BBa_J23101
BBa_B0010
BBa_I13453
BBa_K5149004
Short Description: AquI: Desired homologous region to the genome for CbAgo
Long Description: The AquI Locus is a 1200bp gene that is a putative type II restriction endonuclease that has been hypothesized to be a neutral site in
PCC 11901. In addition, the removal of type II restriction endonucleases has shown to increase transformation efficiency so we decided to use the region as a Neutral Site.
The 1-400 bp region of AquI Locus is implemented into our plasmids to help provide the Argonaute protein with DNA guides. In theory, CbAgo and RecBCD will break and digest
this region into 16-18bp chunks and CbAgo will acquire the chunks and form full complexes. Thus the CbAgo complex will find the AquI Locus in the UTEX3154 genome and produce
breaks in the DNA sequence. To use this 400bp region, you just need to include it into a plasmid which the Argonaute system will recognize as exogenous DNA and break it to
take as guides.
Source of Part: We used Q5 PCR with primers that flanked the AquI-1 Locus to amplify the region out of the genome of our strain Synechoccus sp. UTEX3154.
Design Considerations: We decided to split the 1200bp AquI Locus into three parts to give flexibility in our experiments. We intend to implement Chi Sites between
each 400bp region to promote DNA guide acquisition. To assemble the AquI-1 Locus correctly into our plasmids, we used Modular Cloning (MoClo). MoClo is a type II restriction
enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible we added the correct restriction enzyme site and 4 base pair overhang on flags to the primers we
used to PCR amplify the region out of the genome.
BBa_K5149005
Short Description: Desired homologous region to the genome for CbAgo to cut
Long Description: The AquI Locus is a 1200bp gene that is a putative type II restriction endonuclease that has been hypothesized to be a neutral site in PCC 11901. In addition, the removal of type II restriction endonucleases has shown to increase transformation efficiency so we decided to use the region as a Neutral Site. The 401-800 bp region of AquI Locus is implemented into our plasmids to help provide the Argonaute protein with DNA guides. In theory, CbAgo and RecBCD will break and digest this region into 16-18bp chunks and CbAgo will acquire the chunks and form full complexes. Thus the CbAgo complex will find the AquI Locus in the UTEX3154 genome and produce breaks in the DNA sequence. To use this 400bp regio, you just need to include it into a plasmid which the Argonaute system will recognize as exogenous DNA and break it to take as guides.
Source of Part: We used Q5 PCR with primers that flanked the AquI-2 Locus to amplify the region out of the genome of our strain Synechoccus sp. UTEX3154.
Design Considerations: We decided to split the 1200bp AquI Locus into three parts to give flexibility in our experiments. We intend to implement Chi Sites between each 400bp region to promote DNA guide acquisition. To assemble the AquI-2 Locus correctly into our plasmids, we used Modular Cloning (MoClo). MoClo is a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible we added the correct restriction enzyme site and 4 base pair overhang on flags to the primers we used to PCR amplify the region out of the genome.
BBa_K5149006
Short Description: Desired homologous region to the genome for CbAgo to cut
Long Description: The AquI Locus is a 1200bp gene that is a putative type II restriction endonuclease that has been hypothesized to be a neutral site in PCC 11901. In addition, the removal of type II restriction endonucleases has shown to increase transformation efficiency so we decided to use the region as a Neutral Site. The 801-1200 bp region of AquI Locus is implemented into our plasmids to help provide the Argonaute protein with DNA guides. In theory, CbAgo and RecBCD will break and digest this region into 16-18bp chunks and CbAgo will acquire the chunks and form full complexes. Thus the CbAgo complex will find the AquI Locus in the UTEX3154 genome and produce breaks in the DNA sequence. To use this 400bp region, you just need to include it into a plasmid which the Argonaute system will recognize as exogenous DNA and break it to take as guides.
Source of Part: We used Q5 PCR with primers that flanked the AquI-3 Locus to amplify the region out of the genome of our strain Synechoccus sp. UTEX3154.
Design Considerations: We decided to split the 1200bp AquI Locus into three parts to give flexibility in our experiments. We intend to implement Chi Sites between each 400bp region to promote DNA guide acquisition. To assemble the AquI-3 Locus correctly into our plasmids, we used Modular Cloning (MoClo). MoClo is a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible we added the correct restriction enzyme site and 4 base pair overhang on flags to the primers we used to PCR amplify the region out of the genome.
