LOCUS       sequence-496859-        5657 bp    DNA     circular SYN 12-JAN-2026
DEFINITION  AND gate receiver with bs for sgRNA2 and sgRNA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5657)
  AUTHORS   Kusumawardhani H, Zoppi F, Avendano R, Schaerli Y
  TITLE     Engineering intercellular communication using M13 phagemid and 
            CRISPR-based gene regulation for multicellular computing in 
            Escherichia coli.
  JOURNAL   Nat Commun. 2025 Apr 15;16(1):3569. doi: 10.1038/s41467-025-58760-z.
  PUBMED    40234414
REFERENCE   2  (bases 1 to 5657)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 12, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-04-15"; volume: "16(1):3569. doi"; pages: " 
            10.1038/s41467-025-58760-z"
COMMENT     Sequence Label: pHK408
FEATURES             Location/Qualifiers
     source          1..5657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        52..86
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     protein_bind    230..249
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    356..375
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     terminator      complement(496..525)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     promoter        604..638
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     protein_bind    782..801
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    908..927
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        1266..1300
                     /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    1527..1546
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             1551..1573
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1581..2294
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     2610..2627
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2611..2627
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2640..2674
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     RBS             2679..2701
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2709..3419
                     /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(2854..2872)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3122..3141
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3580..3598)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3594..3641
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3872..3899
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4087..4902)
                     /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             complement(4087..4902)
                     /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     4810..4829
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4903..4994)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5012..5647)
                     /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(5157..5176)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattcatag gatagattct ggaaacttta ccgtcccagc ctgcggtccg gctgacagct
       61 agctcagtcc taggtataat gctagctcgc tgggacgccc gagtgacgac tgcgaagtaa
      121 cctctattta tcagcctgcg gtccggccaa gattacacta gctcagtcat cgctgggacg
      181 cccggggact acacttacga aactattgat tgctcagcct gcggtccggg ttcactgccg
      241 tataggcaga agtgagtctg agcttagatg tttcagagct atgctggaaa cagcatagca
      301 agttgaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcgttca
      361 ctgccgtata ggcagtcgct gggacgcccg cagttgataa gtccctaact ttttgttatc
      421 aataaaaaag gcctcccgat ttgggaggcc ttattgttcg tcctagtaaa ggaatattca
      481 gcaatttgcc cgtgccgaag aaaggcccac ccgtgaaggt gagccagtga gttgattgct
      541 acgtaattag ttagttagag agccgaatcg cacttattta cagtagttca gcctgcggtc
      601 cggctgacag ctagctcagt cctaggtata atgctagctc gctgggacgc ccgtgaacag
      661 ttgctctgat tgaaaccacg attcagcctg cggtccggcc aagtacttag tacacactga
      721 ttcgctggga cgcccggtag tgcttatcag acccaatact gttgaacagc ctgcggtccg
      781 ggttcactgc cgtataggca ggacacatct tagagtatgt agtttcagag ctatgctgga
      841 aacagcatag caagttgaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      901 tcggtgcgtt cactgccgta taggcagtcg ctgggacgcc cgaggcttac cttaccatgg
      961 ttcagccaaa aaacttaaga ccgccggtct tgtccactac cttgcagtaa tgcggtggac
     1021 aggatcggcg gttttctttt ctcttctcaa ttcttctgac ctgtaacgaa taatagatag
     1081 taaagtagtc tccgattgag ttttctctgc cgagtcccac ccagttctgt gatttcagta
     1141 agttggtaat tgatacactg ttgcgagaac tgctgcctgg tagtagatag gttgttattg
     1201 agtaagaagg taaagtgaac gaaatccctg aaactgagac tgtagaaaat cagcctgcgg
     1261 tccggttgac ggctagctca gtcctaggta cagtgctagc tcgctgggac gcccgagata
     1321 gccgttacac aggtgacact tatttcagcc tgcggtccgg ccatacatac tctaagatgt
     1381 gtctcgctgg gacgcccgta gaactctgtg aggataaagt ctcccttaca gcctgcggtc
     1441 cggccaatct aagctcagac tcactttcgc tgggacgccc gaagagattt ctacacgatt
     1501 gagcactgtc tcagcctgcg gtccgggttc actgccgtat aggcagtaat tttgtttaac
