LOCUS       sequence-469662-        4699 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 4699)
  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 4699)
  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..4699
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        372..406
                     /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    556..575
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             580..602
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             610..1323
                     /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     1639..1656
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1640..1656
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1669..1703
                     /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             1708..1730
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1738..2448
                     /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(1883..1901)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2151..2170
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2609..2627)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2623..2670
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2914..2941
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3129..3944)
                     /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(3129..3944)
                     /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     3852..3871
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3945..4036)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4054..4689)
                     /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(4199..4218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattcatag gatagattct ggaaacttta ccgtccgagc tcaggcttac cttaccatgg
       61 ttcagccaaa aaacttaaga ccgccggtct tgtccactac cttgcagtaa tgcggtggac
      121 aggatcggcg gttttctttt ctcttctcaa ttcttctgac ctgtaacgaa taatagatag
      181 taaagtagtc tccgattgag ttttctctgc cgagtcccac ccagttctgt gatttcagta
      241 agttggtaat tgatacactg ttgcgagaac tgctgcctgg tagtagatag gttgttattg
      301 agtaagaagg taaagtgaac gaaatccctg aaactgagac tgtagaaaat aagcttcagc
      361 ctgcggtccg gttgacggct agctcagtcc taggtacagt gctagctcgc tgggacgccc
      421 gagatagccg ttacacaggt gacacttatt tcagcctgcg gtccggccaa gtacttagta
      481 cacactgatt cgctgggacg cccgggatcc aagagatttc tacacgattg agcactgtct
      541 cagcctgcgg tccgggttca ctgccgtata ggcagtaatt ttgtttaact ttaagaagga
      601 gatatacata tgcgtaaagg cgaagaactg tttaccggtg tggttccgat tctggtggaa
      661 ctggacggcg atgttaatgg tcataaattc agtgttcgcg gcgaaggtga aggcgatgcg
      721 acgaacggca aactgaccct gaaatttatc tgcaccacgg gtaaactgcc ggtcccgtgg
      781 ccgacgctgg tgaccacgct gacctatggc gttcaatgtt ttgcgcgtta cccggatcac
      841 atgaaacagc acgacttttt caaatcggcc atgccggaag gctatgtgca ggaacgtacg
      901 attagcttta aagacgatgg tacgtataaa acccgcgcgg aagtgaaatt cgaaggcgat
      961 accctggtta accgtatcga actgaaaggt atcgatttca aagaagacgg caatattctg
     1021 ggtcataaac tggaatataa cttcaattcc cacaacgtgt acatcaccgc ggataaacag
     1081 aaaaacggca ttaaagccaa tttcaaaatc cgccataatg tggaagatgg tagcgttcag
     1141 ctggccgacc actatcagca aaacacgccg attggtgatg gcccggtcct gctgccggac
     1201 aatcactacc tgagtaccca gtccgtgctg tcaaaagatc cgaacgaaaa acgtgaccac
     1261 atggtcctgc tggaatttgt gacggctgcg ggtatcaccc acggcatgga cgaactgtat
     1321 aaataataat cgctgggacg cccgcctgca ggctcggtac caaattccag aaaagaggcc
     1381 tcccgaaagg ggggcctttt ttcgttttgg tcctaataga taaaggatag gtctggtagt
     1441 gttgttcgtt ctcgcaggta aatcaataat actcagcagt tccgtagact tttcagtggg
     1501 acagggtagc gataacagat agattgtaat aagacacagt aggtgctcgt agttgcgtga
     1561 agagaaccgc tcaggaaatc cagtcagaag tattggtaat cgttgaaaac tcagctccta
     1621 ggatgcagtg actcgagttg taaaacgacg gccagtcaat tggaattctt gacggctagc
     1681 tcagtcctag gtacagtgct agctaatttt gtttaacttt aagaaggaga tatacatatg
     1741 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     1801 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1861 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     1921 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     1981 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     2041 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctctct gcaggacggc
     2101 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     2161 cagaagaaga ctatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcg
     2221 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgaa
     2281 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgcgta caacgtcaac
     2341 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     2401 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaaac tagttgcggc
     2461 cgcctgcgca ccacctgcag gacctcagca atcgcttgga ccagctttct taagggtacc
     2521 tattacagct ggatccggcg ccgctcattc gctaatcgcc acgacgcgta gtcgacccgc
     2581 caattcccat ggggcgcgcc tgctgccacc gctgagcaat aactagcata accccttggg
     2641 gcctctaaac gggtcttgag gggttttttg ccaggcatca aataaaacga aaggctcagt
     2701 cggaagactg gggcgaacga cgagtcactg ttgaggataa atactttctc tactaggcgc
     2761 ctgttacaca ggtcctcagc ggcgcgcctt tgtcggtgaa cgctctcctg agtaggacaa
     2821 atccgccggg agcggatttg aacgttgtga agcaacggcc cggagggtgg cgggcaggac
     2881 gcccgccata aactgccagg catcaaacta agcagaaggc catcctgacg gatggccttt
     2941 ttgcgtttca gatctaccgg taaaccagca atagacataa gcggctattt aacgaccctg
     3001 ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt cggggaaatg
     3061 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3121 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     3181 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     3241 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     3301 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     3361 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     3421 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     3481 cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca attacaaaca
     3541 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     3601 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     3661 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     3721 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     3781 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     3841 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     3901 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt cctttttcaa
     3961 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4021 tagaaaaata aacaaatagg catgctagcg cagaaacgtc ctagaagatg ccaggaggat
     4081 acttagcaga gagacaataa ggccggagcg aagccgtttt tccataggct ccgcccccct
     4141 gacgaacatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa
     4201 agataccagg cgtttccccc tgatggctcc ctcttgcgct ctcctgttcc cgtcctgcgg
     4261 cgtccgtgtt gtggtggagg ctttacccaa atcaccacgt cccgttccgt gtagacagtt
     4321 cgctccaagc tgggctgtgt gcaagaaccc cccgttcagc ccgactgctg cgccttatcc
     4381 ggtaactatc atcttgagtc caacccggaa agacacgaca aaacgccact ggcagcagcc
     4441 attggtaact gagaattagt ggatttagat atcgagagtc ttgaagtggt ggcctaacag
     4501 aggctacact gaaaggacag tatttggtat ctgcgctcca ctaaagccag ttaccaggtt
     4561 aagcagttcc ccaactgact taaccttcga tcaaaccgcc tccccaggcg gttttttcgt
     4621 ttacagagca ggagattacg acgatcgtaa aaggatctca agaagatcct ttacggattc
     4681 ccgacaccat cactctaga
//
