Contribution

Registered Number	Name	Description	Sequence
BBa_K5149000	Codon Optimized CbAgo	CbAgo is a part of the Argonaute protein family. They are 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 are then to cut every part of the genome where the DNA guide binds. CbAgo is a mesophile meaning it functions between 20-45 degrees Celsius compared to most thermophile Argonaute proteins.	ATGAATAACTTGACTTTTGAGGCTTTTGAGGGTATTGGTCAACTGAATGAACTGAACTTTTATAAATACCGGCTCATTGGTAAAGGGCAGATAGATAATGTTCATCAGGCTATCTGGTCTGTTAAATATAAATTACAGGCTAACAACTTCTTTAAACCTGTTTTTGTTAAAGGTGAAATTCTGTATTCTTTAGATGAATTAAAGGTTATTCCTGAGTTTGAGAATGTTGAGGTTATTCTTGACGGTAACATTATTTTGTCTATCTCTGAAAATACTGATATTTATAAAGACGTTATTGTTTTTTATATCAATAATGCTTTGAAGAACATTAAAGACATTACGAATTACCGTAAATACATTACTAAGAATACTGATGAAATTATCTGTAAGTCTATCTTGACTACTAACTTGAAGTATCAATATATGAAGAGTGAGAAAGGTTTTAAATTACAACGTAAATTCAAAATATCTCCTGTTGTATTTAGAAACGGTAAGGTTATTTTGTATTTAAACTGTTCTTCTGATTTTTCTACTGATAAAAGTATTTATGAGATGTTGAATGACGGTTTAGGTGTTGTTGGTTTACAGGTTAAGAATAAATGGACTAACGCTAATGGAAATATCTTTATTGAAAAAGTTCTTGATAACACTATTTCTGATCCTGGAACTTCTGGTAAATTAGGGCAGTCTCTCATTGACTACTACATTAACGGTAACCAAAAATATAGAGTTGAGAAATTCACTGATGAAGATAAAAACGCTAAGGTTATTCAGGCAAAAATAAAGAATAAAACCTATAACTATATTCCTCAGGCTTTAACTCCTGTTATAACTAGAGAGTATTTATCTCATACTGATAAAAAGTTCAGCAAACAGATTGAGAACGTTATAAAAATGGACATGAATTATCGGTATCAAACATTAAAGTCTTTTGTTGAAGATATAGGTGTTATAAAAGAATTAAATAACCTACACTTCAAGAACCAATACTATACTAATTTTGACTTTATGGGTTTTGAATCTGGTGTTTTAGAAGAACCTGTTTTAATGGGAGCTAACGGGAAAATAAAAGACAAGAAACAGATATTCATTAATGGTTTCTTTAAGAACCCTAAAGAGAACGTTAAGTTTGGGGTTTTATATCCTGAAGGATGTATGGAGAATGCTCAGTCTATAGCCCGTTCTATCTTAGATTTTGCCACTGCTGGTAAATATAACAAACAGGAAAATAAATATATTTCTAAAAACCTCATGAATATAGGTTTTAAACCCTCTGAATGTATTTTTGAATCCTATAAATTAGGAGACATTACTGAATATAAGGCTACTGCCCGTAAATTAAAAGAACATGAAAAAGTTGGTTTTGTTATTGCTGTTATTCCTGATATGAATGAGTTAGAAGTTGAAAATCCTTACAATCCTTTTAAAAAGGTTTGGGCTAAATTAAACATTCCCTCACAAATGATCACATTAAAAACTACTGAGAAATTCAAAAATATAGTTGATAAAAGCGGTTTATATTATTTACACAATATAGCTTTAAATATCCTGGGTAAGATAGGTGGTATTCCTTGGATCATTAAAGATATGCCTGGAAATATAGACTGTTTTATTGGTTTAGATGTTGGTACTAGAGAAAAAGGTATTCATTTTCCTGCTTGTTCTGTTTTGTTTGATAAATACGGTAAATTGATAAATTACTATAAACCTACCATTCCTCAGTCTGGTGAGAAGATAGCCGAAACCATTCTTCAAGAGATTTTTGATAATGTTTTAATTTCCTATAAAGAAGAGAATGGTGAATACCCTAAAAATATAGTTATTCACCGGGACGGTTTTTCTAGGGAAAATATAGATTGGTATAAAGAGTATTTTGATAAAAAGGGAATTAAATTCAACATTATTGAGGTTAAGAAAAACATTCCTGTTAAGATAGCTAAAGTTGTTGGTAGTAATATCTGTAATCCTATCAAAGGTAGTTATGTTTTGAAAAACGACAAAGCCTTTATTGTTACTACTGATATAAAAGATGGTGTTGCTTCCCCTAATCCTTTAAAAATAGAGAAAACCTATGGTGATGTTGAGATGAAGAGTATTCTTGAACAGATATATTCTTTATCCCAAATTCATGTTGGTTCTACTAAGTCTTTACGTTTACCTATAACTACTGGATATGCTGATAAAATCTGTAAGGCTATTGAATATATTCCTCAAGGTGTTGTTGATAACCGTTTATTTTTTTTATAA
BBa_K5149001	Up Homology Arm to AquI Locus	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. In addition, 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. By doing this, you now have given your organism 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 for GFP to be inserted into the genome. This is how we used the Up Homology Arm in our project.	CCGTTATCCCATCTGCAATATCCATAAAAGGCGATCGCCTACCCTTCGAGAGACTTGATCCACTCAAGGTAGTATTGTCCTGATCCCTAAGAGATAATCTTTAGGTGAGCTGTACAATAACACAAAATTCATAATGGAAAAAAAACTGATAAGCCTATTTGCCGGAGCTGGTGGCATGGACATCGGCTTTCACGCTGCTGGCTTCAGTACGGCGGTAGCTGTGGAACAAGATCCGTCTTGCTGCAATACTCTAAGGCTTAATATGCCGGACACTCCAGTCATTGAAGGCGACATCACCTCAATTACAACACAGGCTATCTTAGAAGCGGCAAAGGTCAATCCTCTTGAAATTGACCTAGTTATTGGTGGCCCCCCTTGCCAAAGTTTCAGCCTAGCAGGTAGACGTATGGGAATGGATGATCCGAGAGGAATGCTGGTACTTGAGTTTCTACGTGTAGTCAGGGAGGCATTGCCAAAGTGTTTTGTTATGGAGAATGTCAAAGGAATGATCAATTGGTCAAAGGGAAAGGCTCTTGAGGCTATCATGACAGAAGCATCGCAGCCTATAAAATACGCTGGAAAAGAATATAAATACGCTGTTTCGTATCATGTCCTAAATGCTGCTGATTTTGGTGTTCCGCAATTTAGAGAAAGAGTATTCATCGTAGGTAATCGTTTGGGCAAAACATTCCAATTTCCTGAACCAACTCATGGGCCTAGCAACCAAGCGAGACAGATAGATCTTTTTGGCAAGCAGCTAAAACCTTACAAAACTGTTCAAGATGCAATTAGCACTCTCCCCCCTGCAACCCCTCCTTCAGCGATGGCACTAAGAGTTTCGCAGACTATAAAAGATAGGATAAAGAATCATGGATATTAAAAACGTTCATATCAAAAATCACGAACAAACAGCTCATGCACCTTCCACTCTAGAAAAAATTCGTAAAGTCAAACAAGGGGGTAAACTCTCAGAACAGACAAAGACATTTGGTTCAACCTACCGCAGGTTAGATCCGAACCAGCCATCTCCTACAGTGACCCGTAGTGGTTATCGAGATTTTATTCATCCTTTTGAAGATCGAATGCTCACAGTTCGTGAACTGGCTTGTTTGCAAACCTTTCCCCTTGATTGGGAGTTTACCGGAACTCGACTTGATTCTTATAGTAGTAAACGTAAAGTGGCGATGACTCAGTTTGGACAAGTGGGTAATGCAGTACCACCGTTACTTGCTGAAGCTGTTGCTAAAGCGGTTAGCGAACAGCTTCTGGATGTCATT
BBa_K5149002	Down Homology Arm to AquI Locus	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. In addition, 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. By doing this, you now have given your organism 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 for GFP to be inserted into the genome. This is how we used the Down Homology Arm in our project.	CAAAATCAAACCCGATCGCCTCTCTATTTTGATAAATCTATGTCTACTCCCTCTGTTACCCCTGTAGAATCTAGCACCCTAATCAAAACCCCTGAACTGCTGGCTCCAGCGGGAAATTGGGACTGTGCGATCGCCGCCGTAGAAAATGGAGCCGATGCGATTTATTTTGGGCTGGATAAATTTAATGCCCGGATGCGATCGCAAAACTTTGTCGAGTCAGATTTGCCGGAGTTAATGGCATACTTACATCGGCGTGGCGTGAAGGGCTATGTGACGTTAAATACGCTGATTTTCACCTCGGAATTGGCGGCAGTCGAACAATATTTGCGGTCGATTATTGCGGCAGGAGTCGATGCGGCGATTGTCCAGGATGTGGGGCTATGTCAGTTAATTCGGCAATTGTCCCCCGATTTTCCGATCCATGGTTCCACCCAGATGACCGTCACCAGCGCCGCAGGGGTCGAGTTCGCGCAAAACTTGGGTTGTGATTTGGTGGTATTGGCGCGGGAATGTTCGATCAAGGAAATCAATAAAATCCAGCAGGAATTGGGTCAACAAAAGATCTCGATGCCGCTGGAGGTGTTTGTTCATGGAGCGTTGTGTGTCGCCTATTCCGGGCAATGTTTAACCAGTGAATCCCTCGGTGGACGGTCGGCCAATCGCGGGGAATGCGCCCAGGCTTGCCGGATGCCCTACGAAATGATTGTCGATGGTAAGCCATTTGATCTGGGTGACAGACGTTACCTACTAAGCCCCCAAGATTTGGCGGGATTACCTGTTTTGCCGGACTTAATTAAAGCGGGCGTCGTGTCGCTAAAAATCGAAGGACGCTTAAAACAACCGGAATATGTGGCCAGTGTCACCCAAGTGTATCGCCAGGCCATTGATCGGGCGGTGCAGGGCATTGAGCAAGAGGTTTCAGACCAGGAAAAATATCAATTGGAAATGGCCTTTTCGCGTGGGTTGTACACGGGTTGGCTTGACGGCATCGATAATCAAAGCCTTGTCCATGGTCGCTTTGGCAAGAAAAG
BBa_K5149003	Chi Site	Chi Sites are repeated DNA sequences stopping recognition sites for RecBCD complexes. For our project, Chi Sites regulates the complete digestion of the dsDNA region we are attempting to edit. By implementing Chi Sites in 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. To test this, the speed at which the AquI Locus is cut will determine whether or not CbAgo will depend on Chi Sites for more efficient guide acquisition. DNA acquisition for CbAgo is only possible with RecBCD or a RecBCD-like system.	GCGATCGC
BBa_K5149004	AquI-1 - 1-400 bp region of AquI Locus	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.	AACAATTTCAGTGACCTTGTGACGACAAAAGATGCTCGCAGAAGTGGTTTTCTTGAATATGCTCTTCGTCGCAATAAAGAGTCTATTCCATTTATCGATAAGGCGAAAGCTCTACAAGTATCTCTTCAGAAGAATACAAAGTGTGCAGAGGATATTCTCAAACTCTATGACATCCGAGAAACACTCATTGAGGCAGCGGGTGTATCTGTCAAGGCGAAGGCTCACCTCGATGACATGGACAAAAATGAAATTCTTGCAGGCTTTGTTAAGGAGGTTCTTGCACCTTGTGGCAAAAAGTATATCGATGAGATTGTCTATCGTTACTTGCTCACACTTG
BBa_K5149005	AquI-2 - 401-800 bp region of AquI Locus	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 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.	TCGTTACTTGCTCACACTTGGAGATGCTCTTGGTGGTAAAATGCGCAACATTGTTGGTTCTATCGCAGAAGAGAAATTCGTCAGATTTATCGTTGCCCAGTTACAGGTGCATGATATTGAATTTGAACTATTTCCAAAACGTTCGCAATGGATTTCAAACAAAAACTATAGAGCTGAGATGGCTGCACGAACGAAAGCCTTAAGATGGAAAAATAAAACAACTTATCGATCTCTAATTTTCAACATTAATGTTCCACAGGTCAAAAAGAATATTGATATCGTGCTGCTAAATGACAAGATTGAAGGAGTAACCTCGTCTATTTTGGCGCT