     1561 tttaagaagg agatatacat atgcgtaaag gcgaagaact gtttaccggt gtggttccga
     1621 ttctggtgga actggacggc gatgttaatg gtcataaatt cagtgttcgc ggcgaaggtg
     1681 aaggcgatgc gacgaacggc aaactgaccc tgaaatttat ctgcaccacg ggtaaactgc
     1741 cggtcccgtg gccgacgctg gtgaccacgc tgacctatgg cgttcaatgt tttgcgcgtt
     1801 acccggatca catgaaacag cacgactttt tcaaatcggc catgccggaa ggctatgtgc
     1861 aggaacgtac gattagcttt aaagacgatg gtacgtataa aacccgcgcg gaagtgaaat
     1921 tcgaaggcga taccctggtt aaccgtatcg aactgaaagg tatcgatttc aaagaagacg
     1981 gcaatattct gggtcataaa ctggaatata acttcaattc ccacaacgtg tacatcaccg
     2041 cggataaaca gaaaaacggc attaaagcca atttcaaaat ccgccataat gtggaagatg
     2101 gtagcgttca gctggccgac cactatcagc aaaacacgcc gattggtgat ggcccggtcc
     2161 tgctgccgga caatcactac ctgagtaccc agtccgtgct gtcaaaagat ccgaacgaaa
     2221 aacgtgacca catggtcctg ctggaatttg tgacggctgc gggtatcacc cacggcatgg
     2281 acgaactgta taaataataa tcgctgggac gcccgcctgc aggctcggta ccaaattcca
     2341 gaaaagaggc ctcccgaaag gggggccttt tttcgttttg gtcctaatag ataaaggata
     2401 ggtctggtag tgttgttcgt tctcgcaggt aaatcaataa tactcagcag ttccgtagac
     2461 ttttcagtgg gacagggtag cgataacaga tagattgtaa taagacacag taggtgctcg
     2521 tagttgcgtg aagagaaccg ctcaggaaat ccagtcagaa gtattggtaa tcgttgaaaa
     2581 ctcagctcct aggatgcagt gactcgagtt gtaaaacgac ggccagtcaa ttggaattct
     2641 tgacggctag ctcagtccta ggtacagtgc tagctaattt tgtttaactt taagaaggag
     2701 atatacatat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     2761 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     2821 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     2881 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     2941 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     3001 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctctc
     3061 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     3121 gccccgtaat gcagaagaag actatgggct gggaggcctc ctccgagcgg atgtaccccg
     3181 aggacggcgc gctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     3241 acgacgctga agtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcgt
     3301 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     3361 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaaa
     3421 ctagttgcgg ccgcctgcgc accacctgca ggacctcagc aatcgcttgg accagctttc
     3481 ttaagggtac ctattacagc tggatccggc gccgctcatt cgctaatcgc cacgacgcgt
     3541 agtcgacccg ccaattccca tggggcgcgc ctgctgccac cgctgagcaa taactagcat
     3601 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gccaggcatc aaataaaacg
     3661 aaaggctcag tcggaagact gggcactgtt gaggataaat actttctcta ctaggcgcct
     3721 gttacacagg tcctcagcgg cgcgcctttg tcggtgaacg ctctcctgag taggacaaat
     3781 ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg ggcaggacgc
     3841 ccgccataaa ctgccaggca tcaaactaag cagaaggcca tcctgacgga tggccttttt
     3901 gcgtttcaga tctaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc
     3961 ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg gcacttttcg gggaaatgtg
     4021 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     4081 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     4141 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     4201 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     4261 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     4321 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     4381 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     4441 tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg
     4501 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     4561 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     4621 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     4681 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     4741 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     4801 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     4861 tcgcggccta gagcaagacg tttcccgttg aatatggctc atactcttcc tttttcaata
     4921 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4981 gaaaaataaa caaataggca tgctagcgca gaaacgtcct agaagatgcc aggaggatac
     5041 ttagcagaga gacaataagg ccggagcgaa gccgtttttc cataggctcc gcccccctga
     5101 cgaacatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
     5161 ataccaggcg tttccccctg atggctccct cttgcgctct cctgttcccg tcctgcggcg
     5221 tccgtgttgt ggtggaggct ttacccaaat caccacgtcc cgttccgtgt agacagttcg
     5281 ctccaagctg ggctgtgtgc aagaaccccc cgttcagccc gactgctgcg ccttatccgg
     5341 taactatcat cttgagtcca acccggaaag acacgacaaa acgccactgg cagcagccat
     5401 tggtaactga gaattagtgg atttagatat cgagagtctt gaagtggtgg cctaacagag
     5461 gctacactga aaggacagta tttggtatct gcgctccact aaagccagtt accaggttaa
     5521 gcagttcccc aactgactta accttcgatc aaaccgcctc cccaggcggt tttttcgttt
     5581 acagagcagg agattacgac gatcgtaaaa ggatctcaag aagatccttt acggattccc
     5641 gacaccatca ctctaga
//
