Resistance cassette for selection of mammalian cells. The cassette contains a SV40 promoter, the neomycin resistance gene, and the SV40 poly A as a terminator.
Compatible with the Plug'n'Play assembly standard
pCMV_[AAV2]-VP123ex (ViralBrick-587KO-RGD)
Pepsinogen from Homo sapiens optimized for E. coli
Pepsinogen is the precursor protein for the enzyme pepsin, one of the primary digestive enzymes located in the human stomach. Pepsinogen activates into pepsin in the presence of HCl and other pepsinogen molecules. It is a protease, being used in the human digestive system to digest proteins. This sequence has been codon optimized for expression in E. coli.
Codon optimized for expression in E. coli using Gene Designer from DNA 2.0.
pCMV_CFP_Middle-Linker_[AAV2]-VP23 (ViralBrick-587KO-Empty)
Amino acids 2-64 from the zebrafish CBX2 protein. This is the conserved Polycomb chromodomain predicted to bind trimethylated histone H3 lysine 27 in chromatin.
Codons were optimized for expression in cultured human cells.
Danio rerio (zebrafish) CBX2 amino acids 2-64. The human codon-optimized coding sequence was synthesized by Genscript.
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right" ! colspan="2" style="background:#FFBF00;"|VP64 |- |'''Function''' |transcription activator |- |'''RFC standard''' |[http://parts.igem.org/Help:Assembly_standard_10 RFC 10] |- |'''Backbone''' |pSB1C3 |- |'''Organism''' |''synthetic construct'' |- |'''Source''' |synthetic construct |- |'''Submitted by''' |[http://2016.igem.org/Team:NEU-China NEU-China 2016] |} Tetrameric VP16 transcription activator domain
This part is the SNAP-mCHERRY-CYAN UNIT of cyclebow including a SNAP UNIT, a mCHERRY UNIT and a plain CYAN. The SNAP UNIT, which can be cut off by CRE, includes an sv40 nuclear location sequence (NLS), a covalent labeling of fusion proteins named SNAP-tag?? and an sv40 terminator flanked by homodromous recognition site of recombinase CRE named loxP. The mCHERRY UNIT, which can be cut off by VIKA, includes a degradation tag named DBOX, a fluorescent gene mCherry and an sv40 terminator flanked by homodromous recognition site of recombinase VIKA named Vox. You may use this part to: 1) Test the cut-off efficiency of recombinase CRE and VIKA; 2) Estimate the time needed for genome repair because mCherry can only be expressed after the cut off of SNAP UNIT and genome repair; 3) Fast cut off this part by transient transfecting recombinase gene into nucleus; 4) When there is no CRE, you can use this part as the SNAP UNIT, which can help localize proteins in vivo with BLOCK and dye from NEB company.
The SNAP-tag?? was brought from NEB company. NLS and sv40 terminator were got from commercial plasmid: pentry nls GFP/pcdna3.1. The sequence of Vox and DBOX was retrieved from Addgene and got through denovo synthesis. mCHERRY was got from commercial plasmid: pentry dcas9 mcherry bira. CYAN was got from commercial plasmid: puc18 cfp-yfp.
The mCHERRY UNIT: mCherry flanked by Vox
This part is the mCHERRY UNIT of cyclebow system including a degradation tag named DBOX, a fluorescent gene mCherry and an sv40 terminator flanked by homodromous recognition site of recombinase VIKA named Vox. The whole part can be cut off by VIKA. You may use this part to: 1) Provide fluorescent red which can be quickly cut off by transient transfecting recombinase gene into nucleus; 2) Use it as a part of the flp-out system[5]. 5. Voutev, R. and E.J. Hubbard, A "FLP-Out" system for controlled gene expression in Caenorhabditis elegans. Genetics, 2008. 180(1): p. 103-19.
