Voytas Lab - MoClo Plant Parts III: Transformation & Genome Engineering Kit
(Kit # 1000000236 )

Depositing Lab:   Daniel Voytas

The MoClo Plant Parts III: Transformation & Genome Engineering Kit contains 94 Level 0 parts, compatible with the Phytobrick/MoClo system for hierarchical golden gate assembly of plant transformation constructs. All parts are domesticated for BsaI and PaqCI, and the majority are also domesticated for Esp3I, BbsI, and SapI sites.

This kit will be sent as bacterial glycerol stocks in 96-well plate format.

Original Publication

An extensible vector toolkit and parts library for advanced engineering of plant genomes. Chamness JC, Kumar J, Cruz AJ, Rhuby E, Holum MJ, Cody JP, Tibebu R, Gamo ME, Starker CG, Zhang F, Voytas DF. Plant Genome. 2023 Mar 9:e20312. doi: 10.1002/tpg2.20312. PubMed Article .

Description

The kit includes regulatory elements, coding sequences, basic CRISPR parts, and parts for modular geminiviral replicon assembly.

• Regulatory elements: 21 promoters (J1-J4 or J1-J6 part classes), including constitutive, housekeeping, viral, and inducible promoters for both monocot and dicot expression, 19 monocot and dicot terminators (J9-J11), 5 enhancer-blocking insulator sequences (J1-J11), and 1 hairpin transcription insulator sequence (J1-J4)
• Coding sequences (J6-J9): 8 different site-specific recombinases, 5 fluorescent reporters (GFP, RFP, CFP), 4 luciferase reporters (Firefly, Renilla, Emerald, RedF), 2 metabolic reporters (Ruby, fungal luciferase), 2 selectable markers (HptII, NptII), 5 developmental regulators (IPT, AtPLT5, TaGRF-GIF, ZmBbm, ZmWus2), and AtSpCas9
• CRISPR reagents: Arabidopsis and rice U6 promoters and SpCas9 guide scaffold, for direct assembly of sgRNA expression cassettes into Level 1 module
• Geminiviral replicons: parts for Wheat Dwarf Virus (WDV) and Bean Yellow Dwarf Virus (BeYDV), including long intergenic region (LIR), short intergenic region (SIR), and replication initiator protein (Rep)/RepA sequences, compatible with the JMC Toolkit (Addgene Kit # 1000000233) for multigene assembly

• Figure 1: Hierarchical assembly of constructs from Phytobrick parts. a: New parts are assembled into Level 0 destination vectors using BbsI with the Phytobrick (J1-J11) overhangs. b: Phytobricks can occupy one of 10 atomic part classes, but commonly span simplified classes using J1, J6, J9, and J11. c: Level 0 parts are assembled into Level 1 destination vectors using BsaI with J1 and J11. d: Level 1 modules are assembled into Level 2 destination vectors using PaqCI with a new set of overhangs (M1-MΩ). e: The pMIN vectors are Level 2 destination vectors, including a minimal backbone and 2 binary vector backbones.

• Figure 2: Modular expansion of binary vectors. a: Expansion linkers are Level 1 modules at any position with end-paired Esp3I or BsaI sites. b: Level 2 vectors with expansion linkers can be used directly as transformation constructs. c: Destination donor vectors restore a cloning marker and Golden Gate sites into an expansion linker. d: Both BsaI and Esp3I linkers can be used to create Level 2 destination vectors. e: Esp3I linkers can also be used to create Level 1 destination vectors.

Kit Documentation

Protocols and supporting materials: Detailed cloning protocols for use of these parts, as well as expression benchmarking data for many of the regulatory elements, available as part of the associated publication (Chamness et al. 2023).

Related materials: For construct assembly resources, see also the JMC Toolkit and other Phytobrick-compatible cloning toolkits (MoClo, GoldenBraid, Mobius) available on Addgene. For additional Phytobrick parts libraries, see MoClo Plant Parts, MoClo Plant Parts II, MoClo CRISPR/Cas Toolkit, and others.

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

“The Voytas Lab MoClo Plant Parts III: Transformation & Genome Engineering Kit was a gift from Daniel Voytas (Addgene kit #1000000236).”

For your Reference section:

An extensible vector toolkit and parts library for advanced engineering of plant genomes. Chamness JC, Kumar J, Cruz AJ, Rhuby E, Holum MJ, Cody JP, Tibebu R, Gamo ME, Starker CG, Zhang F, Voytas DF. Plant Genome. 2023 Mar 9:e20312. doi: 10.1002/tpg2.20312. PubMed Article .

