LOCUS       sequence-473114-        5010 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BUF/YES gate receiver with bs for sgRNA2.
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: pHK506.4
FEATURES             Location/Qualifiers
     source          1..5010
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        37..71
                     /label=BBa_J23115
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.15"
     protein_bind    206..225
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    332..351
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        696..730
                     /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    880..899
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             904..926
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             934..1647
                     /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     1963..1980
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1964..1980
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1993..2027
                     /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             2032..2054
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2062..2772
                     /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(2207..2225)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2475..2494
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2933..2951)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2947..2994
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3225..3252
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3440..4255)
                     /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(3440..4255)
                     /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     4163..4182
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4256..4347)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4365..5000)
                     /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(4510..4529)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattcatag gatagattct ggaaacttta ccgtccttta tagctagctc agcccttggt
       61 acaatgctag cagatagccg ttacacaggt gacacttatt tcagcctgcg gtccggccaa
      121 gtacttagta cacactgatt cgctgggacg cccgggatcc aagagatttc tacacgattg
      181 agcactgtct cagcctgcgg tccgggttca ctgccgtata ggcaggacac atcttagagt
      241 atgtagtttc agagctatgc tggaaacagc atagcaagtt gaaataaggc tagtccgtta
      301 tcaacttgaa aaagtggcac cgagtcggtg cgttcactgc cgtataggca gtcgctggga
      361 cgcccgaggc ttaccttacc atggttcagc caaaaaactt aagaccgccg gtcttgtcca
      421 ctaccttgca gtaatgcggt ggacaggatc ggcggttttc ttttctcttc tcaattcttc
      481 tgacctgtaa cgaataatag atagtaaagt agtctccgat tgagttttct ctgccgagtc
      541 ccacccagtt ctgtgatttc agtaagttgg taattgatac actgttgcga gaactgctgc
      601 ctggtagtag ataggttgtt attgagtaag aaggtaaagt gaacgaaatc cctgaaactg
      661 agactgtaga aaataagctt cagcctgcgg tccggttgac ggctagctca gtcctaggta
      721 cagtgctagc tcgctgggac gcccgagata gccgttacac aggtgacact tatttcagcc
      781 tgcggtccgg ccatacatac tctaagatgt gtctcgctgg gacgcccggg atccaagaga
      841 tttctacacg attgagcact gtctcagcct gcggtccggg ttcactgccg tataggcagt
      901 aattttgttt aactttaaga aggagatata catatgcgta aaggcgaaga actgtttacc
      961 ggtgtggttc cgattctggt ggaactggac ggcgatgtta atggtcataa attcagtgtt
     1021 cgcggcgaag gtgaaggcga tgcgacgaac ggcaaactga ccctgaaatt tatctgcacc
     1081 acgggtaaac tgccggtccc gtggccgacg ctggtgacca cgctgaccta tggcgttcaa
     1141 tgttttgcgc gttacccgga tcacatgaaa cagcacgact ttttcaaatc ggccatgccg
     1201 gaaggctatg tgcaggaacg tacgattagc tttaaagacg atggtacgta taaaacccgc
     1261 gcggaagtga aattcgaagg cgataccctg gttaaccgta tcgaactgaa aggtatcgat
     1321 ttcaaagaag acggcaatat tctgggtcat aaactggaat ataacttcaa ttcccacaac
     1381 gtgtacatca ccgcggataa acagaaaaac ggcattaaag ccaatttcaa aatccgccat
     1441 aatgtggaag atggtagcgt tcagctggcc gaccactatc agcaaaacac gccgattggt
     1501 gatggcccgg tcctgctgcc ggacaatcac tacctgagta cccagtccgt gctgtcaaaa
     1561 gatccgaacg aaaaacgtga ccacatggtc ctgctggaat ttgtgacggc tgcgggtatc