The sequence of Vox and DBOX was retrieved from Addgene and got through denovo synthesis. mCHERRY and sv40 terminator were got from commercial plasmid: pentry dcas9 mcherry bira and pentry nls GFP/pcdna3.1, respectfully.
pCMV_Z-EGFR-1907_Middle-Linker_[AAV2]-VP23 (ViralBrick-587KO-His-Tag)
This part is the SNAP UNIT of cyclebow system including an sv40 nuclear location sequence (NLS), a covalent labeling of fusion proteins named SNAP-tag?? and an sv40 terminator flanked by homodromous recognition site of recombinase CRE named loxP. The whole part can be cut off by CRE. You may use this part to: 1) Localize proteins in vivo with BLOCK and dye from NEB company; 2) Dye after blocking, you are able to see whether an event have an influence on the localization and expression of the interested protein; 3) Fast cut off this part by transient transfecting recombinase gene into nucleus.
The SNAP-tag?? was brought from NEB company. NLS and sv40 terminator were got from commercial plasmid: pentry nls GFP/pcdna3.1.
pCMV_[AAV2]-VP123ex (ViralBrick-587KO-Empty)
Hygromycin cassette for selection of mammalian cells. The selection cassette contains the SV40 promoter, the hygromycin resistance gene, and the SV40 poly A as terminator
Compatible with the Plu'n'Play assembly standard
From plasmid pcDNA3.1/Hygro from Invitrogen
[AAV2]-Rep-VP123_P5-TATAless (ViralBrick-453-Z34C)
Human phosphodiesterase 5A gene silencing device NO.1
This device is uesd for silencing the human phosphodiesterase 5A (PDE5A) gene.A U6 promoter driving a designed, synthetic shRNA-like miRNA followed by the terminator. PDE5A is a cGMP-binding, cGMP-specific phosphodiesterase, a member of the cyclic nucleotide phosphodiesterase family. This phosphodiesterase specifically hydrolyzes cGMP to 5'-GMP. It is involved in the regulation of intracellular concentrations of cyclic nucleotides and is important for smooth muscle relaxation in the cardiovascular system. We designed 3 silencing device and test their function at the same time.Here is the another two device :BBa_K1720004,BBaK172005 This device with a GFP reporter was then transfected into HEK293 cells by lentiviral vector.Once we silence the PDE5A gene the level of cGMP will be up regulated as a result. The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain sGC subunit. We used Elisa to detect cGMP level. The results are as follow:
1. Kukreja RC, Salloum FN, Das A: Cyclic Guanosine Monophosphate Signaling and Phosphodiesterase-5 Inhibitors in Cardioprotection. Journal Of the American College Of Cardiology 2012, 59(22):1921-1927. 2. Li L, Haider HK, Wang L, Lu G, Ashraf M: Adenoviral short hairpin RNA therapy targeting phosphodiesterase 5a relieves cardiac remodeling and dysfunction following myocardial infarction. American Journal Of Physiology-Heart And Circulatory Physiology 2012, 302(10):H2112-H2121. 3. Unwalla HJ, Li HT, Bahner I, Li MJ, Kohn D, Rossi JJ: Novel Pol II fusion promoter directs human immunodeficiency virus type 1-inducible coexpression of a short hairpin RNA and protein. Journal of virology 2006, 80(4):1863-1873.
We designed the shRNA according to the homology segment sequence of PDE5A from NCBI
This part is the SNAP-mCHERRY UNIT of cyclebow including a SNAP UNIT and a mCHERRY UNIT. The SNAP UNIT, which can be cut off by CRE, includes an sv40 nuclear location sequence (NLS), a covalent labeling of fusion proteins named SNAP-tag?? and an sv40 terminator flanked by homodromous recognition site of recombinase CRE named loxP. The mCHERRY UNIT, which can be cut off by VIKA, includes a degradation tag named DBOX, a fluorescent gene mCherry and an sv40 terminator flanked by homodromous recognition site of recombinase VIKA named Vox. You may use this part to: 1) Test the cut-off efficiency of recombinase CRE and VIKA; 2) Estimate the time needed for genome repair because mCherry can only be expressed after the cut off of SNAP UNIT and genome repair; 3) Fast cut off this part by transient transfecting recombinase gene into nucleus; 4) When there is no CRE, you can use this part as the SNAP UNIT, which can help localize proteins in vivo with BLOCK and dye from NEB company.
