pcDNA3.3-TOPO

pcDNA3.3-TOPO

pcDNA3.3-TOPO载体基本信息

pcDNA3.3-TOPO载体质粒图谱和多克隆位点信息

pcDNA3.3-TOPO载体简介

The pcDNA3.3-TOPO vector is a TOPO-adapted plasmid that allows rapid cloning of a PCR product containing a gene of interest downstream of the CMV promoter. The pcDNA3.3-TOPO vector can be used for high level expression of a native protein in adherent mammalian tissue culture cells following transient transfection, or high level expression of secreted, native protein using the FreeStyle MAX CHO and FreeStyle MAX 293 systems.

The pcDNA3.3-TOPO vector is also included with the OptiCHO Antibody Express System for cloning the light and heavy chains of your antibody of choice.

pcDNA3.3-TOPO载体特征

 Full-length human cytomegalovirus (CMV) immediate-early promoter/enhancer for high-level gene expression in a wide range of mammalian cells

 TOPO Cloning site for rapid and efficient cloning of Taq-amplified PCR products

 Herpes Simplex Virus thymidine kinase polyadenylation signal for proper termination and processing of the recombinant transcript

 Neomycin resistance gene for selection of stable cell lines with Geneticin

 pUC origin for high copy replication and maintenance of the plasmid in E. coli

 Ampicillin (bla) resistance gene for selection in E. coli

CMV Promoter

The human cytomegalovirus immediate-early (HCMV IE1) gene promoter in the pcDNA3.3-TOPO vector is 680 bp and contains the native transcriptional start site (Hennighausen & Fleckenstein, 1986). This sequence results in high levels of transgene expression.

TOPO技术简介：

The pcDNA3.3-TOPO vector is supplied linearized with:

 Single 3′ thymidine (T) overhangs for TA Cloning

 Topoisomerase covalently bound to the vector (this is referred to as“activated” vector) Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′ ends of PCR products. The linearized vector supplied in this kit has single, overhanging 3′ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector. Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5′-CCCTT in one strand (Shuman, 1991). The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3′ phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5′ hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994). TOPO Cloning exploits this reaction to efficiently clone PCR products.Once the PCR product is cloned into the pcDNA3.3-TOPO vector and the transformants are analyzed for correct orientation and reading frame, the expression plasmid may be transfected into your cell line of choice.

实验流程：

To TOPO Clone your gene of interest into pcDNA3.3-TOPO, you will perform the following steps:

1. Generate a PCR product containing your gene of interest with Taq polymerase.

2. TOPO Clone your PCR product into the pcDNA3.3-TOPO vector and use the reaction to transform One Shot TOP10 Chemically Competent E. coli.

3. Pick colonies, isolate plasmid DNA, and screen for insert directionality by sequencing expression clones with the primers provided in the kit.

建立稳定细胞系：

After creation of your four clones, you will then use the OptiCHO Antibody Express Kit to transiently transfect two of each of the plasmid constructs (one of each pcDNA3.3 and pOptiVEC) containing the heavy and light chains into CHO cells. You may also perform several stable transfections directly in DG44 cells with various combinations of pOptiVEC-TOPO and pcDNA3.3-TOPO plasmid constructs.

