LOCUS       sequence-483567-        7522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  inverted BRR1 origin of replication plasmid containing Kva1 
            recombitron with a donor in the ncRNA to target phzM locus in 
            Pseudomonas aeruginosa PAO1 expressed by Pm promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7522)
  AUTHORS   Gonzalez-Delgado A, Bonillo-Lopez L, Johnson MS, Knodlseder N, Ko 
            CC, Lekbach Y, Oh JH, Selvakumar H, Wold MC, Yu Z, Aragon V, 
            Gralnick JA, Guell M, Hatfull GF, Keitz BK, Koskella B, Mutalik VK, 
            van Pijkeren JP, Shipman SL
  TITLE     Genome editing of phylogenetically distinct bacteria using portable 
            retron-mediated recombineering
  JOURNAL   bioRxiv 2025.06.16.660010
REFERENCE   2  (bases 1 to 7522)
  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: "bioRxiv 
            2025.06.16.660010"
COMMENT     Sequence Label: pRECBRR1-Kva1
FEATURES             Location/Qualifiers
     source          1..7522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1100..1108
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        complement(2455..2535)
                     /label=Pm promoter
                     /note="The bacterial Pm promoter is activated by XylS in 
                     the presence of benzoate or m-toluate (Marques et al., 
                     1999)."
     CDS             3435..4400
                     /codon_start=1
                     /gene="xylS"
                     /product="XylS regulator encoded by the Pseudomonas putida 
                     TOL plasmid pWWO"
                     /label=XylS
                     /note="stimulates transcription from the Pm promoter in the
                     presence of aromatic compounds such as m-toluate or 
                     benzoate"
                     /translation="MDFCLLNEKSQIFVHAEPYAVSDYVNQYVGTHSIRLPKGGRPAGR
                     LHHRIFGCLDLCRISYGGSVRVISPGLETCYHLQIILKGHCLWRGHGQEHYFAPGELLL
                     LNPDDQADLTYSEDCEKFIVKLPSVVLDRACSDNNWHKPREGIRFAARHNLQQLDGFIN
                     LLGLVCDEAEHTKSMPRVQEHYAGIIASKLLEMLGSNVSREIFSKGNPSFERVVQFIEE
                     NLKRNISLERLAELAMMSPRSLYNLFEKHAGTTPKNYIRNRKLESIRACLNDPSANVRS
                     ITEIALDYGFLHLGRFAENYRSAFGELPSDTLRQCKKEVA"
     oriT            4610..4718
                     /note="incP origin of transfer"
     CDS             complement(4729..5391)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGETLEPPTRVTLPRR"
     rep_origin      5392..6163
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             complement(6380..6913)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7102..7107,7108..7124,7125..7130))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     terminator      complement(7171..7205)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
ORIGIN
        1 gccgttgatg tcgaccacga tctcggtggt gtagcgatcc tgaccgtcct ggccctgcca
       61 cttgcgggtg cgcaggctgc cttcgacgta gacctgggaa cccttgcgca ggtactcgcc
      121 ggcgatctcc gccaggcggc cgaagaacac cacgcggtgc cattcggtgc gctcctgttg
