Plasmids

From Markus Heitzer, University of Regensburg-Germany, ca. July 2006

Synthetic luciferase reporter gene, controlled by HSP70A/RBCS2

amp resistant

Fuhrmann M, Hausherr A, Ferbitz L, Schödl T, Heitzer M, Hegemann P (2004) Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol 55:869-881

From Markus Heitzer, University of Regensburg-Germany, ca. July 2006

Synthetic gene encoding the green fluorescent protein (GFP) from Aequorea victoria adapted to the nuclear codon usage of Chlamydomonas reinhardtii (cGFP). In vector pBluescriptII-KS (cloned via XhoI/BamHI, ampicillin resistance). 

amp resistant

Sequence and map (scanned)

Fuhrmann M, Oertel W, Hegemann P (1999) A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J 19:353-361

From Carolyn Silflow, University of Minnesota, June 2006

Plasmid for epitope tagging; contains 3 copies of the 9-amino acid influenza hemagglutinin epitope

host strain: DH5 alpha
amp resistant

 

Additional Information

Silflow CD, LaVoie M, Tam LW, Tousey S, Sanders M, Wu W, Borodovsky M, Lefebvre PA (2001) The Vfl1 Protein in Chlamydomonas localizes in a rotationally asymmetric pattern at the distal ends of the basal bodies. J Cell Biol 153:63-74

Received in January 2001

PSAD gene, from which coding region can be removed and replaced with a gene of interest under control of the PSAD flanking sequences; ble insert

host strain: DH5 alpha
amp resistant

Fischer N, Rochaix JD (2001) The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Gen Genet 265:888-894

From Markus Heitzer, University of Regensburg-Germany, March 2006

Synthetic luciferase reporter gene, controlled by HSP70A/RBCS2

host strain: DH5 alpha
amp resistant

Fuhrmann M, Hausherr A, Ferbitz L, Schödl T, Heitzer M, Hegemann P (2004) Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol 55:869-881

Received December 2005

6x-His-tagged PF16 fusion, full length cDNA clone in pRSET B

amp resistant

Smith EF, Lefebvre PA (1996) PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella. J Cell Biol 132:359-370

Received December 2005

Full length genomic clone (in Bluescript) from PF16 gene, capable of rescuing the pf16 mutant by transformation

amp resistant

Smith EF, Lefebvre PA (1996) PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella. J Cell Biol 132:359-370

Received December 2005

Probe for PF20 gene, full length genomic clone in Bluescript

amp resistant

Smith EF, Lefebvre PA (1997) PF20 gene product contains WD repeats and localizes to the intermicrotubule bridges in Chlamydomonas flagella. Mol Biol Cell 8:455-467

Received December 2005

Full length cDNA clone in pUC19

amp resistant

Smith EF, Lefebvre PA (1997) PF20 gene product contains WD repeats and localizes to the intermicrotubule bridges in Chlamydomonas flagella. Mol Biol Cell 8:455-467

Received December 2005

Full length genomic clone in Bluescript

amp resistant

Dymek EE, Lefebvre PA, Smith EF (2004) PF15p is the chlamydomonas homologue of the Katanin p80 subunit and is required for assembly of flagellar central microtubules. Eukaryotic Cell 3:870-879

Received December 2005

Plasmid contains cDNA encoding amino acids 1353-2087, in pCR2.1

amp resistant

Wargo MJ, Dymek EE, Smith EF (2005) Calmodulin and PF6 are components of a complex that localizes to the C1 microtubule of the flagellar central apparatus. J Cell Sci 118:4655-4665

Received December 2005

Plasmid contains cDNA encoding amino acids 4-143, in pCR2.1

amp resistant

Wargo MJ, Dymek EE, Smith EF (2005) Calmodulin and PF6 are components of a complex that localizes to the C1 microtubule of the flagellar central apparatus. J Cell Sci 118:4655-4665

Received December 2005

Plasmid contains cDNA encoding amino acids 2-267, in pCR2.1

amp resistant

Wargo MJ, Dymek EE, Smith EF (2005) Calmodulin and PF6 are components of a complex that localizes to the C1 microtubule of the flagellar central apparatus. J Cell Sci 118:4655-4665

Received December 2005

Plasmid contains cDNA encoding amino acids 24-144, in pCR2.1

amp resistant

Wargo MJ, Dymek EE, Smith EF (2005) Calmodulin and PF6 are components of a complex that localizes to the C1 microtubule of the flagellar central apparatus. J Cell Sci 118:4655-4665

From Karl Johnson, Haverford College, October 2005

RSP3 genomic sequence as originally described by Williams et al. (“pEK RS3”). A 36 bp insertion was cloned into a Stu I site near the end of the RSP3 coding sequence, such that an HA epitope is encoded near the extreme carboxy-terminus of RSP3 (Kozminski et al, 1993) Digestion with Eco R1 releases the entire genomic+tag insert (~4Kb) from the vector (3 Kb). Transformation of this plasmid into pf14 resulted in restoration of wild-type motility and the presence of HA-tagged RSP3 in flagella.

amp resistant

Williams BD, Velleca MA, Curry AM, Rosenbaum JL (1989) Molecular cloning and sequence analysis of the Chlamydomonas gene coding for radial spoke protein 3: flagellar mutation pf-14 is an ochre allele. J Cell Biol 109:235-245 Kozminski KG, Diener DR, Rosenbaum JL (1993) High level expression of nonacetylatable alpha-tubulin in Chlamydomonas reinhardtii. Cell Motil Cytoskeleton 25:158-170

From Peter Hegemann, Humboldt University Berlin, September 2005

Synthetic GFP reporter optimized for nuclear codon bias.