BBa_K5149001
Short Description: Region Flanking AquI for Homologous Recombination
Long Description: The Up Homology Arm is a 1200bp region homologous to the flank of one side of the AquI region.
To use the Up Homology Arm to AquI Locus, you need to pair it with the Down Homology Arm to AquI Locus. Additionally, you need to include some gene in
between the Arms, for example, GFP. The construct will look like this: Up Homology Arm - GFP - Down Homology Arm. This provides your organism with a template
for homologous recombination. When a double-stranded DNA break occurs within the AquI locus, the template will be used for homologous recombination, allowing GFP to
be inserted into the genome. This is how we used the Up Homology Arm in our project.
Source of Part: We used Q5 PCR with primers that flanked the Up Homology Arm region to amplify the region out of the genome of our strain Synechoccus sp. UTEX3154.
Design Considerations: To assemble Up Homology Arm to AquI Locus correctly into our plasmids, we used Modular Cloning (MoClo), a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible, we added the correct restriction enzyme site and 4 base pair overhang on flags to the primers we used to PCR amplify the region out of the genome.
BBa_K5149002
Short Description: Region Flanking AquI for Homologous Recombination
Long Description: The Down Homology Arm is a 1200bp region homologous to the flank of one side of the AquI region. To use the Down
Homology Arm to AquI Locus, you need to pair it with the Up Homology Arm to AquI Locus. Additionally, you need to include some gene in between the Arms,
for example, GFP. The construct will look like this: Up Homology Arm - GFP - Down Homology Arm. This provides your organism with a template for homologous
recombination. When a double-stranded DNA break occurs within the AquI locus, the template will be used for homologous recombination,
allowing GFP to be inserted into the genome. This is how we used the Down Homology Arm in our project.
Source of Part: We used Q5 PCR with primers that flanked the Down Homology Arm region to amplify the region out of the genome of our strain Synechoccus sp. UTEX3154.
Design Considerations: To assemble the Down Homology Arm to AquI Locus correctly into our plasmids, we used Modular Cloning (MoClo), a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible, we added the correct restriction enzyme site and 4 base pair overhang on flags to the primers we used to PCR amplify the region out of the genome.
BBa_K5149000
Short Description: Clostridium butyricum Argonaute protein
Long Description: CbAgo is a part of the Argonaute protein family,
known for gene silencing and protection against exogenous DNA/RNA. In our project, we use CbAgo as a method for genome manipulation. By providing a plasmid with regions
homologous to the target genome and CbAgo, DNA guides are produced by CbAgo and taken in to form full complexes. These complexes then cut every part of the genome where
the DNA guide binds. CbAgo is a mesophile, functioning between 20-45 degrees Celsius, compared to most thermophile Argonaute proteins.
Source of Part: CbAgo comes from the organism Clostridium butyricum. We ordered the sequence through Integrated DNA Technologies (IDT).
Design Considerations: To assemble CbAgo correctly into our plasmids, we used Modular Cloning (MoClo), a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible, we added the correct restriction enzyme site and 4 base pair overhang. Additionally, we codon-optimized CbAgo to Synechoccocus sp. UTEX3154 and removed restriction modification sites by introducing point mutations in the sequence.
BBa_K5149003
Short Description: RecBCD recognition site to stop digesting dsDNA
Long Description: Chi Sites are repeated DNA sequences that act as stopping recognition sites for RecBCD complexes. In our project, Chi Sites
regulate the complete digestion of the dsDNA region we are attempting to edit. By implementing Chi Sites between each of the 400bp regions of AquI Locus, we can
test the efficiency of CbAgo taking in a DNA Guide to form a full complex. The speed at which the AquI Locus is cut will determine whether CbAgo depends on Chi
Sites for more efficient guide acquisition. DNA acquisition for CbAgo is only possible with RecBCD or a RecBCD-like system.
Source of Part: We ordered two primers of linear DNA through Integrated DNA Technologies (IDT): one forward strand and one reverse strand.
To create the part, we put them through a thermocycler at 94 degrees Celsius and allowed the thermocycler to cool down to room temperature, ensuring the annealing
of the two linear fragments.