MoClo Plant Parts III: Transformation & Genome Engineering Kit - #1000000236

Inventory

Well	Plasmid	Resistance
A / 1	pMC-1-EXOB-11	Spectinomycin
A / 2	pMC-1-TBS-11	Spectinomycin
A / 3	pMC-1-Ugi1-11	Spectinomycin
A / 4	pMC-1-Ugi3-11	Spectinomycin
A / 5	pMC-1-Ugi4-11	Spectinomycin
A / 6	pMC-1-Hairpin_Insulator-4	Spectinomycin
A / 7	pMC-1-CmYLCV-4	Spectinomycin
A / 8	pMC-1-DR5v2-4	Spectinomycin
A / 9	pMC-1-OsEf1a-4	Spectinomycin
A / 10	pMC-1-Nos-6	Spectinomycin
A / 11	pMC-1-35S_Long-6	Spectinomycin
A / 12	pMC-1-CmYLCV-6	Spectinomycin
B / 1	pMC-1-AtUbi10-6	Spectinomycin
B / 2	pMC-1-GmHSP17-5E-6	Spectinomycin
B / 3	pMC-1-NtEf1a-6	Spectinomycin
B / 4	pMC-1-OsEf1a-6	Spectinomycin
B / 5	pMC-1-OsUbi-6	Spectinomycin
B / 6	pMC-1-PvUbi1-6	Spectinomycin
B / 7	pMC-1-PvUbi2-6	Spectinomycin
B / 8	pMC-1-SlEf1a-6	Spectinomycin
B / 9	pMC-1-SlRPS5A1-6	Spectinomycin
B / 10	pMC-1-SlUbi-6	Spectinomycin
B / 11	pMC-1-StUbi-6	Spectinomycin
B / 12	pMC-1-ZmAXIG1-6	Spectinomycin
C / 1	pMC-1-ZmEf1a-6	Spectinomycin
C / 2	pMC-1-ZmPLTP-6	Spectinomycin
C / 3	pMC-1-ZmUbi1-6	Spectinomycin
C / 4	pMC-6-BeYDV-Rep-RepA-9	Spectinomycin
C / 5	pMC-6-WDV-Rep-RepA-9	Spectinomycin
C / 6	pMC-1-BeYDV-LS-L-11	Spectinomycin
C / 7	pMC-1-BeYDV-LRS-L-11	Spectinomycin
C / 8	pMC-1-WDV-LS-L-11	Spectinomycin
C / 9	pMC-1-WDV-LRS-L-11	Spectinomycin
C / 10	pMC-6-Cre-9	Spectinomycin
C / 11	pMC-6-Cre-StLS1-9	Spectinomycin
C / 12	pMC-6-Cre-GR-9	Spectinomycin
D / 1	pMC-6-FLPe-9	Spectinomycin
D / 2	pMC-6-PhiC31-9	Spectinomycin
D / 3	pMC-6-R-9	Spectinomycin
D / 4	pMC-6-Integrase4-9	Spectinomycin
D / 5	pMC-6-Integrase13-9	Spectinomycin
D / 6	pMC-6-CinH-9	Spectinomycin
D / 7	pMC-6-Bxb1-9	Spectinomycin
D / 8	pMC-6-AmCyan-9	Spectinomycin
D / 9	pMC-6-Cycle3GFP-9	Spectinomycin
D / 10	pMC-6-EGFP-9	Spectinomycin
D / 12	pMC-6-DsRed2-9	Spectinomycin
E / 1	pMC-6-ELuc-9	Spectinomycin
E / 2	pMC-6-FLuc-9	Spectinomycin
E / 3	pMC-6-RedFLuc-9	Spectinomycin
E / 4	pMC-6-RLuc-9	Spectinomycin
E / 5	pMC-6-Ruby-9	Spectinomycin
E / 6	pMC-6-GFP-P2A-NptII-9	Spectinomycin
E / 7	pMC-6-AmCyan-P2A-NptII-9	Spectinomycin
E / 8	pMC-6-HptII-9	Spectinomycin
E / 9	pMC-6-NptII-9	Spectinomycin
E / 10	pMC-6-AtPLT5-9	Spectinomycin
E / 11	pMC-6-IPT-9	Spectinomycin
E / 12	pMC-6-TaGRF-GIF-9	Spectinomycin
F / 1	pMC-6-ZmBbm-9	Spectinomycin
F / 2	pMC-6-ZmWus2-9	Spectinomycin
F / 3	pMC-6-ZmBbm-P2A-ZmWus2-9	Spectinomycin
F / 4	pMC-6-AsNPGA-9	Spectinomycin
F / 5	pMC-6-NnCPH-9	Spectinomycin
F / 6	pMC-6-NnH3H-9	Spectinomycin
F / 7	pMC-6-NnHISPS-9	Spectinomycin
F / 8	pMC-6-NnLuz-9	Spectinomycin
F / 9	pMC-6-HpaB-9	Spectinomycin
F / 10	pMC-6-HpaC-9	Spectinomycin
F / 11	pMC-6-RcTAL-9	Spectinomycin
F / 12	pMC-9-35S-11	Spectinomycin
G / 1	pMC-9-AtHSP-11	Spectinomycin
G / 2	pMC-9-NbAct3-11	Spectinomycin
G / 3	pMC-9-NbHSP-11	Spectinomycin
G / 4	pMC-9-NtEU-11	Spectinomycin
G / 5	pMC-9-NtEf1a-11	Spectinomycin
G / 6	pMC-9-OsAct2-11	Spectinomycin
G / 7	pMC-9-OsEf1a-11	Spectinomycin
G / 8	pMC-9-OsT28-11	Spectinomycin
G / 9	pMC-9-OsUbi-11	Spectinomycin
G / 10	pMC-9-PinII-11	Spectinomycin
G / 11	pMC-9-RBCS-11	Spectinomycin
G / 12	pMC-9-SlEf1a-11	Spectinomycin
H / 1	pMC-9-SlRPS5A1-11	Spectinomycin
H / 2	pMC-9-SlUbi-11	Spectinomycin
H / 3	pMC-9-StUbi-11	Spectinomycin
H / 4	pMC-9-ZmEf1a-11	Spectinomycin
H / 5	pMC-9-ZmIn2-11	Spectinomycin
H / 6	pMC-9-ZmUbi-11	Spectinomycin
H / 7	pMC-6-AtSpCas9-9	Spectinomycin
H / 8	pMC-1-AtU6-Y	Spectinomycin
H / 9	pMC-1-OsU6-X	Spectinomycin
H / 10	pMC-Z-SpCas9-gRNA-repeat-polyT-11	Spectinomycin

Data calculated @ 2024-05-01