MoClo Toolkit

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Name: PSADpro::aadA::PSAD-T:::: ARpro::PSAN::roGFP2-Tsa2ΔCR::MStop::RPL23-T
Host strain: TOP10
Selection in E. coli: Kanamycin (50 μg/ ml)
Original pL2: pAGM4673
Function: H2O2 reporter-no tag targeted to the chloroplast (Lumen)
TU1: pCM1-01
TU2: pMBS645
End Linker: pICH41744-pL2-EL2

Sequence

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Name: PSADpro::aadA::PSAD-T:::: ARpro::PSBO::roGFP2-Tsa2ΔCR::MStop::RPL23-T
Host strain: TOP10
Selection in E. coli: Kanamycin (50 μg/ ml)
Original pL2: pAGM4673
Function: H2O2 reporter-no tag targeted to the chloroplast (Lumen)
TU1: pCM1-01
TU2: pMBS646
End Linker: pICH41744-pL2-EL2

Sequence

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Name: PSADpro::aadA::PSAD-T:::: ARpro::HSP70C::roGFP2-Tsa2ΔCR::MStop::RPL23-T
Host strain: TOP10
Selection in E. coli: Kanamycin (50 μg/ ml)
Original pL2: pAGM4673
Function: H2O2 reporter-no tag targeted to the mitochondria
TU1: pCM1-01
TU2: pMBS647
End Linker: pICH41744-pL2-EL2

Sequence

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Name: PSADpro::aadA::PSAD-T:::: ARpro::BIP1::roGFP2-Tsa2ΔCR::ER-ret::RPL23-T
Host strain: TOP10
Selection in E. coli: Kanamycin (50 μg/ ml)
Original pL2: pAGM4673
Function: H2O2 reporter-no tag targeted to the endoplasmic reticulum
TU1: pCM1-01
TU2: pMBS648
End Linker: pICH41744-pL2-EL2

Sequence

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Name: PSADpro::aadA::PSAD-T:::: ARpro::roGFP2-Tsa2ΔCR::SV40::RPL23-T
Host strain: TOP10
Selection in E. coli: Kanamycin (50 μg/ ml)
Original pL2: pAGM4673
Function: H2O2 reporter-no tag targeted to the nucleus
TU1: pCM1-01
TU2: pMBS649
End Linker: pICH41744-pL2-EL2

Sequence

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Name: ARpro::roGFP2-Orp1::MStop::RPL23-T
Host strain: TOP10
Selection in E. coli: ampicillin (100 μg/ ml)
Type: 2F
Original pl1: pICH47742
Function: H2O2 reporter-no tag
Brick 1 – pL0: pCM0-017
Brick 2 – pL0: pMBS418
Brick 3 – pL0: pCM0-101
Brick 4 – pL0: pCM0-119
Fusion 5′ lvl2: GCAA
Fusion 3′ lvl2: ACTA

Sequence

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, July 2021

Name: PSADpro::aadA::PSAD-T:::: ARpro::roGFP2-Orp1::MStop::RPL23-T
Host strain: TOP10
Selection in E. coli: Kanamycin (50 μg/ ml)
Original pL2: pAGM4673
Function: H2O2 reporter-no tag
TU1: pCM1-01
TU2: pMBS719
End Linker: pICH41744-pL2-EL2

Sequence

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, March 2022

Host strain: TOP10
Selection in E. coli: kanamycin (50 μg/ ml)
Position L0 (Patron et al):
Brick name: L2-aadA-CRed (SpecR)
Brick type: Destination vector
Sequence reference:
Brick function: Destination vector with L1 aadA (SpecR) and CRed accepting L0 parts
Source of Brick: Niemeyer and Schroda 2022
Acceptor plasmid: pAGM4673
Designer / Builder: J. Niemeyer

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

Sequence

Niemeyer J, Schroda M. New destination vectors facilitate Modular Cloning for Chlamydomonas. Curr Genet. 2022 Aug;68(3-4):531-536. doi: 10.1007/s00294-022-01239-x. Epub 2022 Apr 16. PMID: 35429260; PMCID: PMC9279246.

