LOCUS       sequence-469680-        3828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Message phagemid carrying sgRNA2 (prom. J23110, backbone pBR322).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3828)
  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 3828)
  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: pHK302.2
FEATURES             Location/Qualifiers
     source          1..3828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             268..459
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(453..475)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    561..703
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     616..635
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(718..735)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(889..1477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(969..988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1648..2439,2440..2508))
                     /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     2271..2290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2509..2613)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2681..2699
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2744..2791)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2787..2805
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     protein_bind    complement(2911..2930)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    complement(3037..3056)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        complement(3072..3106)
                     /label=BBa_J23110
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.33"
     primer_bind     complement(3107..3124)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3107..3123)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3116..3138)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3351..3370)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3561..3582
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
       61 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
      121 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
      181 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
      241 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
      301 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
      361 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc
      421 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
      481 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
      541 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
      601 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atatgcggtg
      661 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
      721 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
      781 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
      841 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
      901 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
      961 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1021 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1081 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1141 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1201 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1261 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     1321 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     1381 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     1441 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     1501 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     1561 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     1621 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     1681 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     1741 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     1801 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     1861 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     1921 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc
     1981 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2041 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     2101 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     2161 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     2221 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc
     2281 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     2341 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     2401 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     2461 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     2521 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     2581 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     2641 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     2701 ctttcgtctt caagaattat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga
     2761 cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact
     2821 cagcttcctt tcgggctttg ttagcagccg gatctgctcg agcggccgcc agtgtgatgg
     2881 atatctgcag aatcgggcgt cccagcgagc ctgcctatac ggcagtgaac gcaccgactc
     2941 ggtgccactt tttcaagttg ataacggact agccttattt caacttgcta tgctgtttcc
     3001 agcatagctc tgaaacagta cttagtacac actgatctgc ctatacggca gtgaacccgg
     3061 accgcaggct ggctagcatt gtacctagga ctgagctagc cgtaaaactg gccgtcgttt
     3121 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     3181 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     3241 tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3301 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3361 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3421 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3481 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3541 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaaatt tcgcgggatc
     3601 gagatctcgg gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct cctgtcgttg
     3661 aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc
     3721 gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg
     3781 gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcg
//
