LOCUS       sequence-469114-        4722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NOT gate receiver with bs for sgRNA2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4722)
  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 4722)
  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: pHK001.2
FEATURES             Location/Qualifiers
     source          1..4722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     280..297
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     281..297
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        310..344
                     /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             349..371
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             379..1089
                     /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(524..542)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     792..811
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1250..1268)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1264..1311
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1542..1569
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(1757..2572)
                     /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(1757..2572)
                     /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     2480..2499
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2573..2664)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2682..3317)
                     /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(2827..2846)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3735..3769
                     /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    3919..3938
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3943..3965
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3973..4686
                     /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"
ORIGIN
        1 ccaaattcca gaaaagaggc ctcccgaaag gggggccttt tttcgttttg gtcctaatag
       61 ataaaggata ggtctggtag tgttgttcgt tctcgcaggt aaatcaataa tactcagcag
      121 ttccgtagac ttttcagtgg gacagggtag cgataacaga tagattgtaa taagacacag
      181 taggtgctcg tagttgcgtg aagagaaccg ctcaggaaat ccagtcagaa gtattggtaa
      241 tcgttgaaaa ctcagctcct aggatgcagt gactcgagtt gtaaaacgac ggccagtcaa
      301 ttggaattct tgacggctag ctcagtccta ggtacagtgc tagctaattt tgtttaactt
      361 taagaaggag atatacatat ggtgagcaag ggcgaggagg ataacatggc catcatcaag
      421 gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc
      481 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
      541 aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc
      601 aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag
      661 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
      721 gactcctctc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc
      781 ccctccgacg gccccgtaat gcagaagaag actatgggct gggaggcctc ctccgagcgg
      841 atgtaccccg aggacggcgc gctgaagggc gagatcaagc agaggctgaa gctgaaggac
      901 ggcggccact acgacgctga agtcaagacc acctacaagg ccaagaagcc cgtgcagctg
      961 cccggcgcgt acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc
     1021 atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg
     1081 tacaagtaaa ctagttgcgg ccgcctgcgc accacctgca ggacctcagc aatcgcttgg
     1141 accagctttc ttaagggtac ctattacagc tggatccggc gccgctcatt cgctaatcgc
     1201 cacgacgcgt agtcgacccg ccaattccca tggggcgcgc ctgctgccac cgctgagcaa
     1261 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gccaggcatc
     1321 aaataaaacg aaaggctcag tcggaagact gggcactgtt gaggataaat actttctcta
     1381 ctaggcgcct gttacacagg tcctcagcgg cgcgcctttg tcggtgaacg ctctcctgag
     1441 taggacaaat ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg
     1501 ggcaggacgc ccgccataaa ctgccaggca tcaaactaag cagaaggcca tcctgacgga
     1561 tggccttttt gcgtttcaga tctaccggta aaccagcaat agacataagc ggctatttaa
     1621 cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg gcacttttcg
     1681 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1741 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     1801 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     1861 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     1921 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     1981 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     2041 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2101 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat
     2161 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2221 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2281 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     2341 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     2401 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     2461 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     2521 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc atactcttcc
     2581 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     2641 aatgtattta gaaaaataaa caaataggca tgctagcgca gaaacgtcct agaagatgcc
     2701 aggaggatac ttagcagaga gacaataagg ccggagcgaa gccgtttttc cataggctcc
     2761 gcccccctga cgaacatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag
     2821 gactataaag ataccaggcg tttccccctg atggctccct cttgcgctct cctgttcccg
     2881 tcctgcggcg tccgtgttgt ggtggaggct ttacccaaat caccacgtcc cgttccgtgt
     2941 agacagttcg ctccaagctg ggctgtgtgc aagaaccccc cgttcagccc gactgctgcg
     3001 ccttatccgg taactatcat cttgagtcca acccggaaag acacgacaaa acgccactgg
     3061 cagcagccat tggtaactga gaattagtgg atttagatat cgagagtctt gaagtggtgg
     3121 cctaacagag gctacactga aaggacagta tttggtatct gcgctccact aaagccagtt
     3181 accaggttaa gcagttcccc aactgactta accttcgatc aaaccgcctc cccaggcggt
     3241 tttttcgttt acagagcagg agattacgac gatcgtaaaa ggatctcaag aagatccttt
     3301 acggattccc gacaccatca ctctagagaa ttcataggat agattctgga aactttaccg
     3361 tccgagctca ggcttacctt actcgagcaa taaacagttg atagggcttc tccgttacca
     3421 tggttcagcc aaaaaactta agaccgccgg tcttgtccac taccttgcag taatgcggtg
     3481 gacaggatcg gcggttttct tttctcttct caattcttct gacctgtaac gaataataga
     3541 tagtaaagta gtctccgatt gagttttctc tgccgagtcc cacccagttc tgtgatttca
     3601 gtaagttggt aattgataca ctgttgcgag aactgctgcc tggtagtaga taggttgtta
     3661 ttgagtaaga aggtaaagtg aacgaaatcc ctgaaactga gactgtagaa aataagcttc
     3721 agcctgcggt ccggttgacg gctagctcag tcctaggtac agtgctagct cgctgggacg
     3781 cccgagatag ccgttacaca ggtgacactt atttcagcct gcggtccggc caagtactta
     3841 gtacacactg attcgctggg acgcccggga tccaagagat ttctacacga ttgagcactg
     3901 tctcagcctg cggtccgggt tcactgccgt ataggcagta attttgttta actttaagaa
     3961 ggagatatac atatgcgtaa aggcgaagaa ctgtttaccg gtgtggttcc gattctggtg
     4021 gaactggacg gcgatgttaa tggtcataaa ttcagtgttc gcggcgaagg tgaaggcgat
     4081 gcgacgaacg gcaaactgac cctgaaattt atctgcacca cgggtaaact gccggtcccg
     4141 tggccgacgc tggtgaccac gctgacctat ggcgttcaat gttttgcgcg ttacccggat
     4201 cacatgaaac agcacgactt tttcaaatcg gccatgccgg aaggctatgt gcaggaacgt
     4261 acgattagct ttaaagacga tggtacgtat aaaacccgcg cggaagtgaa attcgaaggc
     4321 gataccctgg ttaaccgtat cgaactgaaa ggtatcgatt tcaaagaaga cggcaatatt
     4381 ctgggtcata aactggaata taacttcaat tcccacaacg tgtacatcac cgcggataaa
     4441 cagaaaaacg gcattaaagc caatttcaaa atccgccata atgtggaaga tggtagcgtt
     4501 cagctggccg accactatca gcaaaacacg ccgattggtg atggcccggt cctgctgccg
     4561 gacaatcact acctgagtac ccagtccgtg ctgtcaaaag atccgaacga aaaacgtgac
     4621 cacatggtcc tgctggaatt tgtgacggct gcgggtatca cccacggcat ggacgaactg
     4681 tataaataat aatcgctggg acgcccgcct gcaggctcgg ta
//
