CyanoGate Kit
(Kit # 1000000146 )

Depositing Lab:   Alistair McCormick

The CyanoGate kit contains 96 parts and acceptor vectors for generating multi-part assemblies in either integrative (suicide) or self-replicating plasmid vectors for working in cyanobacteria.

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

Original Publication

CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax. Vasudevan R, Gale GAR, Schiavon AA, Puzorjov A, Malin J, Gillespie MD, Vavitsas K, Zulkower V, Wang B, Howe CJ, Lea-Smith DJ, McCormick AJ. Plant Physiology.2019 May;180(1):39-55. doi: 10.1104/pp.18.01401. PubMed PMID: 30819783.

Description

The CyanoGate kit contains 96 parts and acceptor vectors for generating multi-part assemblies in either integrative (suicide) or self-replicating plasmid vectors for working in cyanobacteria. The kit is compatible with the plant Modular Cloning (MoClo) syntax (also known as PhytoBricks). You require the backbone acceptor vectors in the MoClo Toolkit (Kit # 1000000044) with this kit to progress from level 0 to level T assemblies. This CyanoGate kit includes promoters, minimal (up to the transcriptional start site (TSS)) promoters, terminators, flanking sequences for HR-mediated genomic integration, selectable marker cassettes, level T (for Transformation) acceptor vectors, and parts for building CRISPRi- and sgRNA-based systems.

Seventeen accessory plasmids that can also be used with this kit are available as individual plasmids. Links to these plasmid pages can be found from the Accessory Plasmids Tab.

Figure 1. Adaptation of the Plant Golden Gate MoClo level 0 syntax for generating level 1 assemblies for transfer to Level T. The format for a level 0 MoClo acceptor vector with the part bordered by two BsaI sites. Typical level 0 parts from the Plant MoClo kit, where parts of the same type are bordered by the same pair of fusion sites (for each fusion site, only the sequence of the top strand is shown). Note that the parts are not drawn to scale. The syntax of the Plant MoClo kit was adapted to generate level 0 parts for engineering marked and unmarked cyanobacterial mutant strains. To generate knock in mutants, short linker parts (30 bp) were constructed to allow assembly of individual flanking sequences, or marker cassettes (AbRor sacB) in level 1 vectors for subsequent assembly in level T. Parts required for generating synthetic srRNA or CRISPRi level 1 constructs. See Supplemental Info 4 and 5 from paper for workflows. Abbreviations: 3U+Ter, 3’UTR and terminator; Ab^R, antibiotic resistance cassette; Ab^R DOWN LINKER, short sequence (~30 bp) to provide CGCT overhang; Ab^R UP LINKER, short sequence (~30 bp) to provide GAGG overhang; CDS2(stop), coding sequence with a stop codon; DOWN FLANK, flanking sequence downstream of target site; DOWN FLANK LINKER, short sequence (~30 bp) to provide GGAG overhang; Prom+5U, promoter and 5’ UTR; Prom TSS, promoter transcription start site; sacB, levansucrase expression cassette; sacB UP LINKER, short sequence (~30 bp) to provide GAGG overhang; sgRNA, single guide RNA; SP, signal peptide; srRNA, small regulatory RNA; UP FLANK, flanking sequence upstream of target site; UP FLANK LINKER, short sequence (~30 bp) to provide CGCT overhang; UNMARK LINKER, short sequence to bridge UP FLANK and DOWN FLANK.

Figure 2. Extension of the Plant Golden Gate MoClo Assembly Standard for cyanobacterial transformation. Assembly relies on one of two Type IIS restriction endonuclease enzymes (BsaI or BpiI). Domesticated level 0 parts are assembled into level 1 vectors. Up to seven level 1 modules can be assembled directly into a level T cyanobacterial transformation vector, which consist of two sub-types (either a replicative or an integrative vector). Alternatively, larger vectors with more modules can be built by assembling level 1 modules into level M, and then cycling assembly between level M and level P, and finally transferred from Level P to level T. Antibiotic selection markers are shown for each level. Level T vectors are supplied with internal antibiotic selection markers (shown), but additional selection markers could be included from level 1 modules as required. See Supplemental Info 3 from paper for full list and maps of level T acceptor vectors.

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 CyanoGate Kit was a gift from Alistair McCormick (Addgene kit #1000000146).”

For your Reference section:

CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax. Vasudevan R, Gale GAR, Schiavon AA, Puzorjov A, Malin J, Gillespie MD, Vavitsas K, Zulkower V, Wang B, Howe CJ, Lea-Smith DJ, McCormick AJ. Plant Physiology.2019 May;180(1):39-55. doi: 10.1104/pp.18.01401. PubMed PMID: 30819783.

