Fluorescent protein vectors for promoter studies

pTimer is a prokaryotic expression vector that encodes DsRed1-E5, a fluorescent protein that changes color as it ages. Shortly after translation, DsRed1-E5 emits green light; its red fluorophore emerges later, hours after translation. Because of its predictable color shift, DsRed1-E5 acts as a timer, useful for monitoring changes in gene activity in living cells.

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Vector map for pTimer vectors

Vector map for pTimer vectors. As the Fluorescent Timer matures, it changes color—in a matter of hours, depending on the expression system used.

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Fluorescent timer under the control of the heat shock promoter hsp16-41 in a transgenic C. elegans embryo

Fluorescent Timer under the control of the heat shock promoter hsp16-41 in a transgenic C. elegans embryo. The embryo was heat shocked in a 33°C water bath. Promoter activity was studied during the heat shock recovery period. Green fluorescence was observed in the embryo as early as two hr into the recovery period. By 50 hr after heat shock, promoter activity had ceased, as indicated by the lack of green color.

pTimer-1 is a promoterless vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the MCS located upstream of the DsRed1-E5 coding sequence. Shortly after translation, DsRed1-E5 emits green light; its red fluorophore emerges later, hours after translation. Because of its predictable color shift, DsRed1-E5 acts as a timer, useful for monitoring changes in gene activity in living cells. pTimer-1 can be used to study the on-off activity of any cis-acting regulatory element cloned into the MCS upstream of DsRed1-E5.

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Vector map for pTimer vectors

Vector map for pTimer vectors. As the Fluorescent Timer matures, it changes color—in a matter of hours, depending on the expression system used.

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Fluorescent timer under the control of the heat shock promoter hsp16-41 in a transgenic C. elegans embryo

Fluorescent Timer under the control of the heat shock promoter hsp16-41 in a transgenic C. elegans embryo. The embryo was heat shocked in a 33°C water bath. Promoter activity was studied during the heat shock recovery period. Green fluorescence was observed in the embryo as early as two hr into the recovery period. By 50 hr after heat shock, promoter activity had ceased, as indicated by the lack of green color.

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632403: pTimer-1 Vector

pDsRed2-1 encodes DsRed2, a variant of the Discosoma sp. red fluorescent protein (DsRed). DsRed2 contains six amino acid substitutions that accelerate chromophore maturation and decrease protein aggregation. In addition, the coding sequence for DsRed2 contains a series of silent base-pair changes for higher expression in mammalian cells. pDsRed2-1 can be used as a reporter to monitor the activity of promoters and promoter/enhancer combinations cloned into the multiple cloning site (MCS) upstream of the promoterless DsRed2 gene. Without the addition of a functional promoter, this vector will not express DsRed2.

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Vector map for promoterless fluorescent protein vectors AcGFP1-1, DsRed2-1, DsRed-Express-1, HcRed1-1 and ZsGreen1-1

Vector map for promoterless fluorescent protein vectors pAcGFP1-1, pDsRed2-1, pDsRed-Express-1, pHcRed1-1 and pZsGreen1-1.

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Excitation and emission spectra of reef coral fluorescent proteins AmCyan1, ZsGreen1, ZsYellow1, DsRed2, AsRed2, and HcRed1

Excitation and emission spectra of reef coral fluorescent proteins AmCyan1, ZsGreen1, ZsYellow1, DsRed2, AsRed2,  and HcRed1.

pHcRed1-1 is a promoterless, mammalian expression vector which can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS) located upstream of the HcRed1 coding sequence. Promoters should be cloned into the pHcRed1-1 MCS upstream from the HcRed1 coding sequences. Without the addition of a functional promoter, this vector will not express HcRed1.

HcRed1 is a far-red fluorescent protein which was generated by mutagenesis of a non-fluorescent chromoprotein from the reef coral Heteractis crispa. The HcRed1 coding sequence has been human codon-optimized for higher expression in mammalian cells. The sequence upstream of HcRed1 has been converted to a Kozak consensus sequence to further increase the translation efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the HcRed1 gene direct proper processing of the 3' end of the HcRed1 mRNA. The vector also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin resistance cassette (Neo^r)-consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene-allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli.

pHcRed1-1 can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418.

