LOCUS       sequence-457260-        6906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mini-Tn7 pGP-Tn7-Cm vector with inducible Pcym cumate promoter for 
            the inducible/controlled expression of target genes inserted into 
            the attTn7 chromosomal site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6906)
  AUTHORS   Chang C, Phan M-D, Schembri MA
  TITLE     Modified Tn7 transposon vectors for controlled chromosomal gene 
            expression.
  JOURNAL   Appl Environ Microbiol. 2024 Oct 23;90(10):e0155624. doi: 
            10.1128/aem.01556-24. Epub 2024 Sep 18.
  PUBMED    39291982
REFERENCE   2  (bases 1 to 6906)
  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: "Appl 
            Environ Microbiol."; date: "2024-10-23"; volume: "90(10):e0155624. 
            doi"; pages: " 10.1128/aem.01556-24. Epub 2024 Sep 18"
COMMENT     Sequence Label: pGP-Tn7-Pcym
FEATURES             Location/Qualifiers
     source          1..6906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(727..1098)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            1131..1240
                     /note="incP origin of transfer"
     primer_bind     complement(1921..1940)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(2079..2101)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2242..2261
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(2318..3109,3110..3178))
                     /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     2941..2960
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3179..3283)
                     /gene="bla"
                     /label=AmpR promoter
     mobile_element  3679..3844
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     protein_bind    3907..3934
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     promoter        complement(join(3942..3947,3948..3964,3965..3970))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             4111..4722
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     protein_bind    complement(4777..4824)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(4964..5623)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5538..5557
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5624..5726)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(5811..5858)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     terminator      complement(5904..5990)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(6093..6187)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(6215..6231)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6216..6232)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     rep_origin      join(6627..6906,1..109)
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
ORIGIN
        1 ttagttcgtt aaacatgaga gcttagtacg ttaaacatga gagcttagta cgtgaaacat
       61 gagagcttag tacgtactat caacaggttg aactgctgat cttcagatcc tctacgccgg
      121 acgcatcgtg gccggatcca gccgaccagg ctttccacgc ccgcgtgccg ctccatgtcg
      181 ttcgcgcggt tctcggaaac gcgctgccgc gtttcgtgat tgtcacgctc aagcccgtag
      241 tcccgttcga gcgtcgcgca gaggtcagcg agggcgcggt aggcccgata cggctcatgg
      301 atggtgtttc gggtcgggtg aatcttgttg atggcgatat ggatgtgcag gttgtcggtg
      361 tcgtgatgca cggcactgac gcgctgatgc tcggcgaagc caagcccagc gcagatgcgg
      421 tcctcaatcg cgcgcaacgt ctccgcgtcg ggcttctctc ccgcgcggaa gctaaccagc
      481 aggtgatagg tcttgtcggc ctcggaacgg gtgttgccgt gctgggtcgc catcacctcg
      541 gccatgacag cgggcagggt gtttgcctcg cagttcgtga cgcgcacgtg acccaggcgc
      601 tcggtcttgc cttgctcgtc ggtgatgtac ttcaccagct ccgcgaagtc gctcttcttg
      661 atggagcgca tggggacgtg cttggcaatc acgcgcaccc cccggccgtt ttagcggcta
      721 aaaaagtcat ggctctgccc tcgggcggac cacgcccatc atgaccttgc caagctcgtc
      781 ctgcttctct tcgatcttcg ccagcagggc gaggatcgtg gcatcaccga accgcgccgt
      841 gcgcgggtcg tcggtgagcc agagtttcag caggccgccc aggcggccca ggtcgccatt
      901 gatgcgggcc agctcgcgga cgtgctcata gtccacgacg cccgtgattt tgtagccctg
      961 gccgacggcc agcaggtagg ccgacaggct catgccggcc gccgccgcct tttcctcaat
     1021 cgctcttcgt tcgtctggaa ggcagtacac cttgataggt gggctgccct tcctggttgg
     1081 cttggtttca tcagccatcc gcttgccctc atctgttacg ccggcggtag ccggccagcc
     1141 tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag tgaagaagga
     1201 acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc gttggataca
     1261 ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga aaaaggatgg
     1321 atataccgaa aaaatcgcta taatgacccc gaagcagggt tatgcagcgg aaaagcgctg
     1381 cttccctgct gttttgtgga atatctaccg actggaaaca ggcaaatgca ggaaattact
     1441 gaactgaggg gacaggcgag agacgatgcc aaagagctac accgacgagc tggccgagtg
     1501 ggttgaatcc cgcgcggcca agaagcgccg gcgtgatgag gctgcggttg cgttcctggc
     1561 ggtgagggcg gatgtcgagg cggcgttagc gtccggctat gcgctcgtca ccatttggga
     1621 gcacatgcgg gaaacgggga aggtcaagtt ctcctacgag acgttccgct cgcacgccag
     1681 gcggcacatc aaggccaagc ccgccgatgt gcccgcaccg caggccaagg ctgcggaacc
     1741 cgcgccggca cccaagacgc cggagccacg gcggccgaag caggggggca aggctgaaaa
     1801 gccggccccc gctgcggccc cgaccggctt caccttcaac ccaacaccgg acaaaaagga
     1861 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     1921 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     1981 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     2041 ccttgctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     2101 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     2161 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     2221 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccataaat
     2281 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2341 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2401 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     2461 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     2521 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     2581 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca
     2641 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2701 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     2761 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     2821 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     2881 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg
     2941 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3001 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3061 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3121 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3181 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3241 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     3301 tgccacctgc agatctgcag gtcgagttta tagtcctgtc gggtttcgcc acctctgact
     3361 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3421 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3481 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3541 ccgcagccga acgaccgagc gcagcgggtc agtgagcgag gaagcggaag agcgcccaat
     3601 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcgccc aaacataaca
     3661 ggaagaaaaa tgccccgctg tgggcggaca aaatagttgg gaactgggag gggtggaaat
     3721 ggagttttta aggattattt agggaagagt gacaaaatag atgggaactg ggtgtagcgt
     3781 cgtaagctaa tacgaaaatt aaaaatgaca aaatagtttg gaactagatt tcacttatct
     3841 ggttggcctg caaggccttc gcgaggtacc gggcccaagc ttctcgagga attcctgcag
     3901 cccgggtaca aacagaccag attgtctgtt tgttgcatta cagtttacga accgaacagg
     3961 cttatgtcaa ctggtacaaa cagaccaagt tgtctgtttg ttggcgatag ctagactggg
     4021 cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg
     4081 ggaaccaacg cctttcgcaa gatggtgatc atgagtccaa agagaagaac acaggcagag
     4141 cgcgcaatgg aaacccaggg caagttgatt gcagcggccc tgggggtttt acgggaaaaa
     4201 ggttacgcgg gattccggat cgcagatgtg cccggtgcag ctggtgtgtc gagaggagcg
     4261 cagagccatc atttcccgac aaagttggag cttctgcttg ccacttttga atggctttac
     4321 gaacagatca ccgaacgcag tcgggctcga ttagcgaaat tgaagccaga ggatgacgtc
     4381 atccagcaaa tgctggacga cgccgccgaa tttttcctcg acgatgactt ctctatcagc
     4441 cttgatttga ttgtggctgc cgaccgtgat ccagcgttac gcgagggtat tcagcgcacg
     4501 gtagagagga atcggtttgt cgtcgaggat atgtggcttg gtgttctggt gagccgtggt
     4561 ctttcgcgtg atgatgcaga agacatcctt tggttgatat tcaattcggt gcgtggactt
     4621 gctgttcgta gcctatggca gaaggacaaa gaacgctttg agcgtgtcag gaactcgaca
     4681 ctcgaaattg cgcgagagcg gtacgcgaaa ttcaagcgct agtgggggat ccactagtga
     4741 gctcatgcat gatcgaatta gcttcaaaag cgctctgaag ttcctatact ttctagagaa
     4801 taggaacttc ggaataggaa cttcaagatc ccctgattcc ctttgtcaac agcaatggat
     4861 gggtcgaatt tgctttcgaa tttctgccat tcatccgctt attatcactt attcaggcgt
     4921 agcaaccagg cgtttaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc
     4981 actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa
     5041 cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt
     5101 tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac
     5161 tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag
     5221 ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact
     5281 gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga
     5341 aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca
     5401 tacggaactc cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa
     5461 acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct
     5521 ggttataggt acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt
     5581 gggatatatc aacggtggta tatccagtga tttttttctc cattttagct tccttagctc
     5641 ctgaaaatct cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa
     5701 agttggaacc tcttacgtgc cgatcaacgt ctcattttcg ccaaaagttg gcccatctag
     5761 aattattcca ttgagtaagt ttttaagcac atcagcttca aaagcgctct gaagttccta
     5821 tactttctag agaataggaa cttcggaata ggtacttcaa gatccccaat tcgatcgtcc
     5881 gggccgcaag ctcctagcgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa
     5941 cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctcaag
     6001 ctagagagtc attaccccag gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc
     6061 cccgccctgc cactcatcgc agtctagctt ggattctcac caataaaaaa cgcccggcgg
     6121 caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac
     6181 aggagtccaa gctcagctaa ttaagctagc ttatcgatac cgtcgacctc gaaccccacg
     6241 cccctcttta atacgacggg caatttgcac ttcagaaaat gaagagtttg ctttagccat
     6301 aacaaaagtc cagtatgctt tttcacagca taactggact gatttcagtt tacaactatt
     6361 ctgtctagtt taagacttta ttgtcatagt ttagatctat tttgttcagt ttaagacttt
     6421 attgtccgcc cacacccgct tacgcagggc atccatttat tactcaaccg taaccgattt
     6481 tgccaggtta cgcggctggt cctctagctg gcgtaatagc gaagaggccc gcaccgatcg
     6541 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     6601 tacgcatctg tgcggtacaa ttcccatgtc agccgttaag tgttcctgtg tcactcaaaa
     6661 ttgctttgag aggctctaag ggcttctcag tgcgttacat ccctggcttg ttgtccacaa
     6721 ccgttaaacc ttaaaagctt taaaagcctt atatattctt ttttttctta taaaacttaa
     6781 aaccttagag gctatttaag ttgctgattt atattaattt tattgttcaa acatgagagc
     6841 ttagtacgtg aaacatgaga gcttagtacg ttagccatga gagcttagta cgttagccat
     6901 gagggt
//
