LOCUS       sequence-469670-        3826 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Message phagemid carrying sgRNA2 (prom. J23119, backbone pBR322).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3826)
  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 3826)
  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: pHK002.2
FEATURES             Location/Qualifiers
     source          1..3826
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(617..1408,1409..1477))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1240..1259
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1478..1582)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1650..1668
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(1713..1760)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1756..1774
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     protein_bind    complement(1878..1897)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    complement(2004..2023)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        complement(2039..2073)
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     primer_bind     complement(2074..2091)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2074..2090)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2083..2105)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2318..2337)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2528..2549
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             3063..3254
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3248..3270)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3356..3498
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3411..3430
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3513..3530)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(3684..3826,1..446))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3764..3783)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
       61 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
      121 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
      181 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
      241 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
      301 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
      361 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
      421 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
      481 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
      541 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
      601 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
      661 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
      721 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
      781 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
      841 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
      901 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
      961 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     1021 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     1081 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     1141 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     1201 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     1261 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     1321 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     1381 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     1441 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     1501 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     1561 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     1621 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     1681 aagaattatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
     1741 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
     1801 cgggctttgt tagcagccgg atctgctcga gcggccgcca gtgtgatgga tatctgcaga
     1861 atcgggcgtc ccagcgactg cctatacggc agtgaacgca ccgactcggt gccacttttt
     1921 caagttgata acggactagc cttatttcaa cttgctatgc tgtttccagc atagctctga
     1981 aacagtactt agtacacact gatctgccta tacggcagtg aacccggacc gcaggctggc
     2041 tagcattata cctaggactg agctagctgt caaactggcc gtcgttttac aacgtcgtga
     2101 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     2161 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     2221 tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2281 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2341 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2401 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2461 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2521 tttaatagtg gactcttgtt ccaaactgga acaaatttcg cgggatcgag atctcgggca
     2581 gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag
     2641 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
     2701 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
     2761 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
     2821 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
     2881 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
     2941 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
     3001 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
     3061 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
     3121 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
     3181 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
     3241 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     3301 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     3361 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     3421 gcagattgta ctgagagtgc accatatatg cggtgtgaaa taccgcacag atgcgtaagg
     3481 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3541 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3601 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3661 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3721 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3781 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgc
//
