LOCUS       sequence-458369-        7234 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Light inducible three-node circuit that can generate ring patterns 
            in a growing bacterial colony upon blue light pulses. Heterologous 
            expression of dCas9, Csy4, and VVD-AraC are necessary..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7234)
  AUTHORS   Hollo G, Park JH, Boni E, Schaerli Y
  TITLE     GRN_modeler: An Intuitive Tool for Constructing and Evaluating Gene 
            Regulatory Networks and its Applications to Oscillators and a Light 
            Biosensor
  JOURNAL   bioRxiv 2024.12.18.629005
REFERENCE   2  (bases 1 to 7234)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.12.18.629005"
COMMENT     Sequence Label: pJP_Osc05
FEATURES             Location/Qualifiers
     source          1..7234
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..636)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(146..165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        791..1075
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     998..1017
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    1221..1240
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    1347..1366
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    1433..1452
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             1457..1479
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1487..2194
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1632..1650)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1900..1919
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        2914..2948
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     protein_bind    3092..3111
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    3218..3237
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    3304..3323
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3328..3350
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3358..4071
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     promoter        4434..4525
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4526..5341
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4526..5341
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4599..4618)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(5529..5556)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5711..5745
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     protein_bind    5889..5908
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    6015..6034
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    6101..6120
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             6125..6147
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     7018..7038
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
ORIGIN
        1 aaacgtccta gaagatgcca ggaggatact tagcagagag acaataaggc cggagcgaag
       61 ccgtttttcc ataggctccg cccccctgac gaacatcacg aaatctgacg ctcaaatcag
      121 tggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctga tggctccctc
      181 ttgcgctctc ctgttcccgt cctgcggcgt ccgtgttgtg gtggaggctt tacccaaatc
      241 accacgtccc gttccgtgta gacagttcgc tccaagctgg gctgtgtgca agaacccccc
      301 gttcagcccg actgctgcgc cttatccggt aactatcatc ttgagtccaa cccggaaaga
      361 cacgacaaaa cgccactggc agcagccatt ggtaactgag aattagtgga tttagatatc
      421 gagagtcttg aagtggtggc ctaacagagg ctacactgaa aggacagtat ttggtatctg
      481 cgctccacta aagccagtta ccaggttaag cagttcccca actgacttaa ccttcgatca
      541 aaccgcctcc ccaggcggtt ttttcgttta cagagcagga gattacgacg atcgtaaaag
      601 gatctcaaga agatccttta cggattcccg acaccatcac tctagatttc agtgcaattt
      661 atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt
      721 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gctcactttt catactcccg
      781 ccattcagag aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt
