LOCUS       sequence-382388-        6998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of Tse5-CT (1169-1317) in P. putida for biological 
            function studies.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6998)
  AUTHORS   Gonzalez-Magana A, Altuna J, Queralt-Martin M, Largo E, Velazquez C,
            Montanchez I, Bernal P, Alcaraz A, Albesa-Jove D
  TITLE     The P. aeruginosa effector Tse5 forms membrane pores disrupting the 
            membrane potential of intoxicated bacteria.
  JOURNAL   Commun Biol. 2022 Nov 5;5(1):1189. doi: 10.1038/s42003-022-04140-y.
  PUBMED    36335275
REFERENCE   2  (bases 1 to 6998)
  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: "Commun 
            Biol."; date: "2022-11-5"; volume: "5(1):1189. doi"; pages: " 
            10.1038/s42003-022-04140-y"
COMMENT     Sequence Label: pS238D1::tse5-CT
FEATURES             Location/Qualifiers
     source          1..6998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(19..984)
                     /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"
     promoter        1884..1964
                     /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             2018..3100
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2037..2056)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3597..3614
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      complement(3731..3758)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(3774..3844)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4030..4124
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(4245..4264)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4256..5071
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     oriT            5238..5346
                     /note="incP origin of transfer"
     CDS             complement(5357..6019)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      6020..6791
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     terminator      complement(6906..6992)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaattaaac gttcgtaatc aagccacttc ctttttgcat tgacgcaggg tgtcggaagg
       61 caactcgccg aacgcgctcc tatagttttc agcgaagcgt cccaaatgta agaagccgta
      121 gtctagggct atctcagtta tactacgcac attggcactg ggatcgttca agcaggcgcg
      181 gatgctttcg agcttgcggt tgcggatgta gttcttcggc gtggtgccgg cgtgcttctc
      241 gaacaaattg tagagcgagc gtggactcat catcgccagc tccgctaacc gctcaaggct
      301 gatattccgt ttgagattct cctcaatgaa ttgaacgact cgctcgaaag acgggttacc
      361 tttgctgaaa atttcacggc tgacattgct gcccagcatt tcgagcagct tggaagcgat
      421 gatccccgca tagtgctctt ggacccgagg catcgacttt gtatgttccg cttcgtcaca
      481 aactaacccg agtagattga taaagccatc gagttgctgg agattgtgtc gcgcggcgaa
      541 acggataccc tccctcggct tgtgccaatt gttgtcactg cacgcccgat caaggaccac
      601 tgagggcaat ttaacgataa atttctcgca atcttctgaa taggtcaggt cggcttggtc
      661 atccggattg agcagcaata gttcgcccgg cgcaaaatag tgctcctggc catggccacg
      721 ccacaggcaa tggcctttga gtattatttg cagatgataa caggtctcta atccaggcga
      781 gattaccctc acgctaccgc cgtagctgat tcgacacaga tcgaggcatc cgaagattct
      841 gtggtgcagc ctgcctgccg ggcgcccgcc cttgggcagg cgaatagagt gcgtaccgac
      901 atactggtta acataatcgg agactgcata gggctcggcg tggacgaaga tctgactttt
      961 ctcgttcaat aagcaaaaat ccatagttca cggttctctt attttaatgt gggctgcttg
     1021 gtgtgatgta gaaaggcgcc aagtcgatga aaatgcatct cgacgtgatg cgtatacggg
     1081 ttacccccat tgccacgttg cgccatcctt tttgcaatca gtgaccactt ttccaagcaa
     1141 aaataacgcc aagcagaacg aagacgttct ttttaagaag cgagaacacc agaagttcgt
     1201 gctgtcgggg catggggcga cgaattggcg gataaagggg atctgctgga tattacggcc
     1261 tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt
     1321 gcccgcctga tgaatgctca tccgtaatta cgtatggcaa tgaaagacgg tgagctggtg
     1381 atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca
     1441 tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat
     1501 gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt
     1561 ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg
     1621 gacaacttct tcgcccccgt tttcaccatg catgggaatt agcttgatct gaccaacgac
     1681 cggtagcgga gctatccaac ggcggtatac caggaaaaca cacagcaggt acatcagaac
     1741 agtaccatga ctgaagaaca aatagttttt tcctgatcca taaagcagaa cggcctgctc
     1801 catgacaaat ctggctcccc aactaatgcc ccatgcagcc agcataacca gcataaagtg
     1861 cagtgtccgg tttgataggg ataagtccag ccttgcaaga agcggataca ggagtgcaaa
     1921 aaatggctat ctctagtaag gcctacccct taggctttat gcaacagaaa caataataat
     1981 gggagtcatg accatgccta ggaggaaaaa acatatggtg aaaccagtaa cgttatacga
     2041 tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag
     2101 ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat
     2161 tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac
     2221 ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga
     2281 tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa
     2341 agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct
     2401 ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct
     2461 tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg
     2521 actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc
     2581 attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa
     2641 tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca
     2701 aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca
     2