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, March 2022

Host strain: TOP10
Selection in E. coli: kanamycin (50 μg/ ml)
Position L0 (Patron et al):
Brick name: L2-AphVIII-CRed (ParoR)
Brick type: Destination vector
Sequence reference:
Brick function: Destination vector with L1 aphVIII (ParoR) and CRed accepting L0 parts
Source of Brick: Niemeyer and Schroda 2022
Acceptor plasmid: pAGM4673
Designer / Builder: J. Niemeyer

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

Sequence

Niemeyer J, Schroda M. New destination vectors facilitate Modular Cloning for Chlamydomonas. Curr Genet. 2022 Aug;68(3-4):531-536. doi: 10.1007/s00294-022-01239-x. Epub 2022 Apr 16. PMID: 35429260; PMCID: PMC9279246.

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, March 2022

Host strain: TOP10
Selection in E. coli: kanamycin (50 μg/ ml)
Position L0 (Patron et al):
Brick name: L2-NAT-CRed (NTCR)
Brick type: Destination vector
Sequence reference:
Brick function: Destination vector with L1 nat1 (NTCR) and CRed accepting L0 parts
Source of Brick: Niemeyer and Schroda 2022
Acceptor plasmid: pAGM4673
Designer / Builder: J. Niemeyer

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

Sequence

Niemeyer J, Schroda M. New destination vectors facilitate Modular Cloning for Chlamydomonas. Curr Genet. 2022 Aug;68(3-4):531-536. doi: 10.1007/s00294-022-01239-x. Epub 2022 Apr 16. PMID: 35429260; PMCID: PMC9279246.

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, March 2022

Host strain: TOP10
Selection in E. coli: kanamycin (50 μg/ ml)
Position L0 (Patron et al):
Brick name: L2-AphVII-CRed (HygroR)
Brick type: Destination vector
Sequence reference:
Brick function: Destination vector with L1 aphVII (HygroR) and CRed accepting L0 parts
Source of Brick: Niemeyer and Schroda 2022
Acceptor plasmid: pAGM4673
Designer / Builder: J. Niemeyer

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

Sequence

Niemeyer J, Schroda M. New destination vectors facilitate Modular Cloning for Chlamydomonas. Curr Genet. 2022 Aug;68(3-4):531-536. doi: 10.1007/s00294-022-01239-x. Epub 2022 Apr 16. PMID: 35429260; PMCID: PMC9279246.

From Justus Niemeyer, Michael Schroda lab, University of Kaiserslautern, March 2022

Host strain: TOP10
Selection in E. coli: kanamycin (50 μg/ ml)
Position L0 (Patron et al):
Brick name: L2-Ble-CRed (BleoR)
Brick type: Destination vector
Sequence reference:
Brick function: Destination vector with L1 ble (BleoR) and CRed accepting L0 parts
Source of Brick: Niemeyer and Schroda 2022
Acceptor plasmid: pAGM4673
Designer / Builder: J. Niemeyer

The entire collection of MoClo Toolkit plasmids, distributed as frozen glycerol stocks in a 96-well format, can also be ordered for $400 for academic/non-profit users and $800 for commercial/industry here: https://www.chlamycollection.org/product/moclo-toolkit/

Sequence

Niemeyer J, Schroda M. New destination vectors facilitate Modular Cloning for Chlamydomonas. Curr Genet. 2022 Aug;68(3-4):531-536. doi: 10.1007/s00294-022-01239-x. Epub 2022 Apr 16. PMID: 35429260; PMCID: PMC9279246.

From Pablo Leivar, IQS School of Engineering-Barcelona Spain, December 2024

Host strain: DH5α
Selection in E. coli: Kanamycin (50 µg/ml)
BsaI sites presence for homology arms cloning: No
BbsI sites for Level 2 cloning: Yes: TGCC and GGGA.
LacZα gene flanked by BbsI sites: Yes
Chloroplast 5′ and 3′ homology arms: Amplified from pWF vector (Kuras and Wollman, 1994)
Function: Level 2 Golden Gate compatible destination vector, containing Chlamydomonas plastome 5’ and 3’ homology regions (arms) to allow Chlamydomonas plastome transformation by homologous recombination in the pWF locus. For more information, refer to Melero-Cobo et al.

Sequence

Melero-Cobo X, Gallemí M, Carnicer M, Monte E, Planas A, Leivar P. MoCloro: an extension of the Chlamydomonas reinhardtii modular cloning toolkit for microalgal chloroplast engineering. Physiol Plant. 2025 Jan-Feb;177(1):e70088. doi: 10.1111/ppl.70088. PMID: 39871102; PMCID: PMC11772913.