Design Considerations: To assemble the Chi Site correctly into our plasmids, we used Modular Cloning (MoClo),
a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible, we added the correct restriction
enzyme site and 4 base pair overhang on the IDT-ordered primers.
BBa_K5149100
Short Description: Inducible CbAgo Composite
Long Description: The composite part is used to induce the plasmid expression of CbAgo when transformed into the cell. By doing this, we can study the toxicity of CbAgo based on the amount of Arabinose we provide to our organism. In addition, we can allow our replicative accelerator plasmid to grow in the cell before CbAgo starts to cut and destroy the plasmids.
Source of Part: The araBAD pBAD promoter and rrnB T1 Terminator were sourced from the CyanoGate Kit. The CbAgo was sourced through orders from Integrated DNA Technologies. The basic parts were assembled by Golden Gate Assembly into a level 1 backbone plasmid from the MoClo kit.
Design Considerations: All the parts had to have the correct overhang so they could assemble in the correct orientation
BBa_K5149103
Short Description: Composite GFP with strong promoter BBa_K5149103
Long Description: A genetic transcription unit that consists of promoter, Green Fluorescent Protein (GFP), and a terminator. The J23101 is a strong promoter that will express many GFP units. We use this composite part as an insert into our organisms' chromosomes so that our organism fluoresces green.
Source of Part: Both promoter and terminator originate from the CyanoGate Modular Cloning Kit; the GFP gene is a synthetic gene block that has been codon optimized and run through the BLACKBIRD program.
Design Considerations: All parts were designed to work with either BsaI or BpiI restriction enzymes, so proper recognition sites and overhangs were included to ensure all parts were in the correct order
Our level T Ago construct contains the CbAgo protein and a homology region to the AquI chromosomal locus in a replicative RSF1010 plasmid. The AquI gene is a putative type II restriction endonuclease that has been hypothesized to be a neutral site in PCC 11901. This plasmid will generate our guide DNAs and also express CbAgo which will come together to cut the AquI locus.
Our second level T construct is a suicide vector that contains homology arms to the AquI locus and GFP and a Spectinomycin resistance marker. This vector will act as a repair template for the CbAgo induced double stranded break promoting homologous recombination in the AquI. If our Ago accelerator works, the genome will be repaired with GFP and Spectinomycin resistance markers thus allowing for selection of integrants and ensuring the payload stays within the cell. We intend on using UTEX 3154’s natural competence to transform our cells with these plasmid vectors.
BBa_K5149001
Short Description: Region Flanking AquI for Homologous Recombination
Long Description: The Up Homology Arm is a 1200bp region homologous to the flank of one side of the AquI region.
To use the Up Homology Arm to AquI Locus, you need to pair it with the Down Homology Arm to AquI Locus. Additionally, you need to include some gene in
between the Arms, for example, GFP. The construct will look like this: Up Homology Arm - GFP - Down Homology Arm. This provides your organism with a template
for homologous recombination. When a double-stranded DNA break occurs within the AquI locus, the template will be used for homologous recombination, allowing GFP to
be inserted into the genome. This is how we used the Up Homology Arm in our project.
Source of Part: We used Q5 PCR with primers that flanked the Up Homology Arm region to amplify the region out of the genome of our strain Synechoccus sp. UTEX3154.
Design Considerations: To assemble Up Homology Arm to AquI Locus correctly into our plasmids, we used Modular Cloning (MoClo), a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible, we added the correct restriction enzyme site and 4 base pair overhang on flags to the primers we used to PCR amplify the region out of the genome.
BBa_K5149008
Short Description: Fluorescent marker to show successful integration
Long Description: EGFP is commonly used throughout the world of synthetic biology as a fluorescent marker. Specifically in cyanobacteria, GFP works well due to its emission range not overlapping with that of the native fluorescence that comes from phycocyanin. The excitation wavelength of the fluorescent marker is 488nm while the emission wavelength is 507 nm. This version of EGFP has been optimized by our BLACKBIRD software to ensure the lack of type IIS recognition site and to represent the native codon bias in UTEX 3154.