      841 tactggctct tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa
      901 agcgggacca aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa
      961 gtccacattg attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata
     1021 agattagcgg ttcctacctg acgcttttta tcgcaactct ctactgtttc tccataccct
     1081 gaattcatag gatagattct ggaaacttta ccgtccgagc tccagcctgc ggtccggcca
     1141 tacatactct aagatgtgtc tcgctgggac gcccggggac tacacttacg aaactattga
     1201 ttgctcagcc tgcggtccgg gttcactgcc gtataggcag atcagtgtgt actaagtact
     1261 gtttcagagc tatgctggaa acagcatagc aagttgaaat aaggctagtc cgttatcaac
     1321 ttgaaaaagt ggcaccgagt cggtgcgttc actgccgtat aggcagtcgc tgggacgccc
     1381 gctcgagcaa taaacagttg atagggcttc tccgttacag cctgcggtcc gggttcactg
     1441 ccgtataggc agtaattttg tttaacttta agaaggagat atacatatgg tgagcaaggg
     1501 cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg
     1561 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     1621 cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat
     1681 cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     1741 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     1801 ggacggcggc gtggtgaccg tgacccagga ctcctctctg caggacggcg agttcatcta
     1861 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac
     1921 tatgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgcgc tgaagggcga
     1981 gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgaag tcaagaccac
     2041 ctacaaggcc aagaagcccg tgcagctgcc cggcgcgtac aacgtcaaca tcaagttgga
     2101 catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg
     2161 ccactccacc ggcggcatgg acgagctgta caagatgtcc cgccgtaata ctgacgccat
     2221 cacaatccac agcatcctgg attggattga agacaacttg gagtcgccgt tgagtttaga
     2281 aaaagttagt gaacgtagtg gttactcaaa gtggcacctt cagcgcatgt ttaagaagga
     2341 aacgggtcat tcattgggtc aatatattcg ttctcgcaag atgactgaaa ttgcccagaa
     2401 attgaaagag tctaatgaac ctattttgta cctggcggag cgttacggct ttgaaagtca
     2461 gcaaaccctt acacgtacct tcaagaatta ctttgacgtt ccaccacaca aatatcgtat
     2521 gaccaacatg cagggtgagt cacgtttttt gcatccgttg aatcattaca attcctaata
     2581 atcgctggga cgcccgccat ggttcagcca aaaaacttaa gaccgccggt cttgtccact
     2641 accttgcagt aatgcggtgg acaggatcgg cggttttctt ttctcttctc aattcttctg
     2701 acctgtaacg aataatagat agtaaagtag tctccgattg agttttctct gccgagtccc
     2761 acccagttct gtgatttcag taagttggta attgatacac tgttgcgaga actgctgcct
     2821 ggtagtagat aggttgttat tgagtaagaa ggtaaagtga acgaaatccc tgaaactgag
     2881 actgtagaaa ataagcttca gcctgcggtc cggttgacgg ctagctcagt cctaggtaca
     2941 gtgctagctc gctgggacgc ccgagatagc cgttacacag gtgacactta tttcagcctg
     3001 cggtccggcc acactagagt ctagcttgag atcgctggga cgcccgtgaa cagttgctct
     3061 gattgaaacc acgattcagc ctgcggtccg ggttcactgc cgtataggca ggacacatct
     3121 tagagtatgt agtttcagag ctatgctgga aacagcatag caagttgaaa taaggctagt
     3181 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcgtt cactgccgta taggcagtcg
     3241 ctgggacgcc cgggatccaa gagatttcta cacgattgag cactgtctca gcctgcggtc
     3301 cgggttcact gccgtatagg cagtaatttt gtttaacttt aagaaggaga tatacatatg
     3361 tctaaaggtg aagaattatt cactggtgtt gtcccaattt tggttgaatt agatggtgat
     3421 gttaatggtc acaaattttc tgtctccggt gaaggtgaag gtgatgctac ttacggtaaa
     3481 ttgaccttaa aatttatttg tactactggt aaattgccag ttccgtggcc aaccttagtc
     3541 actactttag gttatggttt gatgtgtttt gctagatacc cagatcatat gaaacaacat
     3601 gactttttca agtctgccat gccagaaggt tatgttcaag aaagaactat ttttttcaaa
     3661 gatgacggta actacaagac cagagctgaa gtcaagtttg aaggtgatac cttagttaat
     3721 agaatcgaat taaaaggtat tgattttaaa gaagatggta acattttagg tcacaaattg
     3781 gaatacaact ataactctca caatgtttac atcatggctg acaaacaaaa gaatggtatc
     3841 aaagttaact tcaaaattag acacaacatt gaagatggtt ctgttcaatt agctgaccat
     3901 tatcaacaaa atactccaat tggtgatggt ccagtcttgt taccagacaa ccattactta
