pSEAP2-Basic

pSEAP2-Basic

pSEAP2-Basic载体基本信息

pSEAP2-Basic载体质粒图谱和多克隆位点信息

pSEAP2-Basic载体描述 

pSEAP2-Basic allows for expression of the reporter gene secreted alkaline phosphatase (SEAP). This vector lacks eukaryotic promoter and enhancer sequences and has an MCS that allows putative promoter DNA fragments to be inserted upstream of the SEAP gene. Enhancers can be cloned into either the MCS or unique downstream sites. 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-stranded 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 increaing 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 EscAPe? SEAP2 Vectors has been designed to provide maximal flexibility in studying regulatory sequences from the gene of interest. pSEAP2-Basic allows for expression of SEAP under control of putative promoters and/or enhancers of interest. 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

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.

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

载体序列

ORIGIN

        1 GGTACCGAGC TCTTACGCGT GCTAGCCCGG GCTCGAGATC TGCGATCTAA GTAAGCTTCG

       61 AATCGCGAAT TCGCCCACCA TGCTGCTGCT GCTGCTGCTG CTGGGCCTGA GGCTACAGCT

      121 CTCCCTGGGC ATCATCCCAG TTGAGGAGGA GAACCCGGAC TTCTGGAACC GCGAGGCAGC

      181 CGAGGCCCTG GGTGCCGCCA AGAAGCTGCA GCCTGCACAG ACAGCCGCCA AGAACCTCAT

      241 CATCTTCCTG GGCGATGGGA TGGGGGTGTC TACGGTGACA GCTGCCAGGA TCCTAAAAGG

      301 GCAGAAGAAG GACAAACTGG GGCCTGAGAT ACCCCTGGCC ATGGACCGCT TCCCATATGT

      361 GGCTCTGTCC AAGACATACA ATGTAGACAA ACATGTGCCA GACAGTGGAG CCACAGCCAC

      421 GGCCTACCTG TGCGGGGTCA AGGGCAACTT CCAGACCATT GGCTTGAGTG CAGCCGCCCG

      481 CTTTAACCAG TGCAACACGA CACGCGGCAA CGAGGTCATC TCCGTGATGA ATCGGGCCAA

      541 GAAAGCAGGG AAGTCAGTGG GAGTGGTAAC CACCACACGA GTGCAGCACG CCTCGCCAGC

      601 CGGCACCTAC GCCCACACGG TGAACCGCAA CTGGTACTCG GACGCCGACG TGCCTGCCTC

      661 GGCCCGCCAG GAGGGGTGCC AGGACATCGC TACGCAGCTC ATCTCCAACA TGGACATTGA

      721 CGTGATCCTA GGTGGAGGCC GAAAGTACAT GTTTCGCATG GGAACCCCAG ACCCTGAGTA

      781 CCCAGATGAC TACAGCCAAG GTGGGACCAG GCTGGACGGG AAGAATCTGG TGCAGGAATG

      841 GCTGGCGAAG CGCCAGGGTG CCCGGTATGT GTGGAACCGC ACTGAGCTCA TGCAGGCTTC

      901 CCTGGACCCG TCTGTGACCC ATCTCATGGG TCTCTTTGAG CCTGGAGACA TGAAATACGA

      961 GATCCACCGA GACTCCACAC TGGACCCCTC CCTGATGGAG ATGACAGAGG CTGCCCTGCG

     1021 CCTGCTGAGC AGGAACCCCC GCGGCTTCTT CCTCTTCGTG GAGGGTGGTC GCATCGACCA

     1081 TGGTCATCAT GAAAGCAGGG CTTACCGGGC ACTGACTGAG ACGATCATGT TCGACGACGC

     1141 CATTGAGAGG GCGGGCCAGC TCACCAGCGA GGAGGACACG CTGAGCCTCG TCACTGCCGA

     1201 CCACTCCCAC GTCTTCTCCT TCGGAGGCTA CCCCCTGCGA GGGAGCTCCA TCTTCGGGCT

     1261 GGCCCCTGGC AAGGCCCGGG ACAGGAAGGC CTACACGGTC CTCCTATACG GAAACGGTCC

     1321 AGGCTATGTG CTCAAGGACG GCGCCCGGCC GGATGTTACC GAGAGCGAGA GCGGGAGCCC

     1381 CGAGTATCGG CAGCAGTCAG CAGTGCCCCT GGACGAAGAG ACCCACGCAG GCGAGGACGT

     1441 GGCGGTGTTC GCGCGCGGCC CGCAGGCGCA CCTGGTTCAC GGCGTGCAGG AGCAGACCTT

     1501 CATAGCGCAC GTCATGGCCT TCGCCGCCTG CCTGGAGCCC TACACCGCCT GCGACCTGGC

     1561 GCCCCCCGCC GGCACCACCG ACGCCGCGCA CCCGGGTTAC TCTAGAGTCG GGGCGGCCGG

     1621 CCGCTTCGAG CAGACATGAT AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG

     1681 CAGTGAAAAA AATGCTTTAT TTGTGAAATT TGTGATGCTA TTGCTTTATT TGTAACCATT

     1741 ATAAGCTGCA ATAAACAAGT TAACAACAAC AATTGCATTC ATTTTATGTT TCAGGTTCAG

     1801 GGGGAGGTGT GGGAGGTTTT TTAAAGCAAG TAAAACCTCT ACAAATGTGG TAAAATCGAT