BBa_K5149006	AquI-1 - 801-1200 bp region of AquI Locus	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.	GGAGTAACCTCGTCTATTTTGGCTAATATCCTTAACGATAAGAATAAGTATCTTGCCATCGGTGAGTTAAAGGGCGGAATTGATCCAGCCGGGGCAGATGAACATTGGAAAACGGCAAATACAGCTCTTAGTAGAGTAAGAGATTCTTTCAAGAACAAAATCCACTTATTTTTCATTGGGGCTGCGATAGAAACCAGTATGTCGGCAGAGATTTACGAACAATGCCAGACAGGTGAACTTAGTAACGCAGCAAATCTCACTGTTGATAATCAATTATCTGCATTGTGTGAGTGGCTAATAACGCAATAACTTTACAAAATCAAACCCGATCGC
BBa_K5149007	Codon Optimized Cpf1	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.	ATGTCTATATATCAAGAGTTTGTGAATAAATATAGTCTGAGTAAAACTCTACGCTTCGAGTTAATTCCACAGGGTAAAACACTTGAGAACATAAAGGCTAGAGGTTTAATTCTTGATGATGAGAAGAGAGCTAAAGACTACAAGAAGGCTAAGCAAATCATTGATAAATATCATCAGTTCTTTATAGAGGAGATACTAAGTTCTGTTTGTATTTCTGAGGATTTACTACAAAACTACTCTGATGTTTACTTTAAACTGAAGAAGAGTGATGATGATAACCTACAGAAAGACTTCAAATCCGCAAAAGATACCATAAAGAAACAAATAAGTGAATATATAAAAGACAGTGAGAAATTCAAGAATTTGTTTAACCAAAACCTCATTGACGCTAAGAAAGGGCAGGAGTCTGATCTAATTCTGTGGCTAAAGCAGTCTAAGGATAACGGTATTGAATTATTTAAGGCTAATTCTGATATAACAGATATAGATGAGGCGTTAGAGATAATCAAGTCTTTTAAAGGCTGGACAACATACTTTAAGGGGTTTCATGAGAATAGGAAGAATGTTTATTCCAGCAACGACATACCTACATCTATTATTTATAGGATAGTAGATGATAATTTGCCTAAATTCCTGGAGAATAAAGCAAAGTATGAGAGTTTGAAAGACAAAGCTCCTGAGGCTATAAATTATGAACAAATTAAGAAGGATTTAGCCGAAGAGCTAACCTTTGATATTGACTACAAGACCTCTGAGGTTAACCAAAGAGTGTTCTCACTTGATGAGGTATTCGAGATAGCTAACTTTAACAACTATCTAAACCAGAGCGGAATTACTAAATTCAACACTATTATTGGTGGTAAATTCGTTAATGGTGAGAATACAAAGCGTAAAGGTATCAATGAATATATAAACCTATACTCACAGCAGATAAACGACAAAACACTCAAGAAATATAAGATGAGTGTCTTATTTAAGCAAATTTTAAGTGATACAGAGTCTAAGTCTTTTGTTATTGATAAACTGGAAGATGACTCTGATGTAGTTACAACGATGCAGTCCTTCTATGAGCAGATAGCCGCTTTTAAAACAGTAGAAGAGAAGAGCATCAAAGAAACACTATCTTTATTGTTTGATGATTTAAAAGCTCAGAAACTGGATTTGTCGAAGATATACTTTAAGAACGATAAATCTCTTACTGATCTAAGTCAGCAGGTATTCGATGACTATAGTGTTATTGGTACAGCGGTACTGGAATATATAACTCAACAAATAGCACCTAAGAACCTTGATAACCCTAGTAAGAAAGAGCAGGAACTGATAGCCAAGAAAACTGAGAAAGCAAAGTACCTATCCCTAGAAACTATAAAGTTGGCGTTGGAAGAGTTTAACAAACATAGAGATATAGATAAACAGTGTAGGTTTGAAGAAATACTTGCCAACTTTGCCGCTATACCGATGATCTTTGATGAAATAGCTCAGAACAAAGATAATTTGGCACAAATATCTATAAAGTATCAAAACCAAGGTAAGAAAGACCTACTTCAGGCTTCTGCGGAAGATGACGTTAAGGCAATCAAGGATCTGTTAGATCAAACCAATAATTTGTTACATAAACTAAAGATATTTCACATCAGTCAGTCTGAAGATAAAGCAAACATCTTAGACAAAGATGAGCATTTCTATCTGGTCTTTGAGGAGTGTTACTTTGAGCTAGCCAATATAGTGCCTCTTTATAACAAAATAAGAAACTATATAACTCAGAAACCATACAGTGATGAGAAATTCAAGCTCAATTTTGAAAACTCGACGTTGGCTAATGGTTGGGATAAGAATAAAGAGCCCGACAATACAGCCATTCTGTTTATCAAAGATGATAAATACTATTTAGGTGTGATGAATAAGAAGAACAACAAGATCTTTGATGACAAAGCTATCAAGGAAAATAAAGGTGAGGGTTATAAGAAGATAGTTTATAAACTTTTGCCTGGGGCAAACAAAATGTTGCCTAAGGTTTTCTTCTCTGCTAAGTCTATAAAGTTCTATAACCCCAGTGAAGATATATTGAGAATAAGAAACCATTCCACACATACAAAGAATGGTTCACCTCAGAAGGGATATGAGAAGTTCGAGTTTAACATTGAGGATTGCCGAAAATTCATAGACTTTTATAAACAGTCTATCAGTAAACATCCTGAATGGAAGGATTTTGGCTTTAGGTTTTCTGATACTCAAAGATATAACTCTATAGATGAGTTTTATAGAGAAGTTGAAAATCAAGGCTACAAACTAACTTTTGAAAATATAAGTGAGAGCTATATTGATAGTGTAGTTAATCAGGGTAAGTTGTACCTATTCCAGATATATAACAAAGACTTTTCAGCCTACAGCAAAGGACGCCCAAACCTACATACTTTATACTGGAAAGCTCTGTTCGATGAGAGAAACCTTCAAGATGTGGTTTATAAGTTGAATGGTGAGGCAGAGCTTTTCTACCGTAAACAGTCAATACCTAAGAAAATAACTCATCCAGCCAAAGAGGCTATAGCCAATAAGAACAAAGATAATCCTAAGAAGGAGAGTGTGTTCGAATATGATTTAATCAAAGATAAACGCTTTACTGAAGATAAATTCTTCTTCCACTGTCCTATAACAATCAACTTTAAAAGTAGTGGAGCTAATAAGTTCAATGATGAAATTAATTTGTTACTAAAAGAGAAAGCTAATGATGTTCACATACTGAGTATAGATAGAGGTGAGAGACATTTAGCCTACTATACCTTGGTTGACGGTAAGGGCAATATCATCAAACAGGATACATTCAACATCATTGGTAACGACAGAATGAAAACAAACTACCATGATAAATTGGCTGCCATTGAAAAGGACAGGGATTCAGCCCGTAAAGACTGGAAGAAGATAAATAACATCAAAGAGATGAAAGAGGGCTATCTATCTCAGGTAGTTCATGAGATAGCTAAGTTGGTTATAGAGTATAACGCCATTGTGGTATTCGAGGATTTAAATTTTGGCTTTAAAAGAGGGCGTTTCAAGGTAGAGAAACAGGTTTATCAAAAGCTGGAGAAAATGCTCATTGAGAAACTAAACTATCTGGTGTTCAAAGATAATGAGTTTGATAAAACAGGAGGAGTTTTAAGAGCCTATCAACTAACAGCACCGTTTGAGACTTTTAAGAAAATGGGTAAACAAACAGGAATTATCTATTATGTACCCGCTGGTTTTACTTCAAAGATCTGTCCTGTAACTGGTTTTGTAAATCAGTTATACCCCAAGTATGAGAGCGTCAGCAAAAGTCAAGAGTTCTTCTCTAAGTTCGACAAAATCTGTTATAACCTTGATAAAGGCTATTTTGAGTTTAGCTTTGACTATAAGAACTTTGGAGACAAGGCTGCCAAAGGTAAATGGACTATAGCTAGCTTTGGCAGTAGGTTGATTAACTTTAGAAATTCTGATAAGAATCATAATTGGGATACTAGAGAGGTTTACCCAACTAAAGAGCTAGAGAAATTGCTAAAAGATTACTCTATCGAGTATGGTCATGGTGAATGTATCAAAGCCGCTATCTGCGGTGAGTCTGATAAGAAGTTCTTTGCCAAACTAACTTCTGTTCTAAATACTATCTTACAAATGCGTAACTCAAAGACAGGAACTGAGTTAGACTATCTAATTTCCCCAGTAGCGGATGTGAATGGCAACTTCTTTGATAGCCGACAAGCGCCCAAGAATATGCCTCAAGATGCTGATGCCAATGGTGCGTATCATATTGGGCTAAAGGGGCTGATGCTCCTGGGTCGTATCAAGAACAACCAAGAGGGCAAGAAGCTCAATCTGGTGATCAAGAATGAAGAGTATTTTGAATTTGTACAGAACAGGAATAACTAA
BBa_K5149008	Codon Optimized EGFP	EGFP is commonly used throughout the world of synthetic biology as a fluorescent market. 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.	ATGGTGAGTAAGGGAGAGGAACTGTTCACAGGGGTAGTGCCTATCCTCGTCGAACTGGACGGAGATGTAAACGGGCATAAGTTCAGCGTTTCCGGTGAGGGTGAAGGTGATGCCACCTACGGCAAACTAACCCTGAAGTTTATCTGTACCACAGGCAAACTGCCCGTGCCTTGGCCTACCCTCGTCACTACCCTAACCTACGGCGTACAGTGCTTCAGCCGCTACCCCGACCACATGAAACAACATGACTTTTTCAAAAGTGCCATGCCCGAAGGCTACGTTCAGGAACGCACTATCTTCTTCAAGGATGACGGCAACTATAAGACTAGAGCCGAGGTGAAGTTCGAGGGTGATACTCTGGTGAACCGCATAGAGTTAAAGGGCATTGACTTTAAAGAGGACGGCAACATCCTGGGGCACAAACTGGAATACAACTACAACAGCCACAACGTCTATATCATGGCAGACAAACAGAAGAACGGCATCAAGGTGAACTTCAAGATACGCCACAACATAGAGGACGGCAGCGTACAGTTAGCCGACCACTATCAGCAGAACACCCCCATCGGTGATGGCCCAGTGCTGCTGCCCGACAACCACTACCTGAGTACCCAGTCTGCCCTGAGCAAAGACCCCAACGAGAAGCGGGATCACATGGTCCTGTTAGAGTTTGTTACCGCAGCAGGAATCACTCTGGGTATGGACGAACTGTATAAATAA
BBa_K5149009	sgRNA Cassette	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.	TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAGCTGATTTAGGCAAAAACGGAATTTCTACTGTTGTAGATGTAAACAATTTCAGTGACTGGCAATTTCTACTGTTGTAGATTAAACAATTTCAGTGACCTTCTAATTTCTACTGTTGTAGATCAGTGACCTTGTGACGACCAATAATTTCTACTGTTGTAGATCTGCGTTACTAAGTTCACTATGAATTTCTACTGTTGTAGATGCTGCGTTACTAAGTTCAAGAAAATTTCTACTGTTGTAGATACCTGTCTGGCATTGTTCGCTGAATTTCTACTGTTGTAGATTTCGTAAATCTCTGCCGAAACGAATTTCTACTGTTGTAGATCTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCC
BBa_K5149010	Up Homology Arm to Editing Vector	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.	ACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGCTCGAGTTACCAATGCTTAATCAG
BBa_K5149011	Down Homology Arm to Editing Vector	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 517 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.	TGTGTACCCGGACAAACACCATTAATCAGCAGGAAGGTAAATTCTTCAATATCCTGTTCAACGGTCAACTGTTCGGTCAGCAGGCGATACCAACAAAAACCAAAAATCATATCCAGCAGCAGTTCACGATTGATATCTTTCGGCAGTTCACCATTGCTAATGGCATCTTCAACCAGTTTTTTCGGTATCTCACGACGACGTTCCATAAACTGATCTTTCAGTTGGGTCAGGGTTACAGGGTCCAACTGTGCTTCTGCAATAACACAACGAAATGCTTCACCACAAATGGTTTCACGCCAAACTTTCCACAGATTATGCAGCAGAAAATCCAGATCGGCTTTAAAGCTACCCAAATCCGGAAATTTACGTACCTGTTCGATTTCATTTTCATACACTTCGGCAATCAGTGCTGCTTTGTTGGTCCACCAACGATAAATGGTCGGTTTGCCTGCACCGGCGCGACGTGC
BBa_K5149012	Codon Optimized Gentamicin	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.	ATGTTACGCAGCAGCAACGACGTTACGCAGCAAGGTAGCCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATCCGCACATGTAGGCTCGGTCCTGACCAAGTTAAATCCATGAGGGCTGCTCTTGACCTTTTTGGTCGTGAGTTTGGAGACGTAGCCACCTACAGTCAACACCAGCCGGACAGTGATTACTTAGGGAACTTGCTCCGTAGTAAGACATTCATTGCGCTTGCAGCCTTTGACCAAGAAGCGGTTGTGGGTGCTCTGGCGGCTTACGTTTTACCTAGGTTTGAGCAGCCGCGTAGTGAAATCTATATCTATGACTTAGCCGTCTCCGGTGAGCACCGGAGACAGGGCATTGCCACCGCTCTCATCAACCTCCTCAAACATGAGGCTAACGCTCTTGGGGCTTATGTGATTTACGTGCAAGCGGATTACGGTGACGACCCCGCAGTGGCTCTCTATACAAAATTGGGCATACGGGAAGAAGTGATGCACTTTGATATTGACCCAAGTACCGCCACCTAA

