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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.

Add to Cart
$350 USD + shipping

Available to academics and nonprofits only.

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.

Cyanogate.png
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; AbR, antibiotic resistance cassette; AbR DOWN LINKER, short sequence (~30 bp) to provide CGCT overhang; AbR 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.
Cyanogate2.png
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

Resistance Color Key

Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.

Inventory

Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.

Kit Plate Map

96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.

Resistance Color Key

Spectinomycin
Ampicillin
Ampicillin and Kanamycin
Kanamycin

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 @ 2020-07-02

Kit Plate Map - #1000000146

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

CDS

  • pC0.286: Chloramphenicol resistance cassette

Insertion Site

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.
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