The SNAP-tag?? was brought from NEB company. NLS and sv40 terminator were got from commercial plasmid: pentry nls GFP/pcdna3.1. The sequence of Vox and DBOX was retrieved from Addgene and got through denovo synthesis. mCHERRY was got from commercial plasmid: pentry dcas9 mcherry bira.
Tetrameric VP16 transc. activator domain
Coding Sequence of IscA1 pigeon homologue (codon-optimized for humans)
Michael Becich, Thomas Lau, Sreyas Misra
The gene Iron-Sulfur cluster assembly 1 (IscA1) is a mitochondrial protein involved in the biogenesis and assembly of iron-sulfur clusters, which play a role in electron-transfer reactions and is theorized to be associated with certain organisms' magnetic perception. The homing pigeon is one such organism with this ability, and here we have codon-optimized the Columba Livia homologue for homo sapiens. The sequence can be inserted into plasmids for expression of the magnetoreceptor IscA gene, which hopefully will create magnetogenetic biosensors in the form of iron-sulfur cluster, for downstream output and applications.
While the plasmid design for this insert can be complicated, the coding sequence itself is quite simple. We only needed to remove the start codon in order to co-express a fluorescent protein, and modify two BbsII restriction enzyme sites by altering the third base pair of codons (wobble effect) at bp locations 75 (C->G) and 309 (A->G).
The predicted IscA amino acid sequence was obtained from NCBI (http://www.ncbi.nlm.nih.gov/protein/XP_005508102.1), and codon-optimized for humans with IDT's tools.
PstI restriction site was mutated. Includes start and stop codon.
Part comes from Origene human cDNA clone (NM_002176.2).
Chemokine (C-C motif) receptor 5, a HIV-1 co-receptor.
The part is cloned into BBa_J52017, a vector with eukaryontic terminator. PstI and EcoRI restriction sites inside CCR5 gene were mutated.
pCMV_VP1up_NLS_mVenus_[AAV2]-VP23 (ViralBrick-587KO-Empty)
[AAV2]-Rep-VP123_P5-TATAless (ViralBrick-587KO-BAP)
dnMyD88 is a dominant negative form of protein MyD88 (an important protein in signaling cascade through TLR4 receptor). dnMyD88 is linked to GFP (acts as a reporter) through six aminoacid long protein linker.
dnMyD88-linker-GFP was made by using PCR overlap extension technique. This part is cloned in BioBrick vector pSB1AK3. Original sequence has PstI restriction site so we have to introduce a mutation without affect on amino acid sequence.
This a flexible linker combined by two former linkers, which can be easily connect to the end of protein with overlap pcr.
when we chose a linker for our fusion protein，we would have wanted to use -gggsgggs- linker. But we found that such a tandem repeating linker is hard to work well with overlap PCR because the OE region should be unique. Therefore, we improved the original part by change the first GGGS to a carefully selected natural linker from linker database to avoid repeating sequence. Besides, we used the selected natural linker to adjust its length to about 50A.
Coding Sequence of IscA2 pigeon homologue (codon-optimized for humans)
The gene Iron-Sulfur cluster assembly 2 (IscA2) is a mitochondrial protein involved in the biogenesis and assembly of iron-sulfur clusters, which play a role in electron-transfer reactions and is theorized to be associated with certain organisms' magnetic perception. The homing pigeon is one such organism with this ability, and here we have codon-optimized the Columba Livia homologue for homo sapiens. The sequence can be inserted into plasmids for expression of the magnetoreceptor IscA gene, which hopefully will create magnetogenetic biosensors in the form of iron-sulfur cluster, for downstream output and applications.
While the plasmid design for this insert can be complicated, the coding sequence itself is quite simple. We only needed to add the start codon since this second isoform did not have methionine built in.
The predicted IscA amino acid sequence was obtained from NCBI (http://www.ncbi.nlm.nih.gov/protein/XP_005502393.1), and codon-optimized for humans with IDT's tools.
A cyclic promoter of Ki-67 from human genome
This part is a cyclic promoter named Ki67 from human genome. It can lead to transcription of the downstream DNA sequence once in every G1 phase[3]. 3. Pei, D.S., et al., Analysis of human Ki-67 gene promoter and identification of the Sp1 binding sites for Ki-67 transcription. Tumour Biol, 2012. 33(1): p. 257-66. You may use this part to: 1) Express something in mammal cell lines particularly in G1 phases or once in every cell cycle by stable transfecting it into cell line; 2) Use it as a human promoter by transient transfecting it into cells.
