HcRed1 fluorescent protein

pHcRed1-N1/1 encodes HcRed1, a far red fluorescent protein that can be used to monitor gene expression and protein localization in vivo. HcRed1 was generated by mutagenesis of a non-fluorescent chromoprotein from the reef coral Heteractis crispa. The coding sequence for HcRed1 is human codon-optimized for higher expression in mammalian cells. Genes cloned into the multiple cloning site (MCS) upstream of the HcRed1 coding sequence are expressed as fusions to the N-terminus of HcRed1. The unmodified vector will express HcRed1 in mammalian cells.

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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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Schematic for fluorescent fusion protein vectors

Schematic for fluorescent fusion protein vectors. Panel A. N-terminal fluorescent protein (FP) vector. Panel B. C-terminal fluorescent protein vector.

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Hygromycin DsRed-Monomer and AcGFP1 expression vectors

Hygromycin DsRed-Monomer and AcGFP1 expression vectors. Vector maps of our DsRed-Monomer and pAcGFP1 Hyg-N1 (Panel A) and Hyg-C1 (Panel B) mammalian expression vectors for fusion applications. Each vector contains a hygromycin selection cassette. These vectors can be used together with our standard N1/C1 vectors containing the neomycin selection cassette to establish double stable cell lines expressing two different Living Colors proteins.

pHcRed1-C1 is a mammalian expression vector designed to express a protein of interest fused to the C-terminus of the far-red fluorescent protein HcRed1. pHcRed1-C1 can be used to monitor gene expression and protein localization in vivo or as a cotransfection marker; the unmodified vector will express HcRed1 in mammalian cells.

HcRed1 was generated by mutagenesis of a non-fluorescent chromoprotein from the reef coral Heteractis crispa. The coding sequence for HcRed1 is human codon-optimized for higher expression in mammalian cells. Genes cloned into the multiple cloning site (MCS) downstream of the HcRed1 coding sequence are expressed as fusions to the C-terminus of HcRed1. The MCS in pHcRed1-C1 is positioned between the HcRed1 coding sequence and the SV40 polyadenylation signal (SV40 poly A). Genes cloned into the MCS will be expressed as fusions to the C-terminus of HcRed1 if they are in the same reading frame as HcRed1 and there are no intervening stop codons. A Kozak consensus sequence upstream of HcRed1 increases the translation efficiency in eukaryotic cells. SV40 poly A signals downstream of the MCS direct proper processing of the 3' end of mRNA transcripts. 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-C1 can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418.

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Schematic for fluorescent fusion protein vectors

Schematic for fluorescent fusion protein vectors. Panel A. N-terminal fluorescent protein (FP) vector. Panel B. C-terminal fluorescent protein vector.

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.

pHcRed1 is a bacterial expression vector which encodes the far-red fluorescent protein, HcRed1. HcRed1 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.

The HcRed1 coding sequence is flanked by distinct multiple cloning sites (MCS) so that the gene can be easily excised from pHcRed1. Alternatively, the HcRed1 coding sequence can be amplified by PCR. In E. coli, HcRed1 is expressed from the lac promoter as a fusion with several additional amino acids, including the first five amino acids of the LacZ protein. Note, however, that if you excise the HcRed1 coding sequence using a restriction site in the 5' MCS, the resulting fragment will encode the native (i.e., non-fusion) HcRed1 protein. pHcRed1 is a pUC 19 derivative (pPD16.43) which provides a high-copy-number origin of replication as well as an ampicillin resistance gene for propagation and selection in E. coli.

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Four-color visualization of Living Colors Fluorescent Proteins

Four-color visualization of Living Colors Fluorescent Proteins. HEK 293 clonal cell lines stably expressing AmCyan1, ZsGreen1, ZsYellow1, or HcRed1 were mixed and plated in the same culture dish and imaged at 20X magnification.

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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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Basic fluorescent protein vector map

Basic fluorescent protein vector map. Use this bacterial expression vector as a source of the fluorescent protein gene. Note: There is a stop codon at the 5' end of the 3' MCS. The 3' MCS should not be used for cloning.

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.