      181 ctggccggtc tgcttgtcct tccagctctc gctggtggcg agggtgatgt tggtcaccgc
      241 attgccgttg ggcatgtagc gggtttccgg gtcaccaccg acgttaccaa ccagaatgac
      301 tttgttaacc ccacgggcca tcatatgttt ttcctcctat tttttgttac gctgctcgat
      361 catcgccggt ctcctcgaat tcaacgtcta taggggcgtc cagcaattct ttcttccgct
      421 ggtccttggc cgccgtaagc tggtcgcgcg cgccctttgt cttataggct ttccaggcat
      481 tgctgaatgc tgcctgcaag tcttccattg ttggggagtc cttgatcagg cagatcgcct
      541 cgctgacgtc ctcgtactgc tctgcgggag tgacgtctcg ttcaacgatc cgctcggcct
      601 cgtcctggtc gtatatgccg gcgaacccga acgcgaggcg tgcgcactgg atcattgcct
      661 tgtggcgaag catccggcgc ggatgggact gccaaggctg ggtgttccgc ttgcactcgg
      721 ccatgtactc agtcgcgctg atggcatggc tgcggtcctt ccgatagatc ttgcaggtgc
      781 attcggttcc ctgctggtcc attgagaatt ccatgccatc gaactgtggg ttctcgttga
      841 tgatccgagc ccagccatcc acaccaacaa ccggcacgat gccgttgttc ttgtcgggga
      901 atgcgtacaa ctccttggtg aaggggttaa gcttgtactg gtctgccacg atcagcaggg
      961 cgaccatctg cgaatcattg acctggccct tgaaacaggt ctgcttgagc gtgttcgcca
     1021 cttcttcagg cgtggtaccc atctcgtagc gcgtggcgaa cttcgtcagg agcggtgtta
     1081 gtgcagttcc catgaaaaaa cctccttaaa ttggcggcta tcagagtgcg gccgcggatc
     1141 atgcaggtgg cagccgctta tgttataaaa aaccccggta aaccggggtt ctgctcttta
     1201 cctagaaggg cacggcctgg gacgggactt ccattccccg gtaacaactt gtatcaacgc
     1261 gcagcagcaa gattcgaatt attcatcttt tcttaggccg tggcataaat gaccacaatc
     1321 aacaagttcc ggtgatacac cagatgaagc agatacgcct ctatcgcgta aagagtatgt
     1381 tatacactaa cttaatgatt tgcatcaatc aagctgcttg tctcaagatt atggatcacc
     1441 tgctctagcc tcatatgaac ggtattagtc attgtttttc tgcaggactg cgaagctatc
     1501 cttacccagt tgtttcagtt tgtcctcacc gttaatttta atcaggctat agtaatagtc
     1561 cggttcgata tttttgatgt gagacagcat gcccaggagg tacggaatgt ccttatgatt
     1621 gattttgtta ttgctgtaca ggtaaaacag tgcgcggatt ttcttacgca gaaaacggcc
     1681 cacacccaga tcgccggtgt tagaaataac cacaccagta atgcgacggc tacgaccttt
     1741 accaataaat ttggttttag aagtgttgat aatcagttct ggtttcttct gcagagacac
     1801 caggttaata acgaagtttt tgacatcgtt cagtttgtca aaagaatcgg aagaaaaaga
     1861 caggtcatca gcgtaacgag tgtagatcac atcgttttct ttacagtagt tgaacagttt
     1921 ttcgtcgaag tcgtacataa cgatgttgga gatcatcgga gagctcggcg caccgatgca
     1981 caggcgcagt ttcttttcac ctttcttgcg acgaaacagg tagtggctaa ttacttcata
     2041 ttcaaattgg ttcagtttaa tatcgttttt gctgataaaa tagttcagat cctgcggttt
     2101 gatgctgtag aaaaaatttt cgaaatccat tttgaggatg tattcgtttt tcttgtgcgg
     2161 ttctgcgtta tcaacaatgg atttttcctc gatatacgcg gtcgcgcact cgtgaaccgg
     2221 caggagagac aggaggctgt taacaatagc acgttgaact ttcttaacgt ccggagacgg
     2281 ctgcgcgatc tcacggaggc ctttaccgtt tttctttttg ataaagaaca ccttgtattt
     2341 cttaggcgcc ttgatgtagc cagagataat agtcttcgga gtgcagttca gagtcttgct
     2401 cagctcatcg atataggaca tatgtatatc tccttcgaat tcggatccta gaggttgcat
     2461 aaagcctaag gggtaggcct tactagagat agccattttt tgcactcctg tatccgcttc
     2521 ttgcaaggct ggacttatcc ctatcaaacc ggacactgca ctttatgctg gttatgctgg
     2581 ctgcatgggg cattagttgg ggagccagat ttgtcatgga gcaggccgtt ctgctttatg