The sequence was retrieved from Addgene. We got it from human genome through PCR using the following primers: pKi67-F: ACCTCTGCCCTCCGCCAGCCG pKi67-R: ACCCGGTGGCCCTACAGGCTACG (Product: 360)
[AAV2]Rep-VP123_p5TATAless (ViralBrick 453 Z34C-587 HSPG-KO)
pCMV_[AAV2]-VP123ex (ViralBrick-587KO-His-Tag)
pCMV_[AAV2]-VP123ex (ViralBrick-587-BAP)
Mitochondrial Leader Sequence in RFC10 standard
Mitochondrial leader sequence (MLS) helps direct protein to the mitochondria. When this sequence is cloned in frame at the N-terminus of the protein, it can target the recombinant protein to the mitochondria in mammalian cells. MLS will be removed upon translocation into the mitochondrial matrix. However, at least four additional amino acids (Ile-His-Ser-Leu) on this sequence are known to be retained on the N-terminus of the protein.
Plasmid from Invitrogen: pCMV/myc/mito (http://tools.lifetechnologies.com/content/sfs/manuals/pshooter_pcmv_man.pdf)
It is an imortant protein in signaling cascade through TLr receptors (MyD88 dependent pathway).
Human phosphodiesterase 5A gene silencing device NO.3
dnMyD88-linker-Rluc-linker-PEST191 is a part where dnMyD88 (very important protein in signaling cascade through TLR4 receptor) is linked to Rluc (reporter) and PEST191 (degradation tag). Both linkers are made of six aminoacids.
dnMyD88-linker-Rluc-linker-PEST191 was made by using PCR ligation technique. It was difficult since we had some problems with primers. This part is cloned in BioBrick vector pSB1AK3.
Keratinocyte growth factor (KGF) is a locally acting epithelial mitogen that is produced by cells of mesenchymal origin and has an important role in protecting and repairing epithelial tissues.Use of recombinant human KGF (palifermin) in patients with hematologic malignancies reduces the incidence and duration of severe oral mucositis experienced after intensive chemoradiotherapy. These results suggest that KGF may be useful in the treatment of patients with other kinds of tumors, including those of epithelial origin.[1]The potential effect of KGF on wound healing was assessed in vitro by measuring randomized migration and plasminogen activator (PA) activity of keratinocytes in response to the growth factor. Incubation of normal human keratinocytes with KGF in modified MCDB 153 medium significantly stimulated cell migration[2] The Keratinocyte Growth Factor (KGF), also known as FGF7, is a growth factor present in the epithelialization-phase of wound healing. In this phase, keratinocytes are covering the wound, forming the epithelium.[3] . KGF is weakly expressed in human skin, but is strongly upregulated in dermal fibroblasts after skin injury. Its binding to a transmembrane receptor on keratinocytes induces proliferation and migration of these cells. Furthermore, KGF has been shown to protect epithelial cells from the toxic effects of reactive oxygen species. We have identified a series of KGF-regulated genes that are likely to play a role in these processes. In addition to KGF, activin seems to be a novel player in wound healing.[4]
The gene is in pMA vector having biobrick restriction sites(Ecor1, Xba1 and Spe1,Pst1,respectively)
pCMV_Z-EGFR-1907_Middle-Linker_[AAV2]-VP23 (ViralBrick-587KO-BAP)
SV40 poly A, terminator for mammalian cells
SV40 poly A, terminator for use in mammalian cells
[AAV2]-Rep-VP123_P5-TATAless (ViralBrick-587KO-RGD)
Toll-like receptor 3 cytoplasmic domain.
Cloned into pSBAK3 vector with terminator. Start codon is NOT included.
Mitochondrial Leader Sequence with RFC25 standard
Mitochondrial leader sequence (MLS) helps direct protein to the mitochondria. When this sequence is cloned in frame at the N-terminus of the protein, it can target the recombinant protein to the mitochondria in mammalian cells. MLS will be removed upon translocation into the mitochondrial matrix. However, at least four additional amino acids (Ile-His-Ser-Leu) on this sequence are known to be retained on the N-terminus of the protein. Since MLS always fuses with protein for function, this BioBrick is in RFC 25 standard, which facilitate protein fusion in order to introduce other protein into mitochondria.
