LOCUS       sequence-473112-        4894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NOR gate receiver with bs for sgRNA2 and sgRNA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4894)
  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 4894)
  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: pHK009
FEATURES             Location/Qualifiers
     source          1..4894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(62..80)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      76..123
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      354..381
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        503..607
                     /gene="bla"
                     /label=AmpR promoter
     CDS             608..1423
                     /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             608..1423
                     /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     complement(681..700)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1494..2129)
                     /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(1639..1658)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2547..2581
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     promoter        2642..2676
                     /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    2903..2922
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2927..2949
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2957..3670
                     /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     3986..4003
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3987..4003
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4016..4050
                     /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             4055..4077
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4085..4795
                     /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(4230..4248)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4498..4517
                     /label=mCherry-F
                     /note="mCherry, forward primer"
ORIGIN
        1 ttcgctaatc gccacgacgc gtagtcgacc cgccaattcc catggggcgc gcctgctgcc
       61 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      121 ttgccaggca tcaaataaaa cgaaaggctc agtcggaaga ctgggcactg ttgaggataa
      181 atactttctc tactaggcgc ctgttacaca ggtcctcagc ggcgcgcctt tgtcggtgaa
      241 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgtga agcaacggcc
      301 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaacta agcagaaggc
      361 catcctgacg gatggccttt ttgcgtttca gatctaccgg taaaccagca atagacataa
      421 gcggctattt aacgaccctg ccctgaaccg acgacaagct gacgaccggg tctccgcaag
      481 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
      541 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
      601 gaagagtatg agccatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa
      661 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc
      721 gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa
      781 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt
      841 tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac
      901 cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga
      961 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa
     1021 ttgtcctttt aacagcgacc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa
     1081 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt
     1141 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga
     1201 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg
     1261 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga
     1321 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat
     1381 gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg
     1441 gatacatatt tgaatgtatt tagaaaaata aacaaatagg catgctagcg cagaaacgtc
     1501 ctagaagatg ccaggaggat acttagcaga gagacaataa ggccggagcg aagccgtttt
     1561 tccataggct ccgcccccct gacgaacatc acgaaatctg acgctcaaat cagtggtggc
     1621 gaaacccgac aggactataa agataccagg cgtttccccc tgatggctcc ctcttgcgct
     1681 ctcctgttcc cgtcctgcgg cgtccgtgtt gtggtggagg ctttacccaa atcaccacgt
     1741 cccgttccgt gtagacagtt cgctccaagc tgggctgtgt gcaagaaccc cccgttcagc
     1801 ccgactgctg cgccttatcc ggtaactatc atcttgagtc caacccggaa agacacgaca
     1861 aaacgccact ggcagcagcc attggtaact gagaattagt ggatttagat atcgagagtc
     1921 ttgaagtggt ggcctaacag aggctacact gaaaggacag tatttggtat ctgcgctcca
     1981 ctaaagccag ttaccaggtt aagcagttcc ccaactgact taaccttcga tcaaaccgcc
     2041 tccccaggcg gttttttcgt ttacagagca ggagattacg acgatcgtaa aaggatctca
     2101 agaagatcct ttacggattc ccgacaccat cactctagag aattcatagg atagattctg
     2161 gaaactttac cgtccgagct caggcttacc ttactcgagc aataaacagt tgatagggct
     2221 tctccgttac catggttcag ccaaaaaact taagaccgcc ggtcttgtcc actaccttgc
     2281 agtaatgcgg tggacaggat cggcggtttt cttttctctt ctcaattctt ctgacctgta
     2341 acgaataata gatagtaaag tagtctccga ttgagttttc tctgccgagt cccacccagt
     2401 tctgtgattt cagtaagttg gtaattgata cactgttgcg agaactgctg cctggtagta
     2461 gataggttgt tattgagtaa gaaggtaaag tgaacgaaat ccctgaaact gagactgtag
     2521 aaaataagct tcagcctgcg gtccggttga cggctagctc agtcctaggt acagtgctag
     2581 ctcgctggga cgcccgagat agccgttaca caggtgacac ttatttcagc ctgcggtccg
     2641 gttgacggct agctcagtcc taggtacagt gctagctcgc tgggacgccc gagatagccg
     2701 ttacacaggt gacacttatt tcagcctgcg gtccggccaa gtacttagta cacactgatt
     2761 cgctgggacg cccgtagaac tctgtgagga taaagtctcc cttacagcct gcggtccggc
     2821 caagagtaca ctagctcagt catcgctggg acgcccgaag agatttctac acgattgagc
     2881 actgtctcag cctgcggtcc gggttcactg ccgtataggc agtaattttg tttaacttta
     2941 agaaggagat atacatatgc gtaaaggcga agaactgttt accggtgtgg ttccgattct
     3001 ggtggaactg gacggcgatg ttaatggtca taaattcagt gttcgcggcg aaggtgaagg
     3061 cgatgcgacg aacggcaaac tgaccctgaa atttatctgc accacgggta aactgccggt
     3121 cccgtggccg acgctggtga ccacgctgac ctatggcgtt caatgttttg cgcgttaccc
     3181 ggatcacatg aaacagcacg actttttcaa atcggccatg ccggaaggct atgtgcagga
     3241 acgtacgatt agctttaaag acgatggtac gtataaaacc cgcgcggaag tgaaattcga
     3301 aggcgatacc ctggttaacc gtatcgaact gaaaggtatc gatttcaaag aagacggcaa
     3361 tattctgggt cataaactgg aatataactt caattcccac aacgtgtaca tcaccgcgga
     3421 taaacagaaa aacggcatta aagccaattt caaaatccgc cataatgtgg aagatggtag
     3481 cgttcagctg gccgaccact atcagcaaaa cacgccgatt ggtgatggcc cggtcctgct
     3541 gccggacaat cactacctga gtacccagtc cgtgctgtca aaagatccga acgaaaaacg
     3601 tgaccacatg gtcctgctgg aatttgtgac ggctgcgggt atcacccacg gcatggacga
     3661 actgtataaa taataatcgc tgggacgccc gcctgcaggc tcggtaccaa attccagaaa
     3721 agaggcctcc cgaaaggggg gccttttttc gttttggtcc taatagataa aggataggtc
     3781 tggtagtgtt gttcgttctc gcaggtaaat caataatact cagcagttcc gtagactttt
     3841 cagtgggaca gggtagcgat aacagataga ttgtaataag acacagtagg tgctcgtagt
     3901 tgcgtgaaga gaaccgctca ggaaatccag tcagaagtat tggtaatcgt tgaaaactca
     3961 gctcctagga tgcagtgact cgagttgtaa aacgacggcc agtcaattgg aattcttgac
     4021 ggctagctca gtcctaggta cagtgctagc taattttgtt taactttaag aaggagatat
     4081 acatatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     4141 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     4201 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     4261 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     4321 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     4381 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctctctgca
     4441 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     4501 cgtaatgcag aagaagacta tgggctggga ggcctcctcc gagcggatgt accccgagga
     4561 cggcgcgctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     4621 cgctgaagtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcgtacaa
     4681 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     4741 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaaactag
     4801 ttgcggccgc ctgcgcacca cctgcaggac ctcagcaatc gcttggacca gctttcttaa
     4861 gggtacctat tacagctgga tccggcgccg ctca
//
