LOCUS       sequence-470106-        4685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dual NOT gate receiver with bs for sgRNA2 and sgRNA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 408 to 442)
  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 4685)
  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: pHK001.23
FEATURES             Location/Qualifiers
     source          1..4685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        408..442
                     /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    592..611
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             616..638
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             646..1359
                     /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"
     promoter        1671..1705
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     protein_bind    1849..1868
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             1873..1895
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1903..2613
                     /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(2048..2066)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2316..2335
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      2900..2927
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3115..3930)
                     /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(3115..3930)
                     /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     3838..3857
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3931..4022)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4040..4675)
                     /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(4185..4204)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattcatag gatagattct ggaaacttta ccgtccgagc tcaggcttac cttactcgag
       61 caataaacag ttgatagggc ttctccgtta ccatggttca gccaaaaaac ttaagaccgc
      121 cggtcttgtc cactaccttg cagtaatgcg gtggacagga tcggcggttt tcttttctct
      181 tctcaattct tctgacctgt aacgaataat agatagtaaa gtagtctccg attgagtttt
      241 ctctgccgag tcccacccag ttctgtgatt tcagtaagtt ggtaattgat acactgttgc
      301 gagaactgct gcctggtagt agataggttg ttattgagta agaaggtaaa gtgaacgaaa
      361 tccctgaaac tgagactgta gaaaataagc ttcagcctgc ggtccggttg acggctagct
      421 cagtcctagg tacagtgcta gctcgctggg acgcccgaga tagccgttac acaggtgaca
      481 cttatttcag cctgcggtcc ggccaagtac ttagtacaca ctgattcgct gggacgcccg
      541 ggatccaaga gatttctaca cgattgagca ctgtctcagc ctgcggtccg ggttcactgc
      601 cgtataggca gtaattttgt ttaactttaa gaaggagata tacatatgcg taaaggcgaa
      661 gaactgttta ccggtgtggt tccgattctg gtggaactgg acggcgatgt taatggtcat
      721 aaattcagtg ttcgcggcga aggtgaaggc gatgcgacga acggcaaact gaccctgaaa
      781 tttatctgca ccacgggtaa actgccggtc ccgtggccga cgctggtgac cacgctgacc
      841 tatggcgttc aatgttttgc gcgttacccg gatcacatga aacagcacga ctttttcaaa
      901 tcggccatgc cggaaggcta tgtgcaggaa cgtacgatta gctttaaaga cgatggtacg
      961 tataaaaccc gcgcggaagt gaaattcgaa ggcgataccc tggttaaccg tatcgaactg
     1021 aaaggtatcg atttcaaaga agacggcaat attctgggtc ataaactgga atataacttc
     1081 aattcccaca acgtgtacat caccgcggat aaacagaaaa acggcattaa agccaatttc
     1141 aaaatccgcc ataatgtgga agatggtagc gttcagctgg ccgaccacta tcagcaaaac
     1201 acgccgattg gtgatggccc ggtcctgctg ccggacaatc actacctgag tacccagtcc
     1261 gtgctgtcaa aagatccgaa cgaaaaacgt gaccacatgg tcctgctgga atttgtgacg
     1321 gctgcgggta tcacccacgg catggacgaa ctgtataaat aataatcgct gggacgcccg
     1381 cctgcaggct cggtaccaaa ttccagaaaa gaggcctccc gaaagggggg ccttttttcg
     1441 ttttggtcct aatagataaa ggataggtct ggtagtgttg ttcgttctcg caggtaaatc
     1501 aataatactc agcagttccg tagacttttc agtgggacag ggtagcgata acagatagat
     1561 tgtaataaga cacagtaggt gctcgtagtt gcgtgaagag aaccgctcag gaaatccagt
     1621 cagaagtatt ggtaatcgtt gaaaactcag tcgaccagcc tgcggtccgg ttgacagcta
     1681 gctcagtcct aggtataatg ctagctcgct gggacgcccg gtagtgctta tcagacccaa
     1741 tactgttgaa cagcctgcgg tccggccaag agtacactag ctcagtcatc gctgggacgc
     1801 ccgtagaact ctgtgaggat aaagtctccc ttacagcctg cggtccgggt tcactgccgt
     1861 ataggcagta attttgttta actttaagaa ggagatatac atatggtgag caagggcgag
     1921 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     1981 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     2041 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     2101 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     2161 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     2221 ggcggcgtgg tgaccgtgac ccaggactcc tctctgcagg acggcgagtt catctacaag
     2281 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagactatg
     2341 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgcgctgaa gggcgagatc
     2401 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaagtcaa gaccacctac
     2461 aaggccaaga agcccgtgca gctgcccggc gcgtacaacg tcaacatcaa gttggacatc
     2521 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     2581 tccaccggcg gcatggacga gctgtacaag taataatcgc tgggacgccc ggcggccgcg
     2641 gaaacacaga aaaaagcccg cacctgacag tgcgggcttt ttttttcgac caaaggtagc
     2701 gaacgacgag tcactgttga ggataaatac tttctctact aggcgcctgt tacacaggtc
     2761 ctcagcggcg cgcctttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg
     2821 gatttgaacg ttgtgaagca acggcccgga gggtggcggg caggacgccc gccataaact
     2881 gccaggcatc aaactaagca gaaggccatc ctgacggatg gcctttttgc gtttcagatc
     2941 taccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga
     3001 caagctgacg accgggtctc cgcaagtggc acttttcggg gaaatgtgcg cggaacccct
     3061 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     3121 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     3181 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     3241 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     3301 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     3361 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     3421 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     3481 agcgagacga aatacgcggt cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     3541 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     3601 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     3661 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     3721 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     3781 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     3841 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     3901 gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt attgaagcat
     3961 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4021 aataggcatg ctagcgcaga aacgtcctag aagatgccag gaggatactt agcagagaga
     4081 caataaggcc ggagcgaagc cgtttttcca taggctccgc ccccctgacg aacatcacga
     4141 aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4201 tccccctgat ggctccctct tgcgctctcc tgttcccgtc ctgcggcgtc cgtgttgtgg
     4261 tggaggcttt acccaaatca ccacgtcccg ttccgtgtag acagttcgct ccaagctggg
     4321 ctgtgtgcaa gaaccccccg ttcagcccga ctgctgcgcc ttatccggta actatcatct
     4381 tgagtccaac ccggaaagac acgacaaaac gccactggca gcagccattg gtaactgaga
     4441 attagtggat ttagatatcg agagtcttga agtggtggcc taacagaggc tacactgaaa
     4501 ggacagtatt tggtatctgc gctccactaa agccagttac caggttaagc agttccccaa
     4561 ctgacttaac cttcgatcaa accgcctccc caggcggttt tttcgtttac agagcaggag
     4621 attacgacga tcgtaaaagg atctcaagaa gatcctttac ggattcccga caccatcact
     4681 ctaga
//
