pSEAP2-Control

pSEAP2-Control

pSEAP2-Control载体基本信息

pSEAP2-Control载体质粒图谱和多克隆位点信息

pSEAP2-Control载体描述

pSEAP2-Control is a positive control vector expressing secreted alkaline phosphatase (SEAP) under the control of the SV40 early promoter and the SV40 enhancer. The SEAP coding sequence is followed by the SV40 late polyadenylation signal to ensure proper, efficient processing of the SEAP transcript in eukaryotic cells. A synthetic transcription blocker (TB), composed of adjacent polyadenylation and transcription pause sites, located upstream of the MCS reduces background transcription (1). The vector backbone also contains an f1 origin for single-stran ed DNA production, a pUC origin of replication, and an ampicillin resistance gene for propagation and selection in E. coli. The SEAP2 Vectors incorporate a number of features that improve the sensitivity of SEAP by increasing the efficiency of SEAP expression or that enhance the utility of the vectors. These include: an improved Kozak consensus translation initiation site (2); the removal of the SV40 small-t intron, which can cause cryptic splicing and reduced expression in some genes and/or cell types (3, 4); switching from the early to late polyadenylation signal of SV40, which typically causes a five-fold increase in mRNA levels (5); an expanded multiple cloning site (MCS); compact plasmid size; and removal of extraneous sequences from the 3' untranslated region of the SEAP mRNA.

The integrated set of Great EscAPeTM SEAP2 Vectors has been designed to provide maximal flexibility in studying regulatory sequences from the gene of interest. pSEAP2-Control expresses SEAP in most cell types and provides an important control. The secreted SEAP enzyme is assayed directly from the culture medium and permits time-course studies not possible with assays dependent on cell lysates. Furthermore, the cells can be used for further investigations such as RNA or protein studies. The SEAP2 Vectors can be transfected into mammalian cells by any standard method.

Propagation in E. coli

 Suitable host strains: DH5α and other general purpose strains. Single-stranded DNA production requires a host containing an F' episome such as JM109.

 Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) to E. coli hosts.

 E. coli replication origin: pUC

 Copy number: ~500

 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 obtai ned by Clontech. This vector has not been completely sequenced.

