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pSEAP2-Basic

pSEAP2-Basic

pSEAP2-Basic

 

编号

载体名称

北京华越洋生物VECT6050

pSEAP2-Basic

pSEAP2-Basic载体基本信息

载体名称:

pSEAP2-Basic

质粒类型:

哺乳动物细胞表达载体;无启动子载体;分泌型碱性磷酸酶报告载体

高拷贝/低拷贝:

高拷贝

克隆方法:

限制性内切酶,多克隆位点

启动子:

载体大小:

4677 bp

5' 测序引物及序列:

5'-CTAGCAAAATAGGCTGTCCC-3'

3' 测序引物及序列:

5'-CCTCGGCTGCCTCGCGGTTCC-3'

载体标签:

--

载体抗性:

氨苄青霉素

筛选标记:

克隆菌株:

DH5α 等通用菌株

宿主细胞(系):

常规细胞系(293CV-1CHO等)

备注:

哺乳动物细胞表达载体pSEAP2-Basic没有启动子元件;
 
利用分泌型碱性磷酸酶定量定性研究外源启动子及其它顺式转录元件的活性。

稳定性:

瞬表达

组成型/诱导型:

--

病毒/非病毒:

非病毒

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

//

 

其他哺乳动物表达载体:

pEBVHis   A

pcDNA5/TO

pDsRed2-Bid

pNFκB-MetLuc2-Reporter

pGL4.10

pBApo-CMV-neo

pAcGFP1-N1

pEF1α-IRES-DsRed-Express2

pGL4.29

pDsRed-Monomer

pSecTag2   A

pCMV-DsRed-Express2

pGL4.13

pIRES

pGL4.27

pcDNA3.1/NT-GFP-TOPO

pG5   luciferase

pIRES-hrGFP-1a

pGL4.26

pEF1α-IRES-ZsGreen1

pCMV-AD

pDsRed-Express2-N1

pACT

pCMV-Tag   2A

pRevTet-Off

pCMV-Tag   3B

pBIND-Id   Control

pCMV-Tag   5B

pTet-Off

pCRE-hrGFP

pTRE2

pAcGFP1-C   In-Fusion Ready

pTRE2-hygro

pDsRED2-Mito

pRevTRE

p3XFLAG-CMV-14

pVgRxR

pAcGFP1-F

pTK-hyg

p3XFLAG-CMV-8

pOPI3CAT

pAcGFP1-C1

pTRE3G-Luc

pFLAG-CMV-2

pBK-RSV

pAsRed2-C1

pSwitch

pcDNA3.3-TOPO

pIRES2-DsRed2

pAsRed2-N1

pcDNA4/His   C

pcDNA6.2/cLumio-DEST

pCMV-Myc

pAcGFP1-Lam

c-Flag   pcDNA3

pCMV-tdTomato

pCMV-Tag   2C

pAcGFP1-C

pcDNA4/TO/Myc-His   A

pAcGFP1-Mito

pCMV-Tag   5A

pSEAP2-Basic

pcDNA6/myc-His   B

pAcGFP1-N   In-Fusion Ready

pCMV-Tag   3C

pBI-CMV3

pcDNA6/V5-His   B

pDsRed-Monomer-N   In-Fusion Ready

p3XFLAG-CMV-7

pNFkB-DD-tdTomato

pcDNA6.2/nTC-Tag-DEST

pcDNA4/TO/Myc-His   B

p3XFLAG-CMV-9

pcDNA3.1/His   A

pOptiVEC-TOPO

pIRES2-EGFP

pFLAG-CMV-4

pEBVHis   B

pcDNA5/FRT

pcDNA3.1/His   C

pBI-CMV4

pGL4.75

pGL4.30

pcDNA3.1/CT-GFP-TOPO

pcDNA4/His   A

pGL4.20

pGL4.19

pEF1α-IRES-AcGFP1

pcDNA4/myc-His   B

pCMV-SPORT6

pACT-MyoD

pcDNA3.2/V5/GW/D-TOPO

pcDNA4/HisMax   C

pCMV-SPORT6

pCMV-BD

pcDNA4/TO/Myc-His/LacZ

pCMV-Tag   4A

pBIND

pCMV-Tet3G

pcDNA4/HisMax-TOPO

pcDNA6/myc-His   C

pBD-NF-κB

pTet   on advanced

p3XFLAG-CMV-13

pCMV-Tag   2B

pRevTet-On

pTRE-Tight

p3xFLAG-CMV-10

pGRN145

pTet-On

pIND

pFLAG-CMV-3

pCMV-MEK1

pTRE3G

pGene/V5-His   B

pcDNA4/TO/Myc-His   C

pCMV-Tag   3A

pcDNA4/TO

pOPRSVI

pcDNA6.2/C-YFP-DEST

pCMVLacI

pcDNA4/His   B

pcDNA4/HisMax   A

pcDNA6.2/cTC-Tag-DEST

pBI-CMV1

pcDNA4/HisMax   B

pIRESpuro3

pcDNA6.2/nGeneBLAzer-DEST

pEF1α-AcGFP1-N1

pcDNA4/myc-His   C

pIRESneo3

pCRE-MetLuc2-Reporter

pCMV-LacZ

pcDNA3.1/His   B

pIRESneo2

pEF1α-DsRed-Express2

pCMV-Tag   4B

pcDNA6/V5-His   C

pcDNA4/myc-His   A

pDsRed-Express-C1

pCMV-Tag   5C

pCHO1.0

pCMV-PKA

pEF1α-DsRed-Monomer-N1

plRES2-ZsGreen1

pGL3-Promoter

pAcGFP1-N3

pDD-AmCyan1   Reporter

p3XFLAG-CMV-7.1

pCMV-MEKK1

pcDNA5/FRT/TO

pCRE-DD-AmCyan1

pFLAG-CMV-5a

pFLAG-CMV2

pBApo-CMV-Pur

pIRES2-DsRed-Express2

pBudCE4.1

pAcGFP1-C2

pBApo-EF1α-pur

pDsRed-Express-N1

pREP4

ptdTomato-C1

ptdTomato-N1

pcDNA6.2/nLumio-DEST

pBApo-CMV

pCRE-DD-tdTomato

pAcGFP1-Golgi

pcDNA6/myc-His   A

pIRES2-AcGFP1

pAcGFP1-Hyg-C1

pAcGFP1-p53

pcDNA6/V5-His   A

pIREShyg3

pAcGFP1-Mem

pAcGFP1-Actin

pcDNA5/FRT/TO-TOPO

pcDNA6/TR

pAcGFP1-C3

pBI-CMV2

pcDNA6.2/V5/GW/D-TOPO

pDsRed-Express2-C1

pTT5

pEF1α-tdTomato

pcDNA6.2/cGeneBLAzer-DEST

pBI-CMV5

pSEAP2-Control

pAmCyan1-C1

pcDNA6.2/nGeneBLAzer-GW/D-TOPO