LOCUS       sequence-468580-        3823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Message phagemid carrying sgRNA4 (prom. J23110, backbone pBR322).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3823)
  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 3823)
  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: pHK302.4
FEATURES             Location/Qualifiers
     source          1..3823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      532..1120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1021..1040
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1274..1291
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1306..1445
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1371..1390)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1531..1553
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1547..1738)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2252..2273)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2464..2483
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2696..2718
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2710..2727
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2711..2727
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2728..2762
                     /label=BBa_J23110
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.33"
     protein_bind    2778..2797
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    2904..2923
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     complement(3027..3045)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3041..3088
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3133..3151)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3219..3323
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3324..3392,3393..3823,1..361)
                     /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     complement(3542..3561)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
       61 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
      121 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
      181 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
      241 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
      301 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
      361 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
      421 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
      481 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
      541 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
      601 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
      661 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
      721 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
      781 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      841 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      901 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
      961 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1021 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     1081 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     1141 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     1201 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     1261 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     1321 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     1381 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     1441 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     1501 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     1561 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     1621 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     1681 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     1741 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     1801 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     1861 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     1921 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     1981 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     2041 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     2101 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     2161 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc
     2221 tgcccgagat ctcgatcccg cgaaatttgt tccagtttgg aacaagagtc cactattaaa
     2281 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     2341 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     2401 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     2461 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     2521 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca
     2581 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     2641 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     2701 tcacgacgtt gtaaaacgac ggccagtttt acggctagct cagtcctagg tacaatgcta
     2761 gccagcctgc ggtccgggtt cactgccgta taggcaggac acatcttaga gtatgtagtt
     2821 tcagagctat gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg
     2881 aaaaagtggc accgagtcgg tgcgttcact gccgtatagg cagtcgctgg gacgcccgat
     2941 tctgcagata tccatcacac tggcggccgc tcgagcagat ccggctgcta acaaagcccg
     3001 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     3061 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gataattctt
     3121 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3181 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3241 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3301 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3361 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3421 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3481 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3541 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     3601 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3661 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3721 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3781 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gct
//