Registered Number

Name

Description

Sequence

BBa_K5149000

Codon Optimized CbAgo

CbAgo is a part of the Argonaute protein family. They are 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 are then to cut every part of the genome where the DNA guide binds. CbAgo is a mesophile meaning it functions between 20-45 degrees Celsius compared to most thermophile Argonaute proteins.

ATGAATAACTTGACTTTTGAGGCTTTTGAGGGTATTGGTCAACTGAATGAACTGAACTTTTATAAATACCGGCTCATTGGTAAAGGGCAGATAGATAATGTTCATCAGGCTATCTGGTCTGTTAAATATAAATTACAGGCTAACAACTTCTTTAAACCTGTTTTTGTTAAAGGTGAAATTCTGTATTCTTTAGATGAATTAAAGGTTATTCCTGAGTTTGAGAATGTTGAGGTTATTCTTGACGGTAACATTATTTTGTCTATCTCTGAAAATACTGATATTTATAAAGACGTTATTGTTTTTTATATCAATAATGCTTTGAAGAACATTAAAGACATTACGAATTACCGTAAATACATTACTAAGAATACTGATGAAATTATCTGTAAGTCTATCTTGACTACTAACTTGAAGTATCAATATATGAAGAGTGAGAAAGGTTTTAAATTACAACGTAAATTCAAAATATCTCCTGTTGTATTTAGAAACGGTAAGGTTATTTTGTATTTAAACTGTTCTTCTGATTTTTCTACTGATAAAAGTATTTATGAGATGTTGAATGACGGTTTAGGTGTTGTTGGTTTACAGGTTAAGAATAAATGGACTAACGCTAATGGAAATATCTTTATTGAAAAAGTTCTTGATAACACTATTTCTGATCCTGGAACTTCTGGTAAATTAGGGCAGTCTCTCATTGACTACTACATTAACGGTAACCAAAAATATAGAGTTGAGAAATTCACTGATGAAGATAAAAACGCTAAGGTTATTCAGGCAAAAATAAAGAATAAAACCTATAACTATATTCCTCAGGCTTTAACTCCTGTTATAACTAGAGAGTATTTATCTCATACTGATAAAAAGTTCAGCAAACAGATTGAGAACGTTATAAAAATGGACATGAATTATCGGTATCAAACATTAAAGTCTTTTGTTGAAGATATAGGTGTTATAAAAGAATTAAATAACCTACACTTCAAGAACCAATACTATACTAATTTTGACTTTATGGGTTTTGAATCTGGTGTTTTAGAAGAACCTGTTTTAATGGGAGCTAACGGGAAAATAAAAGACAAGAAACAGATATTCATTAATGGTTTCTTTAAGAACCCTAAAGAGAACGTTAAGTTTGGGGTTTTATATCCTGAAGGATGTATGGAGAATGCTCAGTCTATAGCCCGTTCTATCTTAGATTTTGCCACTGCTGGTAAATATAACAAACAGGAAAATAAATATATTTCTAAAAACCTCATGAATATAGGTTTTAAACCCTCTGAATGTATTTTTGAATCCTATAAATTAGGAGACATTACTGAATATAAGGCTACTGCCCGTAAATTAAAAGAACATGAAAAAGTTGGTTTTGTTATTGCTGTTATTCCTGATATGAATGAGTTAGAAGTTGAAAATCCTTACAATCCTTTTAAAAAGGTTTGGGCTAAATTAAACATTCCCTCACAAATGATCACATTAAAAACTACTGAGAAATTCAAAAATATAGTTGATAAAAGCGGTTTATATTATTTACACAATATAGCTTTAAATATCCTGGGTAAGATAGGTGGTATTCCTTGGATCATTAAAGATATGCCTGGAAATATAGACTGTTTTATTGGTTTAGATGTTGGTACTAGAGAAAAAGGTATTCATTTTCCTGCTTGTTCTGTTTTGTTTGATAAATACGGTAAATTGATAAATTACTATAAACCTACCATTCCTCAGTCTGGTGAGAAGATAGCCGAAACCATTCTTCAAGAGATTTTTGATAATGTTTTAATTTCCTATAAAGAAGAGAATGGTGAATACCCTAAAAATATAGTTATTCACCGGGACGGTTTTTCTAGGGAAAATATAGATTGGTATAAAGAGTATTTTGATAAAAAGGGAATTAAATTCAACATTATTGAGGTTAAGAAAAACATTCCTGTTAAGATAGCTAAAGTTGTTGGTAGTAATATCTGTAATCCTATCAAAGGTAGTTATGTTTTGAAAAACGACAAAGCCTTTATTGTTACTACTGATATAAAAGATGGTGTTGCTTCCCCTAATCCTTTAAAAATAGAGAAAACCTATGGTGATGTTGAGATGAAGAGTATTCTTGAACAGATATATTCTTTATCCCAAATTCATGTTGGTTCTACTAAGTCTTTACGTTTACCTATAACTACTGGATATGCTGATAAAATCTGTAAGGCTATTGAATATATTCCTCAAGGTGTTGTTGATAACCGTTTATTTTTTTTATAA

BBa_K5149001

Up Homology Arm to AquI Locus

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. In addition, 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. By doing this, you now have given your organism 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 for GFP to be inserted into the genome. This is how we used the Up Homology Arm in our project.

CCGTTATCCCATCTGCAATATCCATAAAAGGCGATCGCCTACCCTTCGAGAGACTTGATCCACTCAAGGTAGTATTGTCCTGATCCCTAAGAGATAATCTTTAGGTGAGCTGTACAATAACACAAAATTCATAATGGAAAAAAAACTGATAAGCCTATTTGCCGGAGCTGGTGGCATGGACATCGGCTTTCACGCTGCTGGCTTCAGTACGGCGGTAGCTGTGGAACAAGATCCGTCTTGCTGCAATACTCTAAGGCTTAATATGCCGGACACTCCAGTCATTGAAGGCGACATCACCTCAATTACAACACAGGCTATCTTAGAAGCGGCAAAGGTCAATCCTCTTGAAATTGACCTAGTTATTGGTGGCCCCCCTTGCCAAAGTTTCAGCCTAGCAGGTAGACGTATGGGAATGGATGATCCGAGAGGAATGCTGGTACTTGAGTTTCTACGTGTAGTCAGGGAGGCATTGCCAAAGTGTTTTGTTATGGAGAATGTCAAAGGAATGATCAATTGGTCAAAGGGAAAGGCTCTTGAGGCTATCATGACAGAAGCATCGCAGCCTATAAAATACGCTGGAAAAGAATATAAATACGCTGTTTCGTATCATGTCCTAAATGCTGCTGATTTTGGTGTTCCGCAATTTAGAGAAAGAGTATTCATCGTAGGTAATCGTTTGGGCAAAACATTCCAATTTCCTGAACCAACTCATGGGCCTAGCAACCAAGCGAGACAGATAGATCTTTTTGGCAAGCAGCTAAAACCTTACAAAACTGTTCAAGATGCAATTAGCACTCTCCCCCCTGCAACCCCTCCTTCAGCGATGGCACTAAGAGTTTCGCAGACTATAAAAGATAGGATAAAGAATCATGGATATTAAAAACGTTCATATCAAAAATCACGAACAAACAGCTCATGCACCTTCCACTCTAGAAAAAATTCGTAAAGTCAAACAAGGGGGTAAACTCTCAGAACAGACAAAGACATTTGGTTCAACCTACCGCAGGTTAGATCCGAACCAGCCATCTCCTACAGTGACCCGTAGTGGTTATCGAGATTTTATTCATCCTTTTGAAGATCGAATGCTCACAGTTCGTGAACTGGCTTGTTTGCAAACCTTTCCCCTTGATTGGGAGTTTACCGGAACTCGACTTGATTCTTATAGTAGTAAACGTAAAGTGGCGATGACTCAGTTTGGACAAGTGGGTAATGCAGTACCACCGTTACTTGCTGAAGCTGTTGCTAAAGCGGTTAGCGAACAGCTTCTGGATGTCATT

BBa_K5149002

Down Homology Arm to AquI Locus

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. In addition, 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. By doing this, you now have given your organism 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 for GFP to be inserted into the genome. This is how we used the Down Homology Arm in our project.