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Vector map for promoterless fluorescent protein vectors AcGFP1-1, DsRed2-1, DsRed-Express-1, HcRed1-1 and ZsGreen1-1

Vector map for promoterless fluorescent protein vectors pAcGFP1-1, pDsRed2-1, pDsRed-Express-1, pHcRed1-1 and pZsGreen1-1.

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Excitation and emission spectra of reef coral fluorescent proteins AmCyan1, ZsGreen1, ZsYellow1, DsRed2, AsRed2, and HcRed1

Excitation and emission spectra of reef coral fluorescent proteins AmCyan1, ZsGreen1, ZsYellow1, DsRed2, AsRed2,  and HcRed1.

pDsRed-Express-1 is a promoterless mammalian expression vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into MCS. It encodes DsRed-Express, a variant of Discosoma sp. red fluorescent protein (DsRed). DsRed-Express contains nine amino acid substitutions which improve the solubility of the protein, reduce the time from transfection to detection of red fluorescence, and decrease the level of residual green emission. When DsRed-Express is expressed in mammalian cell cultures, red-emitting cells can be detected by either fluorescence microscopy or flow cytometry 8–12 hours after transfection. Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express displays a reduced tendency to aggregate. The DsRed-Express coding sequence is human codon-optimized for high expression in mammalian cells.

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Vector map for promoterless fluorescent protein vectors AcGFP1-1, DsRed2-1, DsRed-Express-1, HcRed1-1 and ZsGreen1-1

Vector map for promoterless fluorescent protein vectors pAcGFP1-1, pDsRed2-1, pDsRed-Express-1, pHcRed1-1 and pZsGreen1-1.

pHcRed1-DR is a promoterless vector that encodes HcRed1-DR, a destabilized variant of Heteractis crispa far-red fluorescent protein, HcRed1. HcRed1-DR contains a PEST degradation domain at its C-terminus, so it is rapidly turned over in mammalian cell culture. Because if its short half-life, HcRed1-DR is well suited for monitoring the activity of promoters and promoter/enhancer combinations cloned into the multiple cloning site, located upstream of the human codon-optimized HCRed1-DR gene. Without the addition of a functional promoter, this vector will not express HcRed1-DR.

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Vector map for destabilized fluorescent protein vectors pDsRed-Express-DR, pHcRed1-DR, and pZsGreen1-DR

Vector map for destabilized fluorescent protein vectors pDsRed-Express-DR, pHcRed1-DR, and pZsGreen1-DR. These fluorescent protein variants display rapid turnover rates.

pDsRed-Express-DR is a promoterless vector that encodes DsRed-Express-DR, a destabilized variant of Discosoma sp. red fluorescent protein, DsRed-Express. DsRed-Express-DR contains a PEST degradation domain at its C-terminus, so it is rapidly turned over in mammalian cell culture. Because if its short half-life, DsRed-Express-DR is well suited for monitoring the activity of promoters and promoter/enhancer combinations cloned into the multiple cloning site, located upstream of the human codon-optimized DsRed-Express-DR gene. Without the addition of a functional promoter, this vector will not express DsRed-Express-DR.

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Vector map for destabilized fluorescent protein vectors pDsRed-Express-DR, pHcRed1-DR, and pZsGreen1-DR

Vector map for destabilized fluorescent protein vectors pDsRed-Express-DR, pHcRed1-DR, and pZsGreen1-DR. These fluorescent protein variants display rapid turnover rates.

pZsGreen1-DR is a promoterless vector that encodes a destabilized variant of Zoanthus sp. green fluorescent protein, ZsGreen1. ZsGreen1-DR contains a PEST degradation domain at its C-terminus, so it is rapidly turned over in mammalian cell culture. Because of its short half-life, ZsGreen1-DR is well suited for monitoring the activity of promoters and promoter/enhancer combinations cloned into the multiple cloning site, located upstream of the human codon-optimized ZsGreen1-DR gene. Without the addition of a functional promoter, this vector will not express ZsGreen1-DR.

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Vector map for destabilized fluorescent protein vectors pDsRed-Express-DR, pHcRed1-DR, and pZsGreen1-DR

Vector map for destabilized fluorescent protein vectors pDsRed-Express-DR, pHcRed1-DR, and pZsGreen1-DR. These fluorescent protein variants display rapid turnover rates.

