LOCUS       sequence-473100-        5087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  OR/NAND gate receiver with bs for sgRNA2 and sgRNA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5087)
  AUTHORS   Kusumawardhani, H., Zoppi, F., Avendano, R. et al.
  TITLE     Engineering intercellular communication using M13 phagemid and 
            CRISPR-based gene regulation for multicellular computing in 
            Escherichia coli
  JOURNAL   Nat Commun 16, 3569 (2025)
REFERENCE   2  (bases 1 to 5087)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Commun 
            16, 3569 (2025)"
COMMENT     Sequence Label: pHK507
FEATURES             Location/Qualifiers
     source          1..5087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        37..71
                     /label=BBa_J23115
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.15"
     protein_bind    283..302
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    409..428
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        773..807
                     /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    957..976
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             981..1003
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1011..1724
                     /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     2040..2057
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2041..2057
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2070..2104
                     /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             2109..2131
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2139..2849
                     /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(2284..2302)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2552..2571
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3010..3028)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3024..3071
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3302..3329
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3517..4332)
                     /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(3517..4332)
                     /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     4240..4259
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4333..4424)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4442..5077)
                     /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(4587..4606)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattcatag gatagattct ggaaacttta ccgtccttta tagctagctc agcccttggt
       61 acaatgctag cagatagccg ttacacaggt gacacttatt tcagcctgcg gtccggccaa
      121 gtacttagta cacactgatt cgctgggacg cccgtagaac tctgtgagga taaagtctcc
      181 cttacagcct gcggtccggc caagagtaca ctagctcagt catcgctggg acgcccgtga
      241 acagttgctc tgattgaaac cacgattcag cctgcggtcc gggttcactg ccgtataggc
      301 aggacacatc ttagagtatg tagtttcaga gctatgctgg aaacagcata gcaagttgaa
      361 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgcgt tcactgccgt
      421 ataggcagtc gctgggacgc ccgaggctta ccttaccatg gttcagccaa aaaacttaag
      481 accgccggtc ttgtccacta ccttgcagta atgcggtgga caggatcggc ggttttcttt
      541 tctcttctca attcttctga cctgtaacga ataatagata gtaaagtagt ctccgattga
      601 gttttctctg ccgagtccca cccagttctg tgatttcagt aagttggtaa ttgatacact
      661 gttgcgagaa ctgctgcctg gtagtagata ggttgttatt gagtaagaag gtaaagtgaa
      721 cgaaatccct gaaactgaga ctgtagaaaa taagcttcag cctgcggtcc ggttgacggc
      781 tagctcagtc ctaggtacag tgctagctcg ctgggacgcc cgagatagcc gttacacagg
      841 tgacacttat ttcagcctgc ggtccggcca tacatactct aagatgtgtc tcgctgggac
      901 gcccgggatc caagagattt ctacacgatt gagcactgtc tcagcctgcg gtccgggttc
      961 actgccgtat aggcagtaat tttgtttaac tttaagaagg agatatacat atgcgtaaag
     1021 gcgaagaact gtttaccggt gtggttccga ttctggtgga actggacggc gatgttaatg
     1081 gtcataaatt cagtgttcgc ggcgaaggtg aaggcgatgc gacgaacggc aaactgaccc
     1141 tgaaatttat ctgcaccacg ggtaaactgc cggtcccgtg gccgacgctg gtgaccacgc
     1201 tgacctatgg cgttcaatgt tttgcgcgtt acccggatca catgaaacag cacgactttt
     1261 tcaaatcggc catgccggaa ggctatgtgc aggaacgtac gattagcttt aaagacgatg
     1321 gtacgtataa aacccgcgcg gaagtgaaat tcgaaggcga taccctggtt aaccgtatcg
     1381 aactgaaagg tatcgatttc aaagaagacg gcaatattct gggtcataaa ctggaatata