CAAAATCAAACCCGATCGCCTCTCTATTTTGATAAATCTATGTCTACTCCCTCTGTTACCCCTGTAGAATCTAGCACCCTAATCAAAACCCCTGAACTGCTGGCTCCAGCGGGAAATTGGGACTGTGCGATCGCCGCCGTAGAAAATGGAGCCGATGCGATTTATTTTGGGCTGGATAAATTTAATGCCCGGATGCGATCGCAAAACTTTGTCGAGTCAGATTTGCCGGAGTTAATGGCATACTTACATCGGCGTGGCGTGAAGGGCTATGTGACGTTAAATACGCTGATTTTCACCTCGGAATTGGCGGCAGTCGAACAATATTTGCGGTCGATTATTGCGGCAGGAGTCGATGCGGCGATTGTCCAGGATGTGGGGCTATGTCAGTTAATTCGGCAATTGTCCCCCGATTTTCCGATCCATGGTTCCACCCAGATGACCGTCACCAGCGCCGCAGGGGTCGAGTTCGCGCAAAACTTGGGTTGTGATTTGGTGGTATTGGCGCGGGAATGTTCGATCAAGGAAATCAATAAAATCCAGCAGGAATTGGGTCAACAAAAGATCTCGATGCCGCTGGAGGTGTTTGTTCATGGAGCGTTGTGTGTCGCCTATTCCGGGCAATGTTTAACCAGTGAATCCCTCGGTGGACGGTCGGCCAATCGCGGGGAATGCGCCCAGGCTTGCCGGATGCCCTACGAAATGATTGTCGATGGTAAGCCATTTGATCTGGGTGACAGACGTTACCTACTAAGCCCCCAAGATTTGGCGGGATTACCTGTTTTGCCGGACTTAATTAAAGCGGGCGTCGTGTCGCTAAAAATCGAAGGACGCTTAAAACAACCGGAATATGTGGCCAGTGTCACCCAAGTGTATCGCCAGGCCATTGATCGGGCGGTGCAGGGCATTGAGCAAGAGGTTTCAGACCAGGAAAAATATCAATTGGAAATGGCCTTTTCGCGTGGGTTGTACACGGGTTGGCTTGACGGCATCGATAATCAAAGCCTTGTCCATGGTCGCTTTGGCAAGAAAAG

BBa_K5149003

Chi Site

Chi Sites are repeated DNA sequences stopping recognition sites for RecBCD complexes. For our project, Chi Sites regulates the complete digestion of the dsDNA region we are attempting to edit. By implementing Chi Sites in 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. To test this, the speed at which the AquI Locus is cut will determine whether or not CbAgo will depend on Chi Sites for more efficient guide acquisition. DNA acquisition for CbAgo is only possible with RecBCD or a RecBCD-like system.

GCGATCGC

BBa_K5149004

AquI-1 - 1-400 bp region of AquI Locus

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.

AACAATTTCAGTGACCTTGTGACGACAAAAGATGCTCGCAGAAGTGGTTTTCTTGAATATGCTCTTCGTCGCAATAAAGAGTCTATTCCATTTATCGATAAGGCGAAAGCTCTACAAGTATCTCTTCAGAAGAATACAAAGTGTGCAGAGGATATTCTCAAACTCTATGACATCCGAGAAACACTCATTGAGGCAGCGGGTGTATCTGTCAAGGCGAAGGCTCACCTCGATGACATGGACAAAAATGAAATTCTTGCAGGCTTTGTTAAGGAGGTTCTTGCACCTTGTGGCAAAAAGTATATCGATGAGATTGTCTATCGTTACTTGCTCACACTTG

BBa_K5149005

AquI-2 - 401-800 bp region of AquI Locus

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 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.

TCGTTACTTGCTCACACTTGGAGATGCTCTTGGTGGTAAAATGCGCAACATTGTTGGTTCTATCGCAGAAGAGAAATTCGTCAGATTTATCGTTGCCCAGTTACAGGTGCATGATATTGAATTTGAACTATTTCCAAAACGTTCGCAATGGATTTCAAACAAAAACTATAGAGCTGAGATGGCTGCACGAACGAAAGCCTTAAGATGGAAAAATAAAACAACTTATCGATCTCTAATTTTCAACATTAATGTTCCACAGGTCAAAAAGAATATTGATATCGTGCTGCTAAATGACAAGATTGAAGGAGTAACCTCGTCTATTTTGGCGCT

BBa_K5149006

AquI-1 - 801-1200 bp region of AquI Locus

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.

GGAGTAACCTCGTCTATTTTGGCTAATATCCTTAACGATAAGAATAAGTATCTTGCCATCGGTGAGTTAAAGGGCGGAATTGATCCAGCCGGGGCAGATGAACATTGGAAAACGGCAAATACAGCTCTTAGTAGAGTAAGAGATTCTTTCAAGAACAAAATCCACTTATTTTTCATTGGGGCTGCGATAGAAACCAGTATGTCGGCAGAGATTTACGAACAATGCCAGACAGGTGAACTTAGTAACGCAGCAAATCTCACTGTTGATAATCAATTATCTGCATTGTGTGAGTGGCTAATAACGCAATAACTTTACAAAATCAAACCCGATCGC

BBa_K5149007

Codon Optimized Cpf1

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.

