LOCUS       sequence-469658-        4919 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  inducible NOT gate receiver with bs for sgRNA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4919)
  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 4919)
  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: pHK001.3QS
FEATURES             Location/Qualifiers
     source          1..4919
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1538..1557
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    1664..1683
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        1989..2023
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     protein_bind    2167..2186
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2191..2213
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2221..2931
                     /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(2366..2384)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2634..2653
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3218..3245
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3433..4248)
                     /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(3433..4248)
                     /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     4156..4175
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4249..4340)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(4358..4919,1..74))
                     /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(4503..4522)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcgtttaca gagcaggaga ttacgacgat cgtaaaagga tctcaagaag atcctttacg
       61 gattcccgac accatcactc tagagaattc ataggataga ttctggaaac tttaccgtcc
      121 gagctcaggc ttaccttact cgagcaataa acagttgata gggcttctcc gttaccatgg
      181 ttcagccaaa aaacttaaga ccgccggtct tgtccactac cttgcagtaa tgcggtggac
      241 aggatcggcg gttttctttt ctcttctcaa ttcttctgac ctgtaacgaa taatagatag
      301 taaagtagtc tccgattgag ttttctctgc cgagtcccac ccagttctgt gatttcagta
      361 agttggtaat tgatacactg ttgcgagaac tgctgcctgg tagtagatag gttgttattg
      421 agtaagaagg taaagtgaac gaaatccctg aaactgagac tgtagaaaat aagcttctat
      481 taccaattac gtcgcgtcat gcggatccta tagggattga tgatgcgcaa ttgaacagcc
      541 cgcgacgcgg cggccgagat cgtggtgcag ccgagcctga agcgggcggt tttcaggtaa
      601 tcgcgtgtgg tatgctctga tatgcccagg atgaccgaaa tatccttgta atccttgccg
      661 agggcggtcc agtgcagaca ctcgatctcg cgcggcgaca atgccggcac cggatcgttt
      721 tcgccatgca gctcatatac ggccttgcgg tggatcagat gggcgatctc gatccactca
      781 ttgcggcagc ggcgcacgag ctcggtccat tcgtcggccg gtatatgggc attcagcgac
      841 agcagggcgc ggcgctgcgc cttgtcggcg acggggatgg agtagccatt gtcatcgatg
      901 ccgtgtttct gggcgtcgac cagcatggca taggcctccg gcgtcggttc gacctcgctc
      961 cagtcgaagg gcagctggcg ttcgaagccc tgcttgatga tcggatcgac cttcacatag
     1021 cagttgagga ggtaacggga aacccaggca tccggatagg tggtgcgcac gaagggcgaa
     1081 tcgatcttgc tcgcgattgt ctgggcgaga tggtaggtga cgaaatcgag gttatattcc
     1141 gcctggagaa tacggatggc ggcatccacg ttggccgccg ctttgatctg ttcgaacgcg
     1201 gactcgaact tttcgctata ggtattctca atcatattca ccaccctgaa ttgactctct
     1261 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggc gcgccgccat
     1321 tggaccaaaa cgagagccga atcgcactta tttacagtag ttcagcctgc ggtccgggac
     1381 atgctctgat ccccctcatc tgggggggcc tatctgaggg aatttccgat ccggctcgcc
     1441 tgaaccattc tgctttccac gaacttgaaa acgctagtcg ctgggacgcc cgctattaca
     1501 ctcgtcgttg gaaactgaag atcagcctgc ggtccgggtt cactgccgta taggcagtga
     1561 ctgagctagt gtactctgtt tcagagctat gctggaaaca gcatagcaag ttgaaataag
     1621 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcgttcact gccgtatagg
     1681 cagtcgctgg gacgcccgcc tgcaggctcg gtaccaaatt ccagaaaaga ggcctcccga
     1741 aaggggggcc ttttttcgtt ttggtcctaa tagataaagg ataggtctgg tagtgttgtt
     1801 cgttctcgca ggtaaatcaa taatactcag cagttccgta gacttttcag tgggacaggg
     1861 tagcgataac agatagattg taataagaca cagtaggtgc tcgtagttgc gtgaagagaa
     1921 ccgctcagga aatccagtca gaagtattgg taatcgttga aaactcagtc gaccagcctg
     1981 cggtccggtt gacagctagc tcagtcctag gtataatgct agctcgctgg gacgcccggt
     2041 agtgcttatc agacccaata ctgttgaaca gcctgcggtc cggccaagag tacactagct
     2101 cagtcatcgc tgggacgccc gtagaactct gtgaggataa agtctccctt acagcctgcg
     2161 gtccgggttc actgccgtat aggcagtaat tttgtttaac tttaagaagg agatatacat
     2221 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     2281 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     2341 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     2401 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     2461 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     2521 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc tctgcaggac
     2581 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     2641 atgcagaaga agactatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     2701 gcgctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     2761 gaagtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc gtacaacgtc
     2821 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     2881 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta ataatcgctg
     2941 ggacgcccgg cggccgcgga aacacagaaa aaagcccgca cctgacagtg cgggcttttt
     3001 ttttcgacca aaggtagcga acgacgagtc actgttgagg ataaatactt tctctactag
     3061 gcgcctgtta cacaggtcct cagcggcgcg cctttgtcgg tgaacgctct cctgagtagg
     3121 acaaatccgc cgggagcgga tttgaacgtt gtgaagcaac ggcccggagg gtggcgggca
     3181 ggacgcccgc cataaactgc caggcatcaa actaagcaga aggccatcct gacggatggc
     3241 ctttttgcgt ttcagatcta ccggtaaacc agcaatagac ataagcggct atttaacgac
     3301 cctgccctga accgacgaca agctgacgac cgggtctccg caagtggcac ttttcgggga
     3361 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3421 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     3481 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     3541 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     3601 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     3661 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     3721 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     3781 cattcgtgat tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag gacaattaca
     3841 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     3901 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     3961 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     4021 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     4081 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     4141 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     4201 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt
     4261 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4321 tatttagaaa aataaacaaa taggcatgct agcgcagaaa cgtcctagaa gatgccagga
     4381 ggatacttag cagagagaca ataaggccgg agcgaagccg tttttccata ggctccgccc
     4441 ccctgacgaa catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc cgacaggact
     4501 ataaagatac caggcgtttc cccctgatgg ctccctcttg cgctctcctg ttcccgtcct
     4561 gcggcgtccg tgttgtggtg gaggctttac ccaaatcacc acgtcccgtt ccgtgtagac
     4621 agttcgctcc aagctgggct gtgtgcaaga accccccgtt cagcccgact gctgcgcctt
     4681 atccggtaac tatcatcttg agtccaaccc ggaaagacac gacaaaacgc cactggcagc
     4741 agccattggt aactgagaat tagtggattt agatatcgag agtcttgaag tggtggccta
     4801 acagaggcta cactgaaagg acagtatttg gtatctgcgc tccactaaag ccagttacca
     4861 ggttaagcag ttccccaact gacttaacct tcgatcaaac cgcctcccca ggcggtttt
//