Source of Part: BLACKBIRD optimized version of EGFP from pET28:GFP
Design Considerations: To assemble the Chi Site correctly into our plasmids, we used Modular Cloning (MoClo). MoClo is a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible we added the correct restriction enzyme site and 4 base pair overhang on the IDT
BBa_K5149007
Short Description: CRISPR-associated protein complex to cleave DNA/RNA
Long Description: Cpf1 is a type 2 Cas protein complex, also known as Cas12a. Using single stranded guide RNA, this complex is able to induce double breaks in the genome of the host to induce homologous recombination. The guide RNA directs the complex to the desired site through a PAM sequence, allowing high specificity when paired with the proper sgRNA cassette. Cpf1 has been shown to have low toxicity in other strains of cyanobacteria compared to other type 2 Cas systems. This version of Cpf1 has been optimized by our BLACKBIRD software to ensure the lack of type IIS recognition site and to represent the native codon bias in UTEX 3154.
Source of Part: BLACKBIRD optimized version of cpf1 from plasmid pSL2680
Design Considerations: To assemble the Chi Site correctly into our plasmids, we used Modular Cloning (MoClo). MoClo is a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible we added the correct restriction enzyme site and 4 base pair overhang on the IDT
BBa_K5149009
Short Description: Guide RNA cassette targeting AquI locus
Long Description: The sgRNA cassette consists of seven unique sgRNAs targeting the AquI locus. The sgRNAs were generated using the sgRNA tool, CasFinder, and were designed to be oligos that were annealed by their complementarity on the sgRNA fragment.
Source of Part: The sgRNAs themselves were sourced from CasFinder, choosing the sgRNAs with the most microhomology score in order to maximize homologous recombination potential in the gene. The appropriate MoClo overhangs were added through the Geneious Prime software as well as in silico cloning validation for the entire cassette.
Design Considerations: The design of the sgRNA cassette was adapted from Victoria et al (2024); however, one difference lies in the sgRNAs. The Victoria et al publication originally intends for the seven sgRNAs to target different loci within the cyanobacteria whereas our design targets one loci with seven unique sgRNAs. The appropriate MoClo overhangs were adapted from the paper as well as the wet lab protocols for the Level 0 sgRNA part assembly
BBa_K5149101
Short Description: Inducible Cpf1 Composite BBa_K5149101
Long Description: Although Cpf1 has been shown to have a low toxicity level compared to other Cas protein complexes, the introduction of the areBAD promoter allows the user to keep better control of the expression of Cpf1 through the use of arabinose.
Source of Part: The araBAD pBAD promoter and rrnB T1 Terminator were sourced from the CyanoGate Kit. The Cpf1 was sourced through orders from Integrated DNA Technologies. The basic parts were assembled by Golden Gate Assembly into a level 1 backbone plasmid from the MoClo kit.
Design Considerations: All parts were designed to work with either BsaI or BpiI restriction enzymes, so proper recognition sites and overhangs were included to ensure all parts were in the correct order
The goal of our plasmid is to use cpf1 and its sgRNA to accelerate the process of homologous recombination with our insert section, which is directed by the homology arms seen on either side of our insert. This system will rely on a two step process. The first step is to introduce the plasmid into our host then bring it into a spectinomycin selective media. Lactose is then introduced to derepress the lac operon and express both the cpf1 and its respective sgRNA. The use of an inducible promoter allows for regulation over Cas12a expression which is necessary since it has been shown to be toxic to its host in large concentrations. The second step is to introduce erythromycin in an attempt to select for integrated inserts. The main reasoning behind considering a single integrative plasmid system for our purposes as an alternative option to a double plasmid system is relative simplicity and the perks that come as a result. A cellular transformation that is performed with only one plasmid reduces the number of variables that need to be considered otherwise. To be more specific, in order for the cell to uptake both the accelerator and the insert, only one procedure needs to be performed. A single plasmid system also implies reduced overall metabolic burden on the cell. This is due to the fact that uptaking plasmid and allowing for their replication and expression in cells still consumes resources that would otherwise be utilized for cellular growth and native chromosomal replication. Due to this concept, cells with multiple plasmid activity grow at a slower rate than cells without these extra fixings.[4]
BBa_K5149001
Short Description: Region Flanking AquI for Homologous Recombination
Long Description: The Up Homology Arm is a 1200bp region homologous to the flank of one side of the AquI region.