     1621 acccacggca tggacgaact gtataaataa taatcgctgg gacgcccgcc tgcaggctcg
     1681 gtaccaaatt ccagaaaaga ggcctcccga aaggggggcc ttttttcgtt ttggtcctaa
     1741 tagataaagg ataggtctgg tagtgttgtt cgttctcgca ggtaaatcaa taatactcag
     1801 cagttccgta gacttttcag tgggacaggg tagcgataac agatagattg taataagaca
     1861 cagtaggtgc tcgtagttgc gtgaagagaa ccgctcagga aatccagtca gaagtattgg
     1921 taatcgttga aaactcagct cctaggatgc agtgactcga gttgtaaaac gacggccagt
     1981 caattggaat tcttgacggc tagctcagtc ctaggtacag tgctagctaa ttttgtttaa
     2041 ctttaagaag gagatataca tatggtgagc aagggcgagg aggataacat ggccatcatc
     2101 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     2161 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     2221 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     2281 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
     2341 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     2401 caggactcct ctctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
     2461 ttcccctccg acggccccgt aatgcagaag aagactatgg gctgggaggc ctcctccgag
     2521 cggatgtacc ccgaggacgg cgcgctgaag ggcgagatca agcagaggct gaagctgaag
     2581 gacggcggcc actacgacgc tgaagtcaag accacctaca aggccaagaa gcccgtgcag
     2641 ctgcccggcg cgtacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
     2701 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
     2761 ctgtacaagt aaactagttg cggccgcctg cgcaccacct gcaggacctc agcaatcgct
     2821 tggaccagct ttcttaaggg tacctattac agctggatcc ggcgccgctc attcgctaat
     2881 cgccacgacg cgtagtcgac ccgccaattc ccatggggcg cgcctgctgc caccgctgag
     2941 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgccaggc
     3001 atcaaataaa acgaaaggct cagtcggaag actgggcact gttgaggata aatactttct
     3061 ctactaggcg cctgttacac aggtcctcag cggcgcgcct ttgtcggtga acgctctcct
     3121 gagtaggaca aatccgccgg gagcggattt gaacgttgtg aagcaacggc ccggagggtg
     3181 gcgggcagga cgcccgccat aaactgccag gcatcaaact aagcagaagg ccatcctgac
     3241 ggatggcctt tttgcgtttc agatctaccg gtaaaccagc aatagacata agcggctatt
     3301 taacgaccct gccctgaacc gacgacaagc tgacgaccgg gtctccgcaa gtggcacttt
     3361 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3421 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     3481 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     3541 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     3601 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     3661 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     3721 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     3781 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcggtcgctg ttaaaaggac
     3841 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     3901 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     3961 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     4021 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     4081 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     4141 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     4201 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcatactct
     4261 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4321 ttgaatgtat ttagaaaaat aaacaaatag gcatgctagc gcagaaacgt cctagaagat
     4381 gccaggagga tacttagcag agagacaata aggccggagc gaagccgttt ttccataggc
     4441 tccgcccccc tgacgaacat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga
     4501 caggactata aagataccag gcgtttcccc ctgatggctc cctcttgcgc tctcctgttc
     4561 ccgtcctgcg gcgtccgtgt tgtggtggag gctttaccca aatcaccacg tcccgttccg
     4621 tgtagacagt tcgctccaag ctgggctgtg tgcaagaacc ccccgttcag cccgactgct
     4681 gcgccttatc cggtaactat catcttgagt ccaacccgga aagacacgac aaaacgccac
     4741 tggcagcagc cattggtaac tgagaattag tggatttaga tatcgagagt cttgaagtgg
     4801 tggcctaaca gaggctacac tgaaaggaca gtatttggta tctgcgctcc actaaagcca
     4861 gttaccaggt taagcagttc cccaactgac ttaaccttcg atcaaaccgc ctccccaggc
     4921 ggttttttcg tttacagagc aggagattac gacgatcgta aaaggatctc aagaagatcc
     4981 tttacggatt cccgacacca tcactctaga
//