Also known as pMF124cGFP,  it was made by Markus Fuhrmann (his plasmid 124) and was later simplified to the name pCrGFP.

host strain: DH5 alpha
amp resistant

Plasmid Information

Fuhrmann M, Oertel W, Hegemann P (1999) A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J 19:353-361

From Heriberto Cerutti, University of Nebraska-Lincoln, March 2004

Construct for post-transcriptional gene silencing, based on tandem inverted repeat sequences from MAA7

host strain: DH5 alpha
amp resistant

Rohr J, Sarkar N, Balenger S, Jeong BR, Cerutti H (2004) Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas. Plant J 40:611-621

From Michael Allen, Merchant Lab, UCLA, May 2007

ARS2 reporter under control of the iron-responsive FEA1 promoter

amp resistant

Allen MD, del Campo JA, Kropat J, Merchant SS (2007) FEA1, FEA2, and FRE1, encoding two homologous secreted proteins and a candidate ferrireductase, are expressed coordinately with FOX1 and FTR1 in iron-deficient Chlamydomonas reinhardtii. Eukaryotic Cell 6:1841-1852

From Michael Allen, Merchant Lab, UCLA, May 2007

ARS2 reporter under control of the iron-responsive FOX1 promoter

amp resistant

Allen MD, del Campo JA, Kropat J, Merchant SS (2007) FEA1, FEA2, and FRE1, encoding two homologous secreted proteins and a candidate ferrireductase, are expressed coordinately with FOX1 and FTR1 in iron-deficient Chlamydomonas reinhardtii. Eukaryotic Cell 6:1841-1852

From Michael Allen, Merchant Lab, UCLA, May 2007

ARS2 reporter under control of the iron-responsive FTR1 promoter

amp resistant

Allen MD, del Campo JA, Kropat J, Merchant SS (2007) FEA1, FEA2, and FRE1, encoding two homologous secreted proteins and a candidate ferrireductase, are expressed coordinately with FOX1 and FTR1 in iron-deficient Chlamydomonas reinhardtii. Eukaryotic Cell 6:1841-1852

aphVIII (paromomycin resistance) with HSP70A/RBCS2 promoter

host strain: DH5 alpha
amp resistant

Sizova I, Fuhrmann M, Hegemann P (2001) A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277:221-229

host strain: DH10B
amp resistant

Sequence

pSI103-1 sequence + notes

host strain: DH10B
amp resistant

host strain: DH10B
amp resistant

host strain: DH10B
amp resistant

From Laurens Mets, University of Chicago, October 2006

ARS gene, with no promoter

host strain: DH5 alpha
amp resistant

de Hostos EL, Schilling J, Grossman AR (1989) Structure and expression of the gene encoding the periplasmic arylsulfatase of Chlamydomonas reinhardtii. Mol Gen Genet 218:229-239

From Laurens Mets, University of Chicago, October 2006

arylsulfatase reporter with beta-tubulin promoter

host strain: DH5 alpha
amp resistant

 

Sequence

Schematic

Davies JP, Weeks DP, Grossman AR (1992) Expression of the arylsulfatase gene from the beta 2-tubulin promoter in Chlamydomonas reinhardtii. Nucl Acids Res 20:2959-2965

From Carolyn Silflow, University of Minnesota, September 2007

genomic clone of UNI2 gene

The plasmid pML9.7 was constructed by cloning a 9.7 kb Sal I genomic fragment containing the UNI2 gene into the Sal I site of pBlueScript II KS (-).

amp resistant

Piasecki BP, LaVoie M, Tam LW, Lefebvre PA, Silflow CD (2008) The Uni2 phosphoprotein is a cell cycle regulated component of the basal body maturation pathway in Chlamydomonas reinhardtii. Mol Biol Cell 19:262-273

From Carolyn Silflow, University of Minnesota, September 2007

UNI2 gene tagged with 3xHA (3 copies of the 9-amino acid influenza hemagglutinin epitope)

To create pML9.7-3xHA , the pML9.7 plasmid was first modified by removing sequences between the EcoRV site and the Xba I site in the multi-cloning site positioned near the 3’ end of the UNI2 gene. This step removed the Bam HI site from the multi-cloning site. The plasmid was then digested with Bam HI which cleaves the UNI2 gene at a position 13 codons upstream of the stop codon. The Bam HI overhang was filled in using Klenow. A blunt-ended Nru I/Sma I fragment from the p3xHA plasmid was then ligated into the blunted Bam HI site.

amp resistant

 

Additional Information

Silflow CD, LaVoie M, Tam LW, Tousey S, Sanders M, Wu W, Borodovsky M, Lefebvre PA (2001) The Vfl1 Protein in Chlamydomonas localizes in a rotationally asymmetric pattern at the distal ends of the basal bodies. J Cell Biol 153:63-74 Piasecki BP, LaVoie M, Tam LW, Lefebvre PA, Silflow CD (2008) The Uni2phosphoprotein is a cell cycle regulated component of the basal body maturation pathway in Chlamydomonas reinhardtii. Mol Biol Cell 19:262-273

From Markus Heitzer, University of Regensburg-Germany, November 2007

loxP, codon-adapted Renilla luciferase under control of HSP70A/RBCS2 promoter with RBCS2 intron 1 and 3′ UTR

host strain: Pir1 (Invitrogen)
kanamycin resistant

 

Sequence

Heitzer M, Zschoernig B (2007) Construction of modular tandem expression vectors for the green alga Chlamydomonas reinhardtii using the Cre/lox-system. Biotechniques 43:324