Invitrogen: pCMV/myc/mito (http://tools.lifetechnologies.com/content/sfs/manuals/pshooter_pcmv_man.pdf)
pCMV_Z-EGFR-1907_Long-Linker_[AAV2]-VP23 (ViralBrick-587KO-Empty)
pCMV_Z-EGFR-1907_Middle-Linker_[AAV2]-VP23 (ViralBrick-587KO-Empty)
pCMV_Z-EGFR-1907_SEG-Linker_[AAV2]-VP23 (ViralBrick-587KO-Empty)
This part is the SNAP-VIKA UNIT of cyclebow including a SNAP UNIT and a VIKA UNIT. The SNAP UNIT, which can be cut off by CRE, includes an sv40 nuclear location sequence (NLS), a covalent labeling of fusion proteins named SNAP-tag?? and an sv40 terminator flanked by homodromous recognition site of recombinase CRE named loxP. The VIKA UNIT, which can be cut off by VIKA, includes an sv40 nuclear location sequence (NLS), a recombinase gene named VIKA and an sv40 terminator flanked by homodromous recognition site of recombinase VIKA named Vox. You may use this part to: 1) Test the cut-off efficiency of recombinase CRE and VIKA by qRT-PCR; 2) Estimate the time needed for genome repair because VIKA can only be expressed after the cut off of SNAP UNIT and genome repair; 3) Fast cut off this part by transient transfecting recombinase gene into nucleus; 4) When there is no CRE, you can use this part as the SNAP UNIT, which can help localize proteins in vivo with BLOCK and dye from NEB company.
The SNAP-tag?? was brought from NEB company. NLS and sv40 terminator were got from commercial plasmid: pentry nls GFP/pcdna3.1. The recombinase gene VIKA and its RTS Vox were got through denovo synthesis.
pCMV_[AAV2]-VP123ex (ViralBrick-587KO-BAP)
This plasmid contains the tGFP open reading frame under the control of human EF1a promoter. The part was cloned into the pSB1C3 plasmid with chloramphenicol rresistance.
the part cloned between EcoRI and XbaI sites.
Homo sapiens caspase 3, apoptosis-related cysteine peptidase (transcript variant beta).
Includes start and stop codon. The part is cloned into BBa_J52017, a vector with eukaryotic terminator.
Part comes from Origene SC101958 vector (NM_032991).
[AAV2]-Rep-VP123_p5-TATAless (ViralBrick-453-RGD)
Granulysin, a T Cell Product,Kills Bacteria by Altering Membrane Permeability
Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has microbial activity against a broad spectrum of microbial pathogens. The substance released by cytotoxic T cells (CD8) when attached to infected body cells. It creates holes in the target cell membrane and destroy it. That is,Granulysin induces apoptosis in target cells and have antimicrobial action. It is broadly antimicrobial, killing microbes that cause, for example, tuberculosis and malaria, and can destroy some tumors. A series of peptides generated from the amino acid sequence of granulysin are potential antibiotics. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ??-galactosidase to its impermeant substrate.Granulysin functions to create holes in the target cell membrane and destroy it. Granulysin is able to induce apoptosis in target cells and also has antimicrobial action.
Granulysin can be purified via nickel affinity chromatography.
Granulysin is a member of the saposin-like protein (SAPLIP) family. The gene was synthesized at GENEART in pMA vector
[AAV2]-Rep-VP123_P5-TATAless (ViralBrick-587KO-Z34C-Spacer)
Enteropeptidase from Homo sapiens optimized for E. coli
Enteropeptidase is an enzyme secreted in the small intestine necessary for digestion in humans. It is necessary for trypsin activation and acts as a serine protease.