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632428: pZsGreen1-DR Vector

pZsGreen1-1 encodes ZsGreen1, a variant of the Zoanthus sp. green fluorescent protein (ZsGreen1) that has been engineered for brighter fluorescence. The ZsGreen1 coding sequence contains a series of silent base pair changes for optimal expression in mammalian cells. pZsGreen1-1 can be used as a reporter to monitor the activity of promoters and promoter/enhancer combinations cloned into the multiple cloning site (MCS) upstream of the promoterless ZsGreen1 gene. Without the addition of a functional promoter, this vector will not express ZsGreen1.

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Excitation and emission spectra of reef coral fluorescent proteins AmCyan1, ZsGreen1, ZsYellow1, DsRed2, AsRed2, and HcRed1

Excitation and emission spectra of reef coral fluorescent proteins AmCyan1, ZsGreen1, ZsYellow1, DsRed2, AsRed2,  and HcRed1.

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Vector map for promoterless fluorescent protein vectors AcGFP1-1, DsRed2-1, DsRed-Express-1, HcRed1-1 and ZsGreen1-1

Vector map for promoterless fluorescent protein vectors pAcGFP1-1, pDsRed2-1, pDsRed-Express-1, pHcRed1-1 and pZsGreen1-1.

pAcGFP1-1 encodes a green fluorescent protein (AcGFP1) from Aequorea coerulescens. The fluorescent protein coding sequence in this construct has been human-codon-optimized for efficient expression and enhanced brightness in mammalian cells. The vector contains a neomycin resistance gene for the selection of stable transfectants. pAcGFP1-1 can be used as a reporter to monitor the activity of promoters and promoter/enhancer combinations cloned into the multiple cloning site (MCS) upstream of the promoterless AcGFP1 gene. Without the addition of a functional promoter, this vector will not express AcGFP1.

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Vector map for promoterless fluorescent protein vectors AcGFP1-1, DsRed2-1, DsRed-Express-1, HcRed1-1 and ZsGreen1-1

Vector map for promoterless fluorescent protein vectors pAcGFP1-1, pDsRed2-1, pDsRed-Express-1, pHcRed1-1 and pZsGreen1-1.

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632497: pAcGFP1-1 Vector

pmCherry-1 encodes mCherry, a mutant derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed. pmCherry-1 is a promoterless vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS). Promoters should be cloned into the pmCherry-1 MCS upstream of the mCherry coding sequence. Without the addition of a functional promoter, this vector will not express mCherry.

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Vector map for promoterless fluorescent protein vectors AcGFP1-1, DsRed2-1, DsRed-Express-1, HcRed1-1 and ZsGreen1-1

Vector map for promoterless fluorescent protein vectors pAcGFP1-1, pDsRed2-1, pDsRed-Express-1, pHcRed1-1 and pZsGreen1-1.

pDsRed-Express2-1 encodes DsRed-Express2, a variant of Discosoma sp. red fluorescent protein. pDsRed-Express2-1 is a promoterless vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS) upstream of the promoterless DsRed-Express2 gene. Without the addition of a functional promoter, this vector will not express DsRed-Express2.

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Vector map for promoterless fluorescent protein vectors AcGFP1-1, DsRed2-1, DsRed-Express-1, HcRed1-1 and ZsGreen1-1

Vector map for promoterless fluorescent protein vectors pAcGFP1-1, pDsRed2-1, pDsRed-Express-1, pHcRed1-1 and pZsGreen1-1.

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Robust expression of DsRed-Express2 in mouse bone marrow hematopoietic stem and progenitor cells

Robust expression of DsRed-Express and DsRed-Express2 in mouse bone marrow hematopoietic stem and progenitor cells. Mononuclear bone marrow cells were transduced with retroviral vectors encoding DsRed-Express (Panel A), DsRed-Express2 (Panel B), or EGFP (Panel C), and fluorescent cells were sorted 87 hr later. Red and green fluorescence signals were detected using the PE and FITC filter sets, respectively. The lines represent gates defined by analyzing untransduced cells.