomegalovirus immediate early promoter (PCMV IE). SV40 polyadenylation signals downstream of the MetLuc gene direct proper processing of the 3' end of the MetLuc mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, the Tn5 kanamycin/neomycin resistance gene, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene. The vector also contains a synthetic transcription blocker (TB), composed of adjacent polyadenylation and transcription pause sites, that reduces background readthrough transcription (2). A bacterial promoter (PKanr) upstream of the cassette allows kanamycin resistance in E. coli.

The pMetLuc2-Control vector can be used as a positive control for the constitutive expression and secretion of MetLuc in experiments using the pMetLuc2-Reporter vector. pMetLuc2-Control can also be used to monitor the inhibitory effect of drugs, such as Brefeldin A, on protein secretion.

The pMetLuc2-Control Vector can be transfected into mammalian cells using any standard transfection method. Stable transfectants can be selected using G418 when required (3). The presence of MetLuc in the cell culture medium can be easily detected by adding luciferase substrate to a small aliquot of the medium and analyzing the sample with a luminometer.

Propagation in E. coli

 Suitable host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA production

requires a host containing an F plasmid such as JM109 or XL1-Blue.

 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.

 E. coli replication origin: pUC

 Plasmid incompatibility group: pMB1/Col E1

Note: The attached sequence file has been compiled from information in the sequence databases, published literature, and other sources, together with partial sequences obtained by Clontech. This vector has not been completely sequenced.

References

1. Markova, S.V. et al. (2004) J. Bio. Chem. 279(5):3212-3217.

2. Eggermont, J. & Proudfoot, N. (1993) EMBO J. 12(6):2539–2548.

3. Gorman, C. (1985) DNA cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press Oxford, U.K.), pp. 143-190.

pMetLuc2-Control载体序列：

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 atccaccggt cgccaccatg gacatcaagg tggtgttcac cctggtgttc

      661 agcgccctgg tgcaggccaa gagcaccgag ttcgacccca acatcgacat cgtgggcctg

      721 gaaggcaagt tcggcatcac caacctggaa accgacctgt tcaccatctg ggagaccatg

      781 gaagtgatga tcaaggccga catcgccgac accgaccggg ccagcaactt cgtggccacc

      841 gagaccgacg ccaaccgggg caagatgccc ggcaagaagc tgcccctggc cgtcatcatg

      901 gaaatggaag ccaacgcctt caaggccggc tgcacccggg gctgcctgat ctgcctgagc

      961 aagatcaagt gcaccgccaa gatgaaggtg tacatccccg gcaggtgcca cgactacggc

     1021 ggcgacaaga aaaccggcca ggccggcatc gtgggcgcca tcgtggacat ccccgagatc

     1081 agcggcttca aagaaatggc ccccatggaa cagttcatcg cccaggtgga cagatgcgcc

     1141 agctgcacca ccggctgcct gaagggcctg gccaacgtga agtgcagcga gctgctgaag

     1201 aagtggctgc ccgaccgctg cgccagcttc gccgacaaga tccagaaaga ggtgcacaac

     1261 atcaagggca tggccggcga caggtgagcg gccgcgactc tagatcataa tcagccatac

     1321 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa

     1381 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa

     1441 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg

     1501 tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt

     1561 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg

     1621 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt

     1681 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct

     1741 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt

     1801 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa

     1861 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc

     1921 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc

     1981 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat

     2041 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc

     2101 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca

     2161 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct

     2221 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca

     2281 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc

     2341 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta

     2401 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt

     2461 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga

     2521 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct

     2581 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc

     2641 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag

     2701 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg

     2761 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc

     2821 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc

     2881 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg

     2941 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc

     3001 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg

     3061 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc

     3121 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag

     3181 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg

     3241 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg

     3301 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc

     3361 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt

     3421 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca

     3481 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat

     3541 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg

     3601 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata

     3661 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct

     3721 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta

     3781 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa

     3841 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga

     3901 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac

     3961 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt

     4021 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc

     4081 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat

     4141 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag

     4201 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc

     4261 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag

     4321 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac

     4381 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg

     4441 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct

     4501 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc

     4561 tcacatgtaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat

     4621 cgatagtact aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat

     4681 aggctgtccc cagtgcaagt gcaggtgcca gaacatttct ctacatgttc tttcctgcgt

     4741 tatcccctga ttctgtggat aaccgtatta ccgccatgca t

//

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