     2641 gatcaggaaa aaactatttg ttcttcagtc atggtactgt tctgatgtac ctgctgtgtg
     2701 ttttcctggt ataccgccgt tggatagctc cgctaccggt cgttggtcag atcaagctaa
     2761 ttcccatgca tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca
     2821 aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct
     2881 ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga
     2941 aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca
     3001 tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt
     3061 gccatacgta attacggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga
     3121 taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagcagatcc
     3181 cctttatccg ccaattcgtc gccccatgcc ccgacagcac gaacttctgg tgttctcgct
     3241 tcttaaaaag aacgtcttcg ttctgcttgg cgttattttt gcttggaaaa gtggtcactg
     3301 attgcaaaaa ggatggcgca acgtggcaat gggggtaacc cgtatacgca tcacgtcgag
     3361 atgcattttc atcgacttgg cgcctttcta catcacacca agcagcccac attaaaataa
     3421 gagaaccgtg aactatggat ttttgcttat tgaacgagaa aagtcagatc ttcgtccacg
     3481 ccgagcccta tgcagtctcc gattatgtta accagtatgt cggtacgcac tctattcgcc
     3541 tgcccaaggg cgggcgcccg gcaggcaggc tgcaccacag aatcttcgga tgcctcgatc
     3601 tgtgtcgaat cagctacggc ggtagcgtga gggtaatctc gcctggatta gagacctgtt
     3661 atcatctgca aataatactc aaaggccatt gcctgtggcg tggccatggc caggagcact
     3721 attttgcgcc gggcgaacta ttgctgctca atccggatga ccaagccgac ctgacctatt
     3781 cagaagattg cgagaaattt atcgttaaat tgccctcagt ggtccttgat cgggcgtgca
     3841 gtgacaacaa ttggcacaag ccgagggagg gtatccgttt cgccgcgcga cacaatctcc
     3901 agcaactcga tggctttatc aatctactcg ggttagtttg tgacgaagcg gaacatacaa
     3961 agtcgatgcc tcgggtccaa gagcactatg cggggatcat cgcttccaag ctgctcgaaa
     4021 tgctgggcag caatgtcagc cgtgaaattt tcagcaaagg taacccgtct ttcgagcgag
     4081 tcgttcaatt cattgaggag aatctcaaac ggaatatcag ccttgagcgg ttagcggagc
     4141 tggcgatgat gagtccacgc tcgctctaca atttgttcga gaagcacgcc ggcaccacgc
     4201 cgaagaacta catccgcaac cgcaagctcg aaagcatccg cgcctgcttg aacgatccca
     4261 gtgccaatgt gcgtagtata actgagatag ccctagacta cggcttctta catttgggac
     4321 gcttcgctga aaactatagg agcgcgttcg gcgagttgcc ttccgacacc ctgcgtcaat
     4381 gcaaaaagga agtggcttga ttacgaacgt ttaattaaag gcatcaaata aaacgaaatg
     4441 ctcagtcgaa agactgggcc tttcgtgttt gtcggtgaac gctctcctga gtaggacaaa
     4501 tccgccgccc tagacagctg ggcgcgcccc ccctacgggc ttgctctccg ggcttcgccc
     4561 tgcgcggtcg ctgcgctccc ttgccatggt gtatccaacg gcgtcagccg ggcaggatag
     4621 gtgaagtagg cccacccgcg agcgggtgtt ccttcttcac tgtcccttat tcgcacctgg
     4681 cggtgctcaa cgggaatcct gctctgcgag gctggccgta ggccggccct accggcgcgg
     4741 cagcgttacc cgtgtcggcg gctccaacgt ctcgccatcg tccagaaaac acggctcatc
     4801 gggcatcggc aggcgctgct gcccgcgccg ttcccattcc tccgtttcgg tcaaggctgg
     4861 caggtctggt tccatgcccg gaatgccggg ctggctgggc ggctcctcgc cggggccggt
     4921 cggtagttgc tgctcgcccg gatacagggt cgggatgcgg cgcaggtcgc catgccccaa
     4981 cagcgattcg tcctggtcgt cgtgatcaac caccacggcg gcactgaaca ccgacaggcg
     5041 caactggtcg cggggctggc cccacgccac gcggtcattg accacgtagg ccgacacggt