Epidermal growth factor (EGF) is a single chain, 53 aminoacid-residue polypeptide (molecular weight 6045) and three intramolecular disulfide bonds. EGF is a low-molecular-weight polypeptide first purified from the mouse submandibular gland, but since then found in many human tissues including submandibular gland, parotid gland. EGF results in cellular proliferation, differentiation, and survival. EGF stimulates the growth of skin and corneal epithelium in vivo and in organ cultures. Epidermal growth factor (EGF) stimulates the growth of keratinocytes in vivo, and therefore plays an important role in the process of wound healing that depends on mitosis and migration of keratinocytes. Rhinewald and Green showed, in vitro that in the presence of growth factors , higher percentage of cells leave the resting state, enter and remain in the mitotic cycle. Mitogenic effect of EGF requires continuous exposure of target cells to EGF for a minimum 6-12 hours. The stimulation of wound healing by EGF has been confirmed as growth of granulation tissue in sponge implants used as inductive matrices. They observed, in rats, dose-dependent effect of EGF on granulation tissue formation as increased amounts of nucleic acids, and accumulation of collagen and glycosaminoglycans.
EGF with Biobrick standart restriction sites
EGF is synthesized in human and some mammalians. Our EGF was synthesized synthetically at GENEART
CMV promoter - MLS - GFP - hGH polyA tail
pCMV_[AAV2]VP123 (ViralBrick-453-His-Tag)
BGH poly A, terminator for use in mammalian cells
Sequence for chemokine receptor CD4, including signal peptide.
Vector expressing eGFP from the CMV promoter with the BGH polyA tail and with a hygromycin resistance cassette
Compatible with the Plug'n'Play with DNA assembly system.
pCMV_Z-EGFR-1907_[AAV2]-VP23 (ViralBrick-587KO-Empty)
[AAV2]Rep-VP123_p5TATAless (ViralBrick 453 RGD-587 HSPG-KO)
dnMyD88 is a dominant negative form of protein MyD88 (an important protein in signaling cascade through TLR4 receptor), which was our main target for modifying the signaling path. dnMyD88 is linked to Rluc (which acts as a reporter) through six aminoacid long protein linker.
dnMyD88-linker-Rluc was made by using PCR ligation technique. This part is cloned in BioBrick vector pSB1AK3.
Promoter for expression in mammalian cells. The SV40 promoter contains an ori.
[AAV2]-Rep-VP123_P5-TATAless (ViralBrick-587-BAP)
Part is composed of TIR domain of Traf6 and GFP linked with 6 aminoacids long linker. GFP serves as reporter. For usage in eukaryotes.
pCMV_[AAV2]-VP123ex (ViralBrick-587KO-Z34C)
[AAV2]-Rep-VP123_P5-TATAless (ViralBrick-587-RGD)
K157011(His affinity tag; Freiburg standard):K157004(Fluorescin-A-binding )
pCMV_[AAV2]-VP123ex (ViralBrick-587-RGD)
A cyclic promoter of CDK4 from human genome
This part is a cyclic promoter named CDK4 from human genome. It can lead to transcription of the downstream DNA sequence in every G1 phase[4]. 4. Guo, Z.Y., et al., The elements of human cyclin D1 promoter and regulation involved. Clin Epigenetics, 2011. 2(2): p. 63-76. You may use this part to: 1) Express something in mammal cell lines particularly in G1 phases or once in every cell cycle by stable transfecting it into cell line; 2) Use it as a human promoter by transient transfecting it into cells.
The sequence was retrieved from Addgene. We got it from human genome through PCR using the following primers: pCDK4-F: TGGTGGAGCGAAAAGGTGACAGCATC pCDK4-R: GCGGAAACTGGGAGGGCGGGGCGAA (Product: 457)
Chemokine (CXC motif) receptor 4, fused to N-terminal half of ubiquitin.
CXCR4 and NUb fragments were fused with PCR overlap extension method without any linker added. The part is cloned into BBa_J52017 (a vector with eukaryontic terminator).
CXCR4 gene obtained from Origene, NUb gene obtained from Dualsystems.
enhanced green fluorescent protein for use in mammalian cells
hGH poly A, terminator for mammalian cells
From plasmid N-terminal pFLAG-CMV from Sigma-Aldrich
This is a 40-50A flexible separation for fusion domains with a SV40 NLS, and its codon is optimized for mammalian. It can be used to link different protein domains for fusion proteins locating in cell nucleus.