To use the Up Homology Arm to AquI Locus, you need to pair it with the Down Homology Arm to AquI Locus. Additionally, you need to include some gene in
between the Arms, for example, GFP. The construct will look like this: Up Homology Arm - GFP - Down Homology Arm. This provides your organism with a template
for homologous recombination. When a double-stranded DNA break occurs within the AquI locus, the template will be used for homologous recombination, allowing GFP to
be inserted into the genome. This is how we used the Up Homology Arm in our project.
Source of Part: We used Q5 PCR with primers that flanked the Up Homology Arm region to amplify the region out of the genome of our strain Synechoccus sp. UTEX3154.
Design Considerations: To assemble Up Homology Arm to AquI Locus correctly into our plasmids, we used Modular Cloning (MoClo), a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible, we added the correct restriction enzyme site and 4 base pair overhang on flags to the primers we used to PCR amplify the region out of the genome.
BBa_K5149009
Short Description: Guide RNA cassette targeting AquI locus
Long Description: The sgRNA cassette consists of seven unique sgRNAs targeting the AquI locus. The sgRNAs were generated using the sgRNA tool, CasFinder, and were designed to be oligos that were annealed by their complementarity on the sgRNA fragment.
Source of Part: The sgRNAs themselves were sourced from CasFinder, choosing the sgRNAs with the most microhomology score in order to maximize homologous recombination potential in the gene. The appropriate MoClo overhangs were added through the Geneious Prime software as well as in silico cloning validation for the entire cassette.
Design Considerations: The design of the sgRNA cassette was adapted from Victoria et al (2024); however, one difference lies in the sgRNAs. The Victoria et al publication originally intends for the seven sgRNAs to target different loci within the cyanobacteria whereas our design targets one loci with seven unique sgRNAs. The appropriate MoClo overhangs were adapted from the paper as well as the wet lab protocols for the Level 0 sgRNA part assembly
BBa_K5149012
Short Description: Gentamicin Resistance Cassette
Long Description: Codon optimized and STEALTHed Gentamicin Resistance cassette, complete with Level 0 overhangs ready for Level 1 cloning. Gentamicin is an antibiotic that is used for selectivity of our parts. It gives us more flexibility.
Source of Part: BLACKBIRD optimized version of GmR from Victoria et al (2024)
Design Considerations: To maximize time efficiency, the Gentamicin cassette already contains the appropriate overhangs to enter a Level 1 construct. This was designed in silico through the program Geneious Prime. The raw BLACKBIRD gene block was cloned into the level 0 backbone to simulate the generation of a Level 0 GmR part. Then, the sequence flanking the BsaI restriction enzyme sites was extracted (INCLUDING the BsaI site and overhangs) to produce a synthetic gene block that is ready for cloning into a Level 1 Assembly
BBa_K5149006
Short Description: Desired homologous region to the genome for CbAgo to cut
Long Description: The AquI Locus is a 1200bp gene that is a putative type II restriction endonuclease that has been hypothesized to be a neutral site in PCC 11901. In addition, the removal of type II restriction endonucleases has shown to increase transformation efficiency so we decided to use the region as a Neutral Site. The 801-1200 bp region of AquI Locus is implemented into our plasmids to help provide the Argonaute protein with DNA guides. In theory, CbAgo and RecBCD will break and digest this region into 16-18bp chunks and CbAgo will acquire the chunks and form full complexes. Thus the CbAgo complex will find the AquI Locus in the UTEX3154 genome and produce breaks in the DNA sequence. To use this 400bp region, you just need to include it into a plasmid which the Argonaute system will recognize as exogenous DNA and break it to take as guides.
Source of Part: We used Q5 PCR with primers that flanked the AquI-3 Locus to amplify the region out of the genome of our strain Synechoccus sp. UTEX3154.
Design Considerations: We decided to split the 1200bp AquI Locus into three parts to give flexibility in our experiments. We intend to implement Chi Sites between each 400bp region to promote DNA guide acquisition. To assemble the AquI-3 Locus correctly into our plasmids, we used Modular Cloning (MoClo). MoClo is a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible we added the correct restriction enzyme site and 4 base pair overhang on flags to the primers we used to PCR amplify the region out of the genome.