     5101 gccggggccg ttgagcttca cgacggagat ccagcgctcg gccaccaagt ccttgactgc
     5161 gtattggacc gtccgcaaag aacgtccgat gagcttggaa agtgtcttct ggctgaccac
     5221 cacggcgttc tggtggccca tctgcgccac gaggtgatgc agcagcattg ccgccgtggg
     5281 tttcctcgca ataagcccgg cccacgcctc atgcgctttg cgttccgttt gcacccagtg
     5341 accgggcttg ttcttggctt gaatgccgat ttctctggac tgcgtggcca tgcttatctc
     5401 catgcggtag gggtgccgca cggttgcggc accatgcgca atcagctgca acttttcggc
     5461 agcgcgacaa caattatgcg ttgcgtaaaa gtggcagtca attacagatt ttctttaacc
     5521 tacgcaatga gctattgcgg ggggtgccgc aatgagctgt tgcgtacccc ccttttttaa
     5581 gttgttgatt tttaagtctt tcgcatttcg ccctatatct agttctttgg tgcccaaaga
     5641 agggcacccc tgcggggttc ccccacgcct tcggcgcggc tccccctccg gcaaaaagtg
     5701 gcccctccgg ggcttgttga tcgactgcgc ggccttcggc cttgcccaag gtggcgctgc
     5761 ccccttggaa cccccgcact cgccgccgtg aggctcgggg ggcaggcggg cgggcttcgc
     5821 ccttcgactg cccccactcg cataggcttg ggtcgttcca ggcgcgtcaa ggccaagccg
     5881 ctgcgcggtc gctgcgcgag ccttgacccg ccttccactt ggtgtccaac cggcaagcga
     5941 agcgcgcagg ccgcaggccg gaggcttttc cccagagaaa attaaaaaaa ttgatggggc
     6001 aaggccgcag gccgcgcagt tggagccggt gggtatgtgg tcgaaggctg ggtagccggt
     6061 gggcaatccc tgtggtcaag ctcgtgggca ggcgcagcct gtccatcagc ttgtccagca
     6121 gggttgtcca cgggccgagc gaagcgagcc agccggtggc cgctcgcggc catcgtccac
     6181 atatccacgg gcaggaaagt ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa
     6241 aaaggatgga tataccgaaa aaatcgctat aatgaccccg aagcagggtt atgcagcgga
     6301 aaaggacaac gcgcggaccg ttgtccaatt taccgaacaa ctccgcggcc gggaagccga
     6361 tctcggcttg aacgaattgt taggtggcgg tacttgggtc gatatcaaag tgcatcactt
     6421 cttcccgtat gcccaacttt gtatagagag ccactgcggg atcgtcaccg taatctgctt
     6481 gcacgtagat cacataagca ccaagcgcgt tggcctcatg cttgaggaga ttgatgagcg
     6541 cggtggcaat gccctgcctc cggtgctctc cggagactgc gagatcatag atatagatct
     6601 cactacgcgg ctgctcaaac ttgggcagaa cgtaagccgc gagagcgcca acaaccgctt
     6661 cttggtcgaa ggcagcaagc gcgatgaatg tcttactacg gagcaagttc ccgaggtaat
     6721 cggagtccgg ctgatgttgg gagtaggtgg ctacgtctcc gaactcacga ccgaaaagat
     6781 caagagcagc ccgcatggat ttgacttggt cagggccgag cctacatgtg cgaatgatgc
     6841 ccatacttga gccacctaac tttgttttag ggcgactgcc ctgctgcgta acatcgttgc
     6901 tgctgcgtaa catcgttgct gctccataac atcaaacatc gacccacggc gtaacgcgct
     6961 tgctgcttgg atgcccgagg cataggctgt acaaaaaaac agtcataaca agccatgaaa
     7021 accgccactg cgccgttacc accgctgcgt tcggtcaagg ttctggacca gttgcgtgag
     7081 cgcatacgct acttgcatta cagtttacga accgaacagg cttatgtcaa atttaaatcg
     7141 taattattgg ggacccctgg attctcacca ataaaaaacg cccggcggca accgagcgtt
     7201 ctgaacaaat ccagatgtct gaacaaatcc agatggagtt ctgaggtcat tactggatct
     7261 atcaacagga gtccaagact agtcgccagg gttttcccag tcacgacgcg gccgcaagct
     7321 tgcatgcctg caggtcgact ctagattaga acggaatgtc gtcgtcgaag ctgtcgtagt
     7381 cctgggccgg ttgcggcgcc ggttgttgtt gcggggccgg acgcgattgt tgttgcgggg
     7441 cttgttgcgg acgttgcatg ggctcacgcg gggcgcgctg cgaatcatca ccggaggggc
     7501 gaccacccaa caactgcatg tt
//