References

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

2. Kozak, M. (1987) Nucleic Acids Res. 15:8125–8148.

3. Huang, M. T. F. & Gorman, C. M. (1990) Mol. Cell. Biol. 10:1805–1810.

4. Evans, M. J. & Scarpulla, R. C. (1989) Gene 84:135–142.

5. Carswell, S. & Alwine, J. C. (1989) Mol. Cell. Biol. 9:4248–4258.

6. Levitt, N. et al. (1989) Genes Dev. 3:1019–1025.

7. Enriquez-Harris, P. et al. (1991) EMBO J. 10:1833–1842

pSEAP2-Control载体序列

ORIGIN

        1 GGTACCGAGC TCTTACGCGT GCTAGCCCGG GCTCGAGATC TGCGATCTGC ATCTCAATTA

       61 GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG CCCCTAACTC CGCCCAGTTC

      121 CGCCCATTCT CCGCCCCATC GCTGACTAAT TTTTTTTATT TATGCAGAGG CCGAGGCCGC

      181 CTCGGCCTCT GAGCTATTCC AGAAGTAGTG AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG

      241 CAAAAAGCTT CGAATCGCGA ATTCGCCCAC CATGCTGCTG CTGCTGCTGC TGCTGGGCCT

      301 GAGGCTACAG CTCTCCCTGG GCATCATCCC AGTTGAGGAG GAGAACCCGG ACTTCTGGAA

      361 CCGCGAGGCA GCCGAGGCCC TGGGTGCCGC CAAGAAGCTG CAGCCTGCAC AGACAGCCGC

      421 CAAGAACCTC ATCATCTTCC TGGGCGATGG GATGGGGGTG TCTACGGTGA CAGCTGCCAG

      481 GATCCTAAAA GGGCAGAAGA AGGACAAACT GGGGCCTGAG ATACCCCTGG CCATGGACCG

      541 CTTCCCATAT GTGGCTCTGT CCAAGACATA CAATGTAGAC AAACATGTGC CAGACAGTGG

      601 AGCCACAGCC ACGGCCTACC TGTGCGGGGT CAAGGGCAAC TTCCAGACCA TTGGCTTGAG

      661 TGCAGCCGCC CGCTTTAACC AGTGCAACAC GACACGCGGC AACGAGGTCA TCTCCGTGAT

      721 GAATCGGGCC AAGAAAGCAG GGAAGTCAGT GGGAGTGGTA ACCACCACAC GAGTGCAGCA

      781 CGCCTCGCCA GCCGGCACCT ACGCCCACAC GGTGAACCGC AACTGGTACT CGGACGCCGA

      841 CGTGCCTGCC TCGGCCCGCC AGGAGGGGTG CCAGGACATC GCTACGCAGC TCATCTCCAA

      901 CATGGACATT GACGTGATCC TAGGTGGAGG CCGAAAGTAC ATGTTTCGCA TGGGAACCCC

      961 AGACCCTGAG TACCCAGATG ACTACAGCCA AGGTGGGACC AGGCTGGACG GGAAGAATCT

     1021 GGTGCAGGAA TGGCTGGCGA AGCGCCAGGG TGCCCGGTAT GTGTGGAACC GCACTGAGCT

     1081 CATGCAGGCT TCCCTGGACC CGTCTGTGAC CCATCTCATG GGTCTCTTTG AGCCTGGAGA

     1141 CATGAAATAC GAGATCCACC GAGACTCCAC ACTGGACCCC TCCCTGATGG AGATGACAGA

     1201 GGCTGCCCTG CGCCTGCTGA GCAGGAACCC CCGCGGCTTC TTCCTCTTCG TGGAGGGTGG

     1261 TCGCATCGAC CATGGTCATC ATGAAAGCAG GGCTTACCGG GCACTGACTG AGACGATCAT

     1321 GTTCGACGAC GCCATTGAGA GGGCGGGCCA GCTCACCAGC GAGGAGGACA CGCTGAGCCT

     1381 CGTCACTGCC GACCACTCCC ACGTCTTCTC CTTCGGAGGC TACCCCCTGC GAGGGAGCTC

     1441 CATCTTCGGG CTGGCCCCTG GCAAGGCCCG GGACAGGAAG GCCTACACGG TCCTCCTATA

     1501 CGGAAACGGT CCAGGCTATG TGCTCAAGGA CGGCGCCCGG CCGGATGTTA CCGAGAGCGA

     1561 GAGCGGGAGC CCCGAGTATC GGCAGCAGTC AGCAGTGCCC CTGGACGAAG AGACCCACGC

     1621 AGGCGAGGAC GTGGCGGTGT TCGCGCGCGG CCCGCAGGCG CACCTGGTTC ACGGCGTGCA

     1681 GGAGCAGACC TTCATAGCGC ACGTCATGGC CTTCGCCGCC TGCCTGGAGC CCTACACCGC

     1741 CTGCGACCTG GCGCCCCCCG CCGGCACCAC CGACGCCGCG CACCCGGGTT ACTCTAGAGT

     1801 CGGGGCGGCC GGCCGCTTCG AGCAGACATG ATAAGATACA TTGATGAGTT TGGACAAACC

     1861 ACAACTAGAA TGCAGTGAAA AAAATGCTTT ATTTGTGAAA TTTGTGATGC TATTGCTTTA

     1921 TTTGTAACCA TTATAAGCTG CAATAAACAA GTTAACAACA ACAATTGCAT TCATTTTATG

     1981 TTTCAGGTTC AGGGGGAGGT GTGGGAGGTT TTTTAAAGCA AGTAAAACCT CTACAAATGT

     2041 GGTAAAATCG ATAAGGATCT GAACGATGGA GCGGAGAATG GGCGGAACTG GGCGGAGTTA

     2101 GGGGCGGGAT GGGCGGAGTT AGGGGCGGGA CTATGGTTGC TGACTAATTG AGATGCATGC

     2161 TTTGCATACT TCTGCCTGCT GGGGAGCCTG GGGACTTTCC ACACCTGGTT GCTGACTAAT

     2221 TGAGATGCAT GCTTTGCATA CTTCTGCCTG CTGGGGAGCC TGGGGACTTT CCACACCCTA

     2281 ACTGACACAC ATTCCACAGC GGATCCGTCG ACCGATGCCC TTGAGAGCCT TCAACCCAGT

     2341 CAGCTCCTTC CGGTGGGCGC GGGGCATGAC TATCGTCGCC GCACTTATGA CTGTCTTCTT

     2401 TATCATGCAA CTCGTAGGAC AGGTGCCGGC AGCGCTCTTC CGCTTCCTCG CTCACTGACT

     2461 CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC

     2521 GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA

     2581 AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG

     2641 ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA

     2701 GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC

     2761 TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC

     2821 GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC

     2881 CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG

     2941 TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT

     3001 ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA

     3061 CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT

     3121 CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA

     3181 TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG

     3241 CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT

     3301 TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT

     3361 AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC

     3421 TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG

     3481 GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG

     3541 ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT

     3601 TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG

     3661 TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT

     3721 TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA

     3781 TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG

     3841 CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT

     3901 CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA

     3961 TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA

     4021 GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT

     4081 TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT

     4141 CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA

     4201 AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT

     4261 GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA

     4321 ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC GCGCCCTGTA

     4381 GCGGCGCATT AAGCGCGGCG GGTGTGGTGG TTACGCGCAG CGTGACCGCT ACACTTGCCA

     4441 GCGCCCTAGC GCCCGCTCCT TTCGCTTTCT TCCCTTCCTT TCTCGCCACG TTCGCCGGCT

     4501 TTCCCCGTCA AGCTCTAAAT CGGGGGCTCC CTTTAGGGTT CCGATTTAGT GCTTTACGGC

     4561 ACCTCGACCC CAAAAAACTT GATTAGGGTG ATGGTTCACG TAGTGGGCCA TCGCCCTGAT

     4621 AGACGGTTTT TCGCCCTTTG ACGTTGGAGT CCACGTTCTT TAATAGTGGA CTCTTGTTCC

     4681 AAACTGGAAC AACACTCAAC CCTATCTCGG TCTATTCTTT TGATTTATAA GGGATTTTGC

     4741 CGATTTCGGC CTATTGGTTA AAAAATGAGC TGATTTAACA AAAATTTAAC GCGAATTTTA

     4801 ACAAAATATT AACGCTTACA ATTTGCCATT CGCCATTCAG GCTGCGCAAC TGTTGGGAAG

     4861 GGCGATCGGT GCGGGCCTCT TCGCTATTAC GCCAGCCCAA GCTACCATGA TAAGTAAGTA

     4921 ATATTAAGGT ACGGGAGGTA CTTGGAGCGG CCGCAATAAA ATATCTTTAT TTTCATTACA

     4981 TCTGTGTGTT GGTTTTTTGT GTGAATCGAT AGTACTAACA TACGCTCTCC ATCAAAACAA

     5041 AACGAAACAA AACAAACTAG CAAAATAGGC TGTCCCCAGT GCAAGTGCAG GTGCCAGAAC

     5101 ATTTCTCTAT CGATA

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