     1861 AAGGATCCGT CGACCGATGC CCTTGAGAGC CTTCAACCCA GTCAGCTCCT TCCGGTGGGC

     1921 GCGGGGCATG ACTATCGTCG CCGCACTTAT GACTGTCTTC TTTATCATGC AACTCGTAGG

     1981 ACAGGTGCCG GCAGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC

     2041 GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG

     2101 GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA

     2161 AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC

     2221 GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC

     2281 CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG

     2341 CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT

     2401 CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC

     2461 GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC

     2521 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG

     2581 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG AACAGTATTT GGTATCTGCG

     2641 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA

     2701 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG

     2761 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT

     2821 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA

     2881 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT

     2941 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG

     3001 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA

     3061 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC

     3121 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT

     3181 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG

     3241 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA

     3301 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG

     3361 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA

     3421 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG

     3481 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT

     3541 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA

     3601 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA

     3661 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG

     3721 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC

     3781 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT

     3841 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC

     3901 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGCGCCCTG TAGCGGCGCA TTAAGCGCGG

     3961 CGGGTGTGGT GGTTACGCGC AGCGTGACCG CTACACTTGC CAGCGCCCTA GCGCCCGCTC

     4021 CTTTCGCTTT CTTCCCTTCC TTTCTCGCCA CGTTCGCCGG CTTTCCCCGT CAAGCTCTAA

     4081 ATCGGGGGCT CCCTTTAGGG TTCCGATTTA GTGCTTTACG GCACCTCGAC CCCAAAAAAC

     4141 TTGATTAGGG TGATGGTTCA CGTAGTGGGC CATCGCCCTG ATAGACGGTT TTTCGCCCTT

     4201 TGACGTTGGA GTCCACGTTC TTTAATAGTG GACTCTTGTT CCAAACTGGA ACAACACTCA

     4261 ACCCTATCTC GGTCTATTCT TTTGATTTAT AAGGGATTTT GCCGATTTCG GCCTATTGGT

     4321 TAAAAAATGA GCTGATTTAA CAAAAATTTA ACGCGAATTT TAACAAAATA TTAACGCTTA

     4381 CAATTTGCCA TTCGCCATTC AGGCTGCGCA ACTGTTGGGA AGGGCGATCG GTGCGGGCCT

     4441 CTTCGCTATT ACGCCAGCCC AAGCTACCAT GATAAGTAAG TAATATTAAG GTACGGGAGG

     4501 TACTTGGAGC GGCCGCAATA AAATATCTTT ATTTTCATTA CATCTGTGTG TTGGTTTTTT

     4561 GTGTGAATCG ATAGTACTAA CATACGCTCT CCATCAAAAC AAAACGAAAC AAAACAAACT

     4621 AGCAAAATAG GCTGTCCCCA GTGCAAGTGC AGGTGCCAGA ACATTTCTCT ATCGATA

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