LOCUS       sequence-473103-        5010 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BUF/YES gate receiver with bs for sgRNA3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5010)
  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 5010)
  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: pHK506.5
FEATURES             Location/Qualifiers
     source          1..5010
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(66..776)
                     /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(344..363)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     613..631
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     RBS             784..806
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        complement(811..845)
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     primer_bind     complement(858..875)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(858..874)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1191..1904)
                     /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"
     RBS             1912..1934
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1939..1958)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        complement(2108..2142)
                     /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    complement(2487..2506)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    complement(2613..2632)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        complement(2767..2801)
                     /label=BBa_J23115
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.15"
     rep_origin      2848..3483
                     /direction=RIGHT
                     /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     3319..3338
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3501..3592
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3593..4408
                     /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             3593..4408
                     /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(3666..3685)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(4596..4623)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4854..4901)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4897..4915
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 ttaagaaagc tggtccaagc gattgctgag gtcctgcagg tggtgcgcag gcggccgcaa
       61 ctagtttact tgtacagctc gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg
      121 tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt gatgttgacg
      181 ttgtacgcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacttcagcg
      241 tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagcgcgccg
      301 tcctcggggt acatccgctc ggaggaggcc tcccagccca tagtcttctt ctgcattacg
      361 gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc
      421 tgcagagagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc
      481 tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc
      541 ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc
      601 agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc
      661 tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg
      721 aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct caccatatgt
      781 atatctcctt cttaaagtta aacaaaatta gctagcactg tacctaggac tgagctagcc
      841 gtcaagaatt ccaattgact ggccgtcgtt ttacaactcg agtcactgca tcctaggagc
      901 tgagttttca acgattacca atacttctga ctggatttcc tgagcggttc tcttcacgca
      961 actacgagca cctactgtgt cttattacaa tctatctgtt atcgctaccc tgtcccactg
     1021 aaaagtctac ggaactgctg agtattattg atttacctgc gagaacgaac aacactacca
     1081 gacctatcct ttatctatta ggaccaaaac gaaaaaaggc ccccctttcg ggaggcctct
     1141 tttctggaat ttggtaccga gcctgcaggc gggcgtccca gcgattatta tttatacagt
     1201 tcgtccatgc cgtgggtgat acccgcagcc gtcacaaatt ccagcaggac catgtggtca
     1261 cgtttttcgt tcggatcttt tgacagcacg gactgggtac tcaggtagtg attgtccggc
     1321 agcaggaccg ggccatcacc aatcggcgtg ttttgctgat agtggtcggc cagctgaacg
     1381 ctaccatctt ccacattatg gcggattttg aaattggctt taatgccgtt tttctgttta
     1441 tccgcggtga tgtacacgtt gtgggaattg aagttatatt ccagtttatg acccagaata
     1501 ttgccgtctt ctttgaaatc gatacctttc agttcgatac ggttaaccag ggtatcgcct
     1561 tcgaatttca cttccgcgcg ggttttatac gtaccatcgt ctttaaagct aatcgtacgt
     1621 tcctgcacat agccttccgg catggccgat ttgaaaaagt cgtgctgttt catgtgatcc
     1681 gggtaacgcg caaaacattg aacgccatag gtcagcgtgg tcaccagcgt cggccacggg
     1741 accggcagtt tacccgtggt gcagataaat ttcagggtca gtttgccgtt cgtcgcatcg
     1801 ccttcacctt cgccgcgaac actgaattta tgaccattaa catcgccgtc cagttccacc
     1861 agaatcggaa ccacaccggt aaacagttct tcgcctttac gcatatgtat atctccttct
     1921 taaagttaaa caaaattact gcctatacgg cagtgaaccc ggaccgcagg ctgagacagt
     1981 gctcaatcgt gtagaaatct cttggatccc gggcgtccca gcgaaagtga gtctgagctt
     2041 agattggccg gaccgcaggc tgaaataagt gtcacctgtg taacggctat ctcgggcgtc
     2101 ccagcgagct agcactgtac ctaggactga gctagccgtc aaccggaccg caggctgaag
     2161 cttattttct acagtctcag tttcagggat ttcgttcact ttaccttctt actcaataac
     2221 aacctatcta ctaccaggca gcagttctcg caacagtgta tcaattacca acttactgaa
     2281 atcacagaac tgggtgggac tcggcagaga aaactcaatc ggagactact ttactatcta
     2341 ttattcgtta caggtcagaa gaattgagaa gagaaaagaa aaccgccgat cctgtccacc
     2401 gcattactgc aaggtagtgg acaagaccgg cggtcttaag ttttttggct gaaccatggt
     2461 aaggtaagcc tcgggcgtcc cagcgactgc ctatacggca gtgaacgcac cgactcggtg
     2521 ccactttttc aagttgataa cggactagcc ttatttcaac ttgctatgct gtttccagca
     2581 tagctctgaa acatctaagc tcagactcac ttctgcctat acggcagtga acccggaccg
     2641 caggctgaga cagtgctcaa tcgtgtagaa atctcttgga tcccgggcgt cccagcgatg
     2701 actgagctag tgtactcttg gccggaccgc aggctgaaat aagtgtcacc tgtgtaacgg
     2761 ctatctgcta gcattgtacc aagggctgag ctagctataa aggacggtaa agtttccaga
     2821 atctatccta tgaattctct agagtgatgg tgtcgggaat ccgtaaagga tcttcttgag
     2881 atccttttac gatcgtcgta atctcctgct ctgtaaacga aaaaaccgcc tggggaggcg
     2941 gtttgatcga aggttaagtc agttggggaa ctgcttaacc tggtaactgg ctttagtgga
     3001 gcgcagatac caaatactgt cctttcagtg tagcctctgt taggccacca cttcaagact
     3061 ctcgatatct aaatccacta attctcagtt accaatggct gctgccagtg gcgttttgtc
     3121 gtgtctttcc gggttggact caagatgata gttaccggat aaggcgcagc agtcgggctg
     3181 aacggggggt tcttgcacac agcccagctt ggagcgaact gtctacacgg aacgggacgt
     3241 ggtgatttgg gtaaagcctc caccacaaca cggacgccgc aggacgggaa caggagagcg
     3301 caagagggag ccatcagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3361 ccactgattt gagcgtcaga tttcgtgatg ttcgtcaggg gggcggagcc tatggaaaaa
     3421 cggcttcgct ccggccttat tgtctctctg ctaagtatcc tcctggcatc ttctaggacg
     3481 tttctgcgct agcatgccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3541 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagcca
     3601 tattcaacgg gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt
     3661 atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt
     3721 gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa
     3781 tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac
     3841 catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg
     3901 gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc
     3961 gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag
     4021 cgaccgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc
     4081 gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca
     4141 taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa
     4201 ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc
     4261 agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt
     4321 acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt
     4381 tcatttgatg ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat
     4441 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacttg
     4501 cggagacccg gtcgtcagct tgtcgtcggt tcagggcagg gtcgttaaat agccgcttat
     4561 gtctattgct ggtttaccgg tagatctgaa acgcaaaaag gccatccgtc aggatggcct
     4621 tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc
     4681 gttgcttcac aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca
     4741 ccgacaaagg cgcgccgcta aggacctgtg taacaggcgc ctagtagaga aagtatttat
     4801 cctcaacagt gcccagtctt ccgactgagc ctttcgtttt atttgatgcc tggcaaaaaa
     4861 cccctcaaga cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca
     4921 gcaggcgcgc cccatgggaa ttggcgggtc gactacgcgt cgtggcgatt agcgaatgag
     4981 cggcgccgga tccagctgta ataggtaccc
//