ATGTCTATATATCAAGAGTTTGTGAATAAATATAGTCTGAGTAAAACTCTACGCTTCGAGTTAATTCCACAGGGTAAAACACTTGAGAACATAAAGGCTAGAGGTTTAATTCTTGATGATGAGAAGAGAGCTAAAGACTACAAGAAGGCTAAGCAAATCATTGATAAATATCATCAGTTCTTTATAGAGGAGATACTAAGTTCTGTTTGTATTTCTGAGGATTTACTACAAAACTACTCTGATGTTTACTTTAAACTGAAGAAGAGTGATGATGATAACCTACAGAAAGACTTCAAATCCGCAAAAGATACCATAAAGAAACAAATAAGTGAATATATAAAAGACAGTGAGAAATTCAAGAATTTGTTTAACCAAAACCTCATTGACGCTAAGAAAGGGCAGGAGTCTGATCTAATTCTGTGGCTAAAGCAGTCTAAGGATAACGGTATTGAATTATTTAAGGCTAATTCTGATATAACAGATATAGATGAGGCGTTAGAGATAATCAAGTCTTTTAAAGGCTGGACAACATACTTTAAGGGGTTTCATGAGAATAGGAAGAATGTTTATTCCAGCAACGACATACCTACATCTATTATTTATAGGATAGTAGATGATAATTTGCCTAAATTCCTGGAGAATAAAGCAAAGTATGAGAGTTTGAAAGACAAAGCTCCTGAGGCTATAAATTATGAACAAATTAAGAAGGATTTAGCCGAAGAGCTAACCTTTGATATTGACTACAAGACCTCTGAGGTTAACCAAAGAGTGTTCTCACTTGATGAGGTATTCGAGATAGCTAACTTTAACAACTATCTAAACCAGAGCGGAATTACTAAATTCAACACTATTATTGGTGGTAAATTCGTTAATGGTGAGAATACAAAGCGTAAAGGTATCAATGAATATATAAACCTATACTCACAGCAGATAAACGACAAAACACTCAAGAAATATAAGATGAGTGTCTTATTTAAGCAAATTTTAAGTGATACAGAGTCTAAGTCTTTTGTTATTGATAAACTGGAAGATGACTCTGATGTAGTTACAACGATGCAGTCCTTCTATGAGCAGATAGCCGCTTTTAAAACAGTAGAAGAGAAGAGCATCAAAGAAACACTATCTTTATTGTTTGATGATTTAAAAGCTCAGAAACTGGATTTGTCGAAGATATACTTTAAGAACGATAAATCTCTTACTGATCTAAGTCAGCAGGTATTCGATGACTATAGTGTTATTGGTACAGCGGTACTGGAATATATAACTCAACAAATAGCACCTAAGAACCTTGATAACCCTAGTAAGAAAGAGCAGGAACTGATAGCCAAGAAAACTGAGAAAGCAAAGTACCTATCCCTAGAAACTATAAAGTTGGCGTTGGAAGAGTTTAACAAACATAGAGATATAGATAAACAGTGTAGGTTTGAAGAAATACTTGCCAACTTTGCCGCTATACCGATGATCTTTGATGAAATAGCTCAGAACAAAGATAATTTGGCACAAATATCTATAAAGTATCAAAACCAAGGTAAGAAAGACCTACTTCAGGCTTCTGCGGAAGATGACGTTAAGGCAATCAAGGATCTGTTAGATCAAACCAATAATTTGTTACATAAACTAAAGATATTTCACATCAGTCAGTCTGAAGATAAAGCAAACATCTTAGACAAAGATGAGCATTTCTATCTGGTCTTTGAGGAGTGTTACTTTGAGCTAGCCAATATAGTGCCTCTTTATAACAAAATAAGAAACTATATAACTCAGAAACCATACAGTGATGAGAAATTCAAGCTCAATTTTGAAAACTCGACGTTGGCTAATGGTTGGGATAAGAATAAAGAGCCCGACAATACAGCCATTCTGTTTATCAAAGATGATAAATACTATTTAGGTGTGATGAATAAGAAGAACAACAAGATCTTTGATGACAAAGCTATCAAGGAAAATAAAGGTGAGGGTTATAAGAAGATAGTTTATAAACTTTTGCCTGGGGCAAACAAAATGTTGCCTAAGGTTTTCTTCTCTGCTAAGTCTATAAAGTTCTATAACCCCAGTGAAGATATATTGAGAATAAGAAACCATTCCACACATACAAAGAATGGTTCACCTCAGAAGGGATATGAGAAGTTCGAGTTTAACATTGAGGATTGCCGAAAATTCATAGACTTTTATAAACAGTCTATCAGTAAACATCCTGAATGGAAGGATTTTGGCTTTAGGTTTTCTGATACTCAAAGATATAACTCTATAGATGAGTTTTATAGAGAAGTTGAAAATCAAGGCTACAAACTAACTTTTGAAAATATAAGTGAGAGCTATATTGATAGTGTAGTTAATCAGGGTAAGTTGTACCTATTCCAGATATATAACAAAGACTTTTCAGCCTACAGCAAAGGACGCCCAAACCTACATACTTTATACTGGAAAGCTCTGTTCGATGAGAGAAACCTTCAAGATGTGGTTTATAAGTTGAATGGTGAGGCAGAGCTTTTCTACCGTAAACAGTCAATACCTAAGAAAATAACTCATCCAGCCAAAGAGGCTATAGCCAATAAGAACAAAGATAATCCTAAGAAGGAGAGTGTGTTCGAATATGATTTAATCAAAGATAAACGCTTTACTGAAGATAAATTCTTCTTCCACTGTCCTATAACAATCAACTTTAAAAGTAGTGGAGCTAATAAGTTCAATGATGAAATTAATTTGTTACTAAAAGAGAAAGCTAATGATGTTCACATACTGAGTATAGATAGAGGTGAGAGACATTTAGCCTACTATACCTTGGTTGACGGTAAGGGCAATATCATCAAACAGGATACATTCAACATCATTGGTAACGACAGAATGAAAACAAACTACCATGATAAATTGGCTGCCATTGAAAAGGACAGGGATTCAGCCCGTAAAGACTGGAAGAAGATAAATAACATCAAAGAGATGAAAGAGGGCTATCTATCTCAGGTAGTTCATGAGATAGCTAAGTTGGTTATAGAGTATAACGCCATTGTGGTATTCGAGGATTTAAATTTTGGCTTTAAAAGAGGGCGTTTCAAGGTAGAGAAACAGGTTTATCAAAAGCTGGAGAAAATGCTCATTGAGAAACTAAACTATCTGGTGTTCAAAGATAATGAGTTTGATAAAACAGGAGGAGTTTTAAGAGCCTATCAACTAACAGCACCGTTTGAGACTTTTAAGAAAATGGGTAAACAAACAGGAATTATCTATTATGTACCCGCTGGTTTTACTTCAAAGATCTGTCCTGTAACTGGTTTTGTAAATCAGTTATACCCCAAGTATGAGAGCGTCAGCAAAAGTCAAGAGTTCTTCTCTAAGTTCGACAAAATCTGTTATAACCTTGATAAAGGCTATTTTGAGTTTAGCTTTGACTATAAGAACTTTGGAGACAAGGCTGCCAAAGGTAAATGGACTATAGCTAGCTTTGGCAGTAGGTTGATTAACTTTAGAAATTCTGATAAGAATCATAATTGGGATACTAGAGAGGTTTACCCAACTAAAGAGCTAGAGAAATTGCTAAAAGATTACTCTATCGAGTATGGTCATGGTGAATGTATCAAAGCCGCTATCTGCGGTGAGTCTGATAAGAAGTTCTTTGCCAAACTAACTTCTGTTCTAAATACTATCTTACAAATGCGTAACTCAAAGACAGGAACTGAGTTAGACTATCTAATTTCCCCAGTAGCGGATGTGAATGGCAACTTCTTTGATAGCCGACAAGCGCCCAAGAATATGCCTCAAGATGCTGATGCCAATGGTGCGTATCATATTGGGCTAAAGGGGCTGATGCTCCTGGGTCGTATCAAGAACAACCAAGAGGGCAAGAAGCTCAATCTGGTGATCAAGAATGAAGAGTATTTTGAATTTGTACAGAACAGGAATAACTAA

BBa_K5149008

Codon Optimized EGFP

EGFP is commonly used throughout the world of synthetic biology as a fluorescent market. 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.