     3961 tcctatcaat ctaaattatc caaagatcca aacgaaaaga gagaccacat ggtcttgtta
     4021 gaatttgtta ctgctgctgg tattacccac ggtatggatg aattgtacaa aatgtcccgc
     4081 cgtaatactg acgccatcac aatccacagc atcctggatt ggattgaaga ctaataatcg
     4141 ctgggacgcc cgcctgcagg ctcggtacca aattccagaa aagaggcctc ccgaaagggg
     4201 ggcctttttt cgttttggtc ctaatagata aaggataggt ctggtagtgt tgttcgttct
     4261 cgcaggtaaa tcaataatac tcagcagttc cgtagacttt tcagtgggac agggtagcga
     4321 taacagatag attgtaataa gacacagtag gtgctcgtag ttgcgtgaag agaaccgctc
     4381 aggaaatcca gtcagaagta ttggtaatcg ttgtttctgc gctagcatgc ctatttgttt
     4441 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     4501 tcaataatat tgaaaaagga agagtatgag ccatattcaa cgggaaacgt cttgctctag
     4561 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     4621 tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt
     4681 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     4741 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     4801 tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga
     4861 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     4921 ttcgattcct gtttgtaatt gtccttttaa cagcgaccgc gtatttcgtc tcgctcaggc
     4981 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     5041 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     5101 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     5161 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     5221 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     5281 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     5341 agaattaatt catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5401 ttccgcgcac atttccccga aaagtgccac ttgcggagac ccggtcgtca gcttgtcgtc
     5461 ggttcagggc agggtcgtta aatagccgct tatgtctatt gctggtttac cggtagatct
     5521 gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc ctggcagttt
     5581 atggcgggcg tcctgcccgc caccctccgg gccgttgctt cacaacgttc aaatccgctc
     5641 ccggcggatt tgtcctactc aggagagcgt tcaccgacaa agaaactcag tcgaccagcc
     5701 tgcggtccgg ttgacggcta gctcagtcct aggtacagtg ctagctcgct gggacgcccg
     5761 agtgacgact gcgaagtaac ctctatttat cagcctgcgg tccggccaag tacttagtac
     5821 acactgattc gctgggacgc ccggtagtgc ttatcagacc caatactgtt gaacagcctg
     5881 cggtccgggt tcactgccgt ataggcagtc tcaagctaga ctctagtggt ttcagagcta
     5941 tgctggaaac agcatagcaa gttgaaataa ggctagtccg ttatcaactt gaaaaagtgg
     6001 caccgagtcg gtgcgttcac tgccgtatag gcagtcgctg ggacgcccgg acgtcctatt
     6061 acactcgtcg ttggaaactg aagatcagcc tgcggtccgg gttcactgcc gtataggcag
     6121 taattttgtt taactttaag aaggagatat acatatgaat cagtcattca tctcggacat
     6181 cttatatgcc gacatcgaat cgaaggctaa ggaacttaca gtcaattcca acaatactgt
     6241 ccagccggtc gcgcttatgc gcttaggagt tttcgttccc aaaccttcca agagcaaagg
     6301 agaaagtaag gaaattgacg ccaccaaagc cttctctcaa ctggagattg ctaaagcaga
     6361 gggcatgagt aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga
     6421 tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata
     6481 cggaaaactt acccttaaat ttatttgcac tactggaaaa ctacctgttc cgtggccaac
     6541 acttgtcact actttgactt ggggtgttca atgctttgct agatacccag atcatatgaa
     6601 acagcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt
     6661 tttcaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct
     6721 tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca
     6781 caaattggaa tacaacgcta tttcagataa tgtatacatc actgcagaca aacaaaagaa
     6841 tggaatcaaa gctaatttca aaattagaca caacattgaa gatggaagcg ttcaactagc
     6901 agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca
     6961 ttacctgtcc acacaatcta aactttcgaa agatcccaac gaaaagagag accacatggt
     7021 ccttcttgag tttgtaacag ctgctgggat tacactaggc atggatgaac tatacaaata
     7081 ataatcgctg ggacgcccgg cggccgcgga aacacagaaa aaagcccgca cctgacagtg
     7141 cgggcttttt ttttcgacca aaggtagcga acgacgagtc actgttgagg ataaatactt
     7201 tctctactag gcgcctgtta cacaggtcct cagc
//