     1441 acttcaattc ccacaacgtg tacatcaccg cggataaaca gaaaaacggc attaaagcca
     1501 atttcaaaat ccgccataat gtggaagatg gtagcgttca gctggccgac cactatcagc
     1561 aaaacacgcc gattggtgat ggcccggtcc tgctgccgga caatcactac ctgagtaccc
     1621 agtccgtgct gtcaaaagat ccgaacgaaa aacgtgacca catggtcctg ctggaatttg
     1681 tgacggctgc gggtatcacc cacggcatgg acgaactgta taaataataa tcgctgggac
     1741 gcccgcctgc aggctcggta ccaaattcca gaaaagaggc ctcccgaaag gggggccttt
     1801 tttcgttttg gtcctaatag ataaaggata ggtctggtag tgttgttcgt tctcgcaggt
     1861 aaatcaataa tactcagcag ttccgtagac ttttcagtgg gacagggtag cgataacaga
     1921 tagattgtaa taagacacag taggtgctcg tagttgcgtg aagagaaccg ctcaggaaat
     1981 ccagtcagaa gtattggtaa tcgttgaaaa ctcagctcct aggatgcagt gactcgagtt
     2041 gtaaaacgac ggccagtcaa ttggaattct tgacggctag ctcagtccta ggtacagtgc
     2101 tagctaattt tgtttaactt taagaaggag atatacatat ggtgagcaag ggcgaggagg
     2161 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     2221 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     2281 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     2341 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     2401 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     2461 gcgtggtgac cgtgacccag gactcctctc tgcaggacgg cgagttcatc tacaaggtga
     2521 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag actatgggct
     2581 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc gctgaagggc gagatcaagc
     2641 agaggctgaa gctgaaggac ggcggccact acgacgctga agtcaagacc acctacaagg
     2701 ccaagaagcc cgtgcagctg cccggcgcgt acaacgtcaa catcaagttg gacatcacct
     2761 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     2821 ccggcggcat ggacgagctg tacaagtaaa ctagttgcgg ccgcctgcgc accacctgca
     2881 ggacctcagc aatcgcttgg accagctttc ttaagggtac ctattacagc tggatccggc
     2941 gccgctcatt cgctaatcgc cacgacgcgt agtcgacccg ccaattccca tggggcgcgc
     3001 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     3061 ggggtttttt gccaggcatc aaataaaacg aaaggctcag tcggaagact gggcactgtt
     3121 gaggataaat actttctcta ctaggcgcct gttacacagg tcctcagcgg cgcgcctttg
     3181 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgtgaag
     3241 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaactaag
     3301 cagaaggcca tcctgacgga tggccttttt gcgtttcaga tctaccggta aaccagcaat
     3361 agacataagc ggctatttaa cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc
     3421 tccgcaagtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3481 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     3541 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     3601 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     3661 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     3721 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     3781 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     3841 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     3901 gtcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     3961 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     4021 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     4081 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     4141 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     4201 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     4261 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     4321 aatatggctc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4381 catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggca tgctagcgca
     4441 gaaacgtcct agaagatgcc aggaggatac ttagcagaga gacaataagg ccggagcgaa
     4501 gccgtttttc cataggctcc gcccccctga cgaacatcac gaaatctgac gctcaaatca
     4561 gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg atggctccct
     4621 cttgcgctct cctgttcccg tcctgcggcg tccgtgttgt ggtggaggct ttacccaaat
     4681 caccacgtcc cgttccgtgt agacagttcg ctccaagctg ggctgtgtgc aagaaccccc
     4741 cgttcagccc gactgctgcg ccttatccgg taactatcat cttgagtcca acccggaaag
     4801 acacgacaaa acgccactgg cagcagccat tggtaactga gaattagtgg atttagatat
     4861 cgagagtctt gaagtggtgg cctaacagag gctacactga aaggacagta tttggtatct
     4921 gcgctccact aaagccagtt accaggttaa gcagttcccc aactgactta accttcgatc
     4981 aaaccgcctc cccaggcggt tttttcgttt acagagcagg agattacgac gatcgtaaaa
     5041 ggatctcaag aagatccttt acggattccc gacaccatca ctctaga
//