The codon is optimized for mammalian, and we insert a NLS to promote locating in cell nucleus. Besides ,we lengthen this linker to provide better separation of two domains. At last we avoid tandem repeat sequence(like GGGSPKKKRKVGGGS), which will facilitate fusing this linker to your domains via overlap PCR.
This part is improved from BBa_K1486003.
pCMV_VP1up_NLS_Z-EGFR-1907_[AAV2]-VP23 (ViralBrick-587KO-Empty)
[AAV2]-Rep-VP123_P5-TATAless (ViralBrick-453-His-Tag)
Human phosphodiesterase 5A gene silencing device NO.2
This device is uesd for silencing the human phosphodiesterase 5A (PDE5A) gene.A U6 promoter driving a designed, synthetic shRNA-like miRNA followed by the terminator. PDE5A is a cGMP-binding, cGMP-specific phosphodiesterase, a member of the cyclic nucleotide phosphodiesterase family. This phosphodiesterase specifically hydrolyzes cGMP to 5'-GMP. It is involved in the regulation of intracellular concentrations of cyclic nucleotides and is important for smooth muscle relaxation in the cardiovascular system. We designed 3 silencing device and test their function at the same time.Here is the another two device :BBa_K1720003,BBaK172005 This device with a GFP reporter was then transfected into HEK293 cells by lentiviral vector.Once we silence the PDE5A gene the level of cGMP will be up regulated as a result. The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain sGC subunit. We used Elisa to detect cGMP level. The results are as follow:
Promoter for expression of genes in mammalian cells
From plasmid pSuperior.puro from ￼￼pSuper RNAi System, OligoEngine
A cyclic promoter of Cyclin E from human genome
This part is a cyclic promoter named Cyclin E from human genome. It can lead to transcription of the downstream DNA sequence once in every G1 phase[1,2] 1. Hwang, H.C. and B.E. Clurman, Cyclin E in normal and neoplastic cell cycles. Oncogene, 2005. 24(17): p. 2776-86. 2. Ohtani, K., J. DeGregori, and J.R. Nevins, Regulation of the cyclin E gene by transcription factor E2F1. Proc Natl Acad Sci U S A, 1995. 92(26): p. 12146-50.
The sequence was retrieved from Addgene. We got it from human genome through PCR using the following primers: pCCNE-F: CGTGTTTACATTCCACCCGCGCCA pCCNE-R: TGATGGGGCTGCTCCGGCCT (Product: 986)
Human Polycomb chromodomain from the CBX8 protein (a.a. 1-63). See Haynes and Silver, JBC, 2011
The VIKA UNIT: recombinase VIKA flanked by Vox
This part is the VIKA UNIT of cyclebow system including an sv40 nuclear location sequence (NLS), a recombinase gene named VIKA and an sv40 terminator flanked by homodromous recognition site of recombinase VIKA named Vox. The whole part can be cut off by VIKA. This part is the improvement of the BBa_K1641003，which is a plain VIKA. You may use this part to: 1) Test the cut-off efficiency of recombinase VIKA by qRT-PCR; 2) Fast cut off this part by transient transfecting recombinase gene into nucleus; 3) Do lineage tracking.
The recombinase gene VIKA and its RTS Vox were got through denovo synthesis. NLS and sv40 terminator were got from commercial plasmid: pentry nls GFP/pcdn3.1.
pCMV_[AAV2]-VP123ex (ViralBrick-587KO-Z34C-Spacer)
pCMV_His-Tag_Middle-Linker_[AAV2]-VP23 (ViralBrick-587KO-Empty)
pCMV_[AAV2]-VP123ex (ViralBrick-587-His-Tag)
pCMV_VP1up_NLS_His-Tag_[AAV2]-VP23 (ViralBrick-587KO-Empty)
pCMV_[AAV2]-VP123ex (ViralBrick-587-Beta-lactamase)
pCMV_Z-EGFR-1907_Short-Linker_[AAV2]-VP23 (ViralBrick-587KO-Empty)
pCMV_DARPin-E01_Middle-Linker_[AAV2]-VP23 (ViralBrick-587KO-Empty)