ATGGTGAGTAAGGGAGAGGAACTGTTCACAGGGGTAGTGCCTATCCTCGTCGAACTGGACGGAGATGTAAACGGGCATAAGTTCAGCGTTTCCGGTGAGGGTGAAGGTGATGCCACCTACGGCAAACTAACCCTGAAGTTTATCTGTACCACAGGCAAACTGCCCGTGCCTTGGCCTACCCTCGTCACTACCCTAACCTACGGCGTACAGTGCTTCAGCCGCTACCCCGACCACATGAAACAACATGACTTTTTCAAAAGTGCCATGCCCGAAGGCTACGTTCAGGAACGCACTATCTTCTTCAAGGATGACGGCAACTATAAGACTAGAGCCGAGGTGAAGTTCGAGGGTGATACTCTGGTGAACCGCATAGAGTTAAAGGGCATTGACTTTAAAGAGGACGGCAACATCCTGGGGCACAAACTGGAATACAACTACAACAGCCACAACGTCTATATCATGGCAGACAAACAGAAGAACGGCATCAAGGTGAACTTCAAGATACGCCACAACATAGAGGACGGCAGCGTACAGTTAGCCGACCACTATCAGCAGAACACCCCCATCGGTGATGGCCCAGTGCTGCTGCCCGACAACCACTACCTGAGTACCCAGTCTGCCCTGAGCAAAGACCCCAACGAGAAGCGGGATCACATGGTCCTGTTAGAGTTTGTTACCGCAGCAGGAATCACTCTGGGTATGGACGAACTGTATAAATAA

BBa_K5149009

sgRNA Cassette

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.

TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAGCTGATTTAGGCAAAAACGGAATTTCTACTGTTGTAGATGTAAACAATTTCAGTGACTGGCAATTTCTACTGTTGTAGATTAAACAATTTCAGTGACCTTCTAATTTCTACTGTTGTAGATCAGTGACCTTGTGACGACCAATAATTTCTACTGTTGTAGATCTGCGTTACTAAGTTCACTATGAATTTCTACTGTTGTAGATGCTGCGTTACTAAGTTCAAGAAAATTTCTACTGTTGTAGATACCTGTCTGGCATTGTTCGCTGAATTTCTACTGTTGTAGATTTCGTAAATCTCTGCCGAAACGAATTTCTACTGTTGTAGATCTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCC

BBa_K5149010

Up Homology Arm to Editing Vector

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.

ACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGCTCGAGTTACCAATGCTTAATCAG

BBa_K5149011

Down Homology Arm to Editing Vector

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 517 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.

TGTGTACCCGGACAAACACCATTAATCAGCAGGAAGGTAAATTCTTCAATATCCTGTTCAACGGTCAACTGTTCGGTCAGCAGGCGATACCAACAAAAACCAAAAATCATATCCAGCAGCAGTTCACGATTGATATCTTTCGGCAGTTCACCATTGCTAATGGCATCTTCAACCAGTTTTTTCGGTATCTCACGACGACGTTCCATAAACTGATCTTTCAGTTGGGTCAGGGTTACAGGGTCCAACTGTGCTTCTGCAATAACACAACGAAATGCTTCACCACAAATGGTTTCACGCCAAACTTTCCACAGATTATGCAGCAGAAAATCCAGATCGGCTTTAAAGCTACCCAAATCCGGAAATTTACGTACCTGTTCGATTTCATTTTCATACACTTCGGCAATCAGTGCTGCTTTGTTGGTCCACCAACGATAAATGGTCGGTTTGCCTGCACCGGCGCGACGTGC

BBa_K5149012

Codon Optimized Gentamicin

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.

ATGTTACGCAGCAGCAACGACGTTACGCAGCAAGGTAGCCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATCCGCACATGTAGGCTCGGTCCTGACCAAGTTAAATCCATGAGGGCTGCTCTTGACCTTTTTGGTCGTGAGTTTGGAGACGTAGCCACCTACAGTCAACACCAGCCGGACAGTGATTACTTAGGGAACTTGCTCCGTAGTAAGACATTCATTGCGCTTGCAGCCTTTGACCAAGAAGCGGTTGTGGGTGCTCTGGCGGCTTACGTTTTACCTAGGTTTGAGCAGCCGCGTAGTGAAATCTATATCTATGACTTAGCCGTCTCCGGTGAGCACCGGAGACAGGGCATTGCCACCGCTCTCATCAACCTCCTCAAACATGAGGCTAACGCTCTTGGGGCTTATGTGATTTACGTGCAAGCGGATTACGGTGACGACCCCGCAGTGGCTCTCTATACAAAATTGGGCATACGGGAAGAAGTGATGCACTTTGATATTGACCCAAGTACCGCCACCTAA

Registered Number	Name	Description
BBa_K5149100	araBAD pBAD promoter (BBa_I13453), CbAgo, and rrnB T1 Terminator (BBa_B0010)	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.
BBa_K5149101	araBAD pBAD promoter (BBa_I13453), Cpf1 (BBa_K5149007), and rrnB T1 Terminator (BBa_B0010)	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.
BBa_K5149102	PphlF Promoter (BBa_K1725000), Cpf1 (BBa_K5149007), and rrnB T1 Terminator (BBa_B0010)	A genetic transcription unit that consists of promoter, Cas12a block, and terminator. The promoter pPhlF expresses Cas12a in the presence of a de-repressor DAPG.
BBa_K5149103	J23101 Promotor (BBa_J23101), GFP (BBa_K5149008), and rrnB T1 Terminator (BBa_B0010)	A genetic transcription unit that consists of promoter, Green Florescent Protein (GFP), and a terminator. The J23101 is a strong promotor that will express many GFP units. We use this composite part as an insert into our organisms chromosomes so that our organism flouresces green.

Registered Number

Name

Description

BBa_K5149100

araBAD pBAD promoter (BBa_I13453), CbAgo, and rrnB T1 Terminator (BBa_B0010)

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.

BBa_K5149101

araBAD pBAD promoter (BBa_I13453), Cpf1 (BBa_K5149007), and rrnB T1 Terminator (BBa_B0010)

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.

BBa_K5149102

PphlF Promoter (BBa_K1725000), Cpf1 (BBa_K5149007), and rrnB T1 Terminator (BBa_B0010)

A genetic transcription unit that consists of promoter, Cas12a block, and terminator. The promoter pPhlF expresses Cas12a in the presence of a de-repressor DAPG.

BBa_K5149103

J23101 Promotor (BBa_J23101), GFP (BBa_K5149008), and rrnB T1 Terminator (BBa_B0010)

A genetic transcription unit that consists of promoter, Green Florescent Protein (GFP), and a terminator. The J23101 is a strong promotor that will express many GFP units. We use this composite part as an insert into our organisms chromosomes so that our organism flouresces green.