CyanoGate Kit - #1000000146

Inventory

Well	Plasmid	Resistance
A / 1	pCAT.011	Spectinomycin
A / 2	pCAT.334	Spectinomycin
A / 3	pCA0.001	Spectinomycin
A / 4	pCAT.015	Ampicillin
A / 5	pCAT.000	Ampicillin and Kanamycin
A / 6	pCA0.002	Kanamycin
A / 7	pC0.049	Kanamycin
A / 8	pC0.034	Kanamycin
A / 9	pC0.041	Kanamycin
A / 10	pC.059	Kanamycin
A / 11	pC0.057	Kanamycin
A / 12	pC.060	Kanamycin
B / 1	pC0.005	Spectinomycin
B / 2	pC0.050	Spectinomycin
B / 3	pC0.051	Spectinomycin
B / 4	pC0.052	Spectinomycin
B / 5	pC0.053	Spectinomycin
B / 6	pC.061	Kanamycin
B / 7	pC0.056	Spectinomycin
B / 8	pC0.093	Spectinomycin
B / 9	pC0.030	Spectinomycin
B / 10	pC0.031	Spectinomycin
B / 11	pC0.032	Spectinomycin
B / 12	pC0.033	Spectinomycin
C / 1	pC0.035	Spectinomycin
C / 2	pC0.036	Spectinomycin
C / 3	pC0.037	Spectinomycin
C / 4	pC0.038	Spectinomycin
C / 5	pC0.039	Spectinomycin
C / 6	pC0.040	Spectinomycin
C / 7	pC0.043	Spectinomycin
C / 8	pC0.044	Spectinomycin
C / 9	pC0.045	Spectinomycin
C / 10	pC0.046	Spectinomycin
C / 11	pC0.047	Spectinomycin
C / 12	pC0.048	Spectinomycin
D / 1	pC0.083	Spectinomycin
D / 2	pC0.084	Spectinomycin
D / 3	pC0.085	Spectinomycin
D / 4	pC0.088	Spectinomycin
D / 5	pC0.089	Spectinomycin
D / 6	pC0.090	Spectinomycin
D / 7	pC0.091	Spectinomycin
D / 8	pC0.092	Spectinomycin
D / 9	pC0.285	Spectinomycin
D / 10	pC.264	Spectinomycin
D / 11	pC0.203	Spectinomycin
D / 12	pC0.208	Spectinomycin
E / 1	pC0.215	Spectinomycin
E / 2	pC0.219	Spectinomycin
E / 3	pC0.220	Spectinomycin
E / 4	pC0.078	Spectinomycin
E / 5	pC0.081	Spectinomycin
E / 6	pC0.082	Spectinomycin
E / 7	pC0.116	Spectinomycin
E / 8	pC0.117	Spectinomycin
E / 9	pC0.118	Spectinomycin
E / 10	pC0.119	Spectinomycin
E / 11	pC0.120	Spectinomycin
E / 12	pC0.121	Spectinomycin
F / 1	pC0.008	Spectinomycin
F / 2	pC0.009	Spectinomycin
F / 3	pC0.161	Spectinomycin
F / 4	pC0.017	Spectinomycin
F / 5	pC0.018	Spectinomycin
F / 6	pC0.026	Spectinomycin
F / 7	pC0.027	Spectinomycin
F / 8	pC0.028	Spectinomycin
F / 9	pC0.029	Spectinomycin
F / 10	pC0.265	Spectinomycin
F / 11	pC0.122	Spectinomycin
F / 12	pC0.123	Spectinomycin
G / 1	pC0.025	Spectinomycin
G / 2	pC0.024	Spectinomycin
G / 3	pC0.166	Spectinomycin
G / 4	pC0.165	Spectinomycin
G / 5	pC0.169	Spectinomycin
G / 6	pC0.170	Spectinomycin
G / 7	pC0.171	Spectinomycin
G / 8	pC0.172	Spectinomycin
G / 9	pC0.174	Spectinomycin
G / 10	pC0.173	Spectinomycin
G / 11	pC0.175	Spectinomycin
G / 12	pC0.176	Spectinomycin
H / 1	pC0.178	Spectinomycin
H / 2	pC0.177	Spectinomycin
H / 3	pC0.281	Spectinomycin
H / 4	pC0.282	Spectinomycin
H / 5	pC0.283	Spectinomycin
H / 6	pC0.284	Spectinomycin
H / 7	pC0.062	Spectinomycin
H / 8	pC0.070	Spectinomycin
H / 9	pC0.066	Spectinomycin
H / 10	pC0.004	Spectinomycin
H / 11	pC0.006	Spectinomycin
H / 12	pC0.007	Spectinomycin

Data calculated @ 2024-12-21

Accessory Plasmids

Seventeen additional accessory level 0 part plasmids are also available. These plasmids are not included in the kit, but can be ordered separately:

Promoters

• pC0.055: PisiAB
• pC0.054: PpetE

CDS

• pC0.286: Chloramphenicol resistance cassette

Insertion Site

• pC0.168: 6803 NS2 Down Flank
• pC0.167: 6803 NS2 Up Flank

Terminators

• pC0.064: Terminator from hupB gene from E. coli.
• pC0.077: Terminator from rpoH gene from E. coli.
• pC0.072: Terminator from hyaABCDEF operon from E. coli.
• pC0.068: Terminator for clpPX gene from E. coli.
• pC0.065: Terminator araBAD operon from E. coli.
• pC0.073: Terminator from rplJL-rpoBC, rplKAJL-rpoBC, rpoBC gene cluster from E. coli.
• pC0.069: Terminator from arcA gene from E. coli.
• pC0.071: Terminator from tatABCD operon from E. coli.
• pC0.074: Terminator from Cysteine synthase A gene (cysK) from E. coli.
• pC0.063: Terminator derived from Biobricks plasmid pSB1AK3.
• pC0.080: Terminator for the cpcG1 gene Synechocystis sp. PCC 6803.
• pC0.079: Terminator for the psbA2 gene Synechocystis sp. PCC 6803.