BBa_K5149004
Short Description: AquI: Desired homologous region to the genome for CbAgo
Long Description: The AquI Locus is a 1200bp gene that is a putative type II restriction endonuclease that has been hypothesized to be a neutral site in
PCC 11901. In addition, the removal of type II restriction endonucleases has shown to increase transformation efficiency so we decided to use the region as a Neutral Site.
The 1-400 bp region of AquI Locus is implemented into our plasmids to help provide the Argonaute protein with DNA guides. In theory, CbAgo and RecBCD will break and digest
this region into 16-18bp chunks and CbAgo will acquire the chunks and form full complexes. Thus the CbAgo complex will find the AquI Locus in the UTEX3154 genome and produce
breaks in the DNA sequence. To use this 400bp region, you just need to include it into a plasmid which the Argonaute system will recognize as exogenous DNA and break it to
take as guides.
Source of Part: We used Q5 PCR with primers that flanked the AquI-1 Locus to amplify the region out of the genome of our strain Synechoccus sp. UTEX3154.
Design Considerations: We decided to split the 1200bp AquI Locus into three parts to give flexibility in our experiments. We intend to implement Chi Sites between
each 400bp region to promote DNA guide acquisition. To assemble the AquI-1 Locus correctly into our plasmids, we used Modular Cloning (MoClo). MoClo is a type II restriction
enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible we added the correct restriction enzyme site and 4 base pair overhang on flags to the primers we
used to PCR amplify the region out of the genome.
BBa_K5149010
Short Description: Region Flanking Editing Vector for Homologous Recombination
Long Description: This homology arm allows for recombination between the Editing Strain and the Hybrid Suicide Plasmid to select for integrants with the new antibiotic resistance. To generate this part, flagged primers were designed flanking a 507 bp region on the pC1.509 construct from the Victoria et al (2024) publication. To obtain the part, PCR was carried out to amplify the desired homology arm.
Source of Part: RSF1010 Editing Strain pC1.509
Design Considerations: The MoClo overhangs of this homology arm were adapted to match the pC0.425 construct from Victoria et al (2024)
BBa_K5149008
Short Description: Fluorescent marker to show successful integration
Long Description: EGFP is commonly used throughout the world of synthetic biology as a fluorescent marker. Specifically in cyanobacteria, GFP works well due to its emission range not overlapping with that of the native fluorescence that comes from phycocyanin. The excitation wavelength of the fluorescent marker is 488nm while the emission wavelength is 507 nm. This version of EGFP has been optimized by our BLACKBIRD software to ensure the lack of type IIS recognition site and to represent the native codon bias in UTEX 3154.
Source of Part: BLACKBIRD optimized version of EGFP from pET28:GFP
Design Considerations: To assemble the Chi Site correctly into our plasmids, we used Modular Cloning (MoClo). MoClo is a type II restriction enzyme system utilizing Bbs1 and Bsa1. To make the sequence MoClo compatible we added the correct restriction enzyme site and 4 base pair overhang on the IDT
The system relies on a two step homologous recombination process to achieve full markerless integration. First, the editing RSF1010 plasmid is introduced into our chosen strain through conjugation. This plasmid, which has a high copy number, generates multiple copies within the cell while also carrying our repressed Cas12a cassette. To prevent transient expression of Cas12a, its expression is tightly regulated by a highly repressed promoter (phlF). Another promoter (pJ23101) controls the expression of the repressor phlF, which is derepressed by DAPG to enable Cas12a expression.
[1] A. J. Victoria et al., “A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901,” PLANT PHYSIOLOGY, May 2024, doi: https://doi.org/10.1093/plphys/kiae261.
[2] R. Vasudevan et al., “CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax,” PLANT PHYSIOLOGY, vol. 180, no. 1, pp. 39–55, Feb. 2019, doi: https://doi.org/10.1104/pp.18.01401.
[3] “Addgene: Modular Cloning Guide,” Addgene.org, 2014. https://www.addgene.org/cloning/moclo/ (accessed Oct. 02, 2024).
[4]G. Wegrzyn and A. Wegrzyn, “Stress responses and replication of plasmids in bacterial cells.,” Microbial Cell Factories, vol. 1, no. 1, pp. 2–2, Jan. 2002, doi: https://doi.org/10.1186/1475-2859-1-2.