pcDNA3.3-TOPO载体序列

ORIGIN

    1 GTTAGGCGTT TTGCGCTGCT TCGCGATGTA CGGGCCAGAT ATACGCGTTG ACATTGATTA

   61 TTGACTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC ATATATGGAG

  121 TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT GACCGCCCAA CGACCCCCGC

  181 CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC CAATAGGGAC TTTCCATTGA

  241 CGTCAATGGG TGGAGTATTT ACGGTAAACT GCCCACTTGG CAGTACATCA AGTGTATCAT

  301 ATGCCAAGTA CGCCCCCTAT TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC

  361 CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT AGTCATCGCT

  421 ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG GTTTGACTCA

  481 CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA GTTTGTTTTG GCACCAAAAT

  541 CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT TGACGCAAAT GGGCGGTAGG

  601 CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTCGTTTAG TGAACCGTCA GATCGCCTGG

  661 AGACGCCATC CACGCTGTTT TGACCTCCAT AGAAGACACC GGGACCGATC CAGCCTCCGG

  721 ACTCTAGAGG ATCGAACCCT TAAGGGTTCG ATCCCTACCG GTTAGTAATG AGTTTAAACG

  781 GGGGAGGCTA ACTGAAACAC GGAAGGAGAC AATACCGGAA GGAACCCGCG CTATGACGGC

  841 AATAAAAAGA CAGAATAAAA CGCACGGGTG TTGGGTCGTT TGTTCATAAA CGCGGGGTTC

  901 GGTCCCAGGG CTGGCACTCT GTCGATACCC CACCGAGACC CCATTGGGGC CAATACGCCC

  961 GCGTTTCTTC CTTTTCCCCA CCCCACCCCC CAAGTTCGGG TGAAGGCCCA GGGCTCGCAG

 1021 CCAACGTCGG GGCGGCAGGC CCTGCCATAG CAGATCTGCG CAGCTGGGGC TCTAGGGGGT

 1081 ATCCCCACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG TGTGGTGGTT ACGCGCAGCG

 1141 TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC CCTTCCTTTC

 1201 TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGCATCCCT TTAGGGTTCC

 1261 GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA TTAGGGTGAT GGTTCACGTA

 1321 GTGGGCCATC GCCCTGATAG ACGGTTTTTC GCCCTTTGAC GTTGGAGTCC ACGTTCTTTA

 1381 ATAGTGGACT CTTGTTCCAA ACTGGAACAA CACTCAACCC TATCTCGGTC TATTCTTTTG

 1441 ATTTATAAGG GATTTTGGGG ATTTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA

 1501 AATTTAACGC GAATTAATTC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG

 1561 CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG

 1621 AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC

 1681 AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA

 1741 TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCTGC

 1801 CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAAA

 1861 GCTCCCGGGA GCTTGTATAT CCATTTTCGG ATCTGATCAA GAGACAGGAT GAGGATCGTT

 1921 TCGCATGATT GAACAAGATG GATTGCACGC AGGTTCTCCG GCCGCTTGGG TGGAGAGGCT

 1981 ATTCGGCTAT GACTGGGCAC AACAGACAAT CGGCTGCTCT GATGCCGCCG TGTTCCGGCT

 2041 GTCAGCGCAG GGGCGCCCGG TTCTTTTTGT CAAGACCGAC CTGTCCGGTG CCCTGAATGA

 2101 ACTGCAGGAC GAGGCAGCGC GGCTATCGTG GCTGGCCACG ACGGGCGTTC CTTGCGCAGC

 2161 TGTGCTCGAC GTTGTCACTG AAGCGGGAAG GGACTGGCTG CTATTGGGCG AAGTGCCGGG

 2221 GCAGGATCTC CTGTCATCTC ACCTTGCTCC TGCCGAGAAA GTATCCATCA TGGCTGATGC

 2281 AATGCGGCGG CTGCATACGC TTGATCCGGC TACCTGCCCA TTCGACCACC AAGCGAAACA

 2341 TCGCATCGAG CGAGCACGTA CTCGGATGGA AGCCGGTCTT GTCGATCAGG ATGATCTGGA

 2401 CGAAGAGCAT CAGGGGCTCG CGCCAGCCGA ACTGTTCGCC AGGCTCAAGG CGCGCATGCC

 2461 CGACGGCGAG GATCTCGTCG TGACCCATGG CGATGCCTGC TTGCCGAATA TCATGGTGGA

 2521 AAATGGCCGC TTTTCTGGAT TCATCGACTG TGGCCGGCTG GGTGTGGCGG ACCGCTATCA

 2581 GGACATAGCG TTGGCTACCC GTGATATTGC TGAAGAGCTT GGCGGCGAAT GGGCTGACCG

 2641 CTTCCTCGTG CTTTACGGTA TCGCCGCTCC CGATTCGCAG CGCATCGCCT TCTATCGCCT

 2701 TCTTGACGAG TTCTTCTGAG CGGGACTCTG GGGTTCGCGA AATGACCGAC CAAGCGACGC

 2761 CCAACCTGCC ATCACGAGAT TTCGATTCCA CCGCCGCCTT CTATGAAAGG TTGGGCTTCG

 2821 GAATCGTTTT CCGGGACGCC GGCTGGATGA TCCTCCAGCG CGGGGATCTC ATGCTGGAGT

 2881 TCTTCGCCCA CCCCAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA

 2941 TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC

 3001 TCATCAATGT ATCTTATCAT GTCTGTATAC CGTCGACCTC TAGCTAGAGC TTGGCGTAAT

 3061 CATGGTCATA GCTGTTTCCT GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC

 3121 GAGCCGGAAG CATAAAGTGT AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA

 3181 TTGCGTTGCG CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT

 3241 GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC

 3301 TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG

 3361 CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG

 3421 GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC

 3481 GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG

 3541 GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA

 3601 CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC

 3661 AATGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG

 3721 TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT

 3781 CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA

 3841 GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA

 3901 CTAGAAGGAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG

 3961 TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA

 4021 AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG

 4081 GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA

 4141 AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA

 4201 TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG

 4261 CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA

 4321 TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC

 4381 CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC

 4441 CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA

 4501 GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC TACAGGCATC GTGGTGTCAC

 4561 GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT

 4621 GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA

 4681 GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG

 4741 TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG

 4801 AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AATACGGGAT AATACCGCGC

 4861 CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT

 4921 CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT

 4981 CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG

 5041 CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC

 5101 AATATTATTG AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA

 5161 TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG

 5221 TCGACGGATC GGGAGATCTC CCGATCCCCT ATGGTCGACT CTCAGTACAA TCTGCTCTGA

 5281 TGCCGCATAG TTAAGCCAGT ATCTGCTCCC TGCTTGTGTG TTGGAGGTCG CTGAGTAGTG

 5341 CGCGAGCAAA ATTTAAGCTA CAACAAGGCA AGGCTTGACC GACAATTGCA TGAAGAATCT

 5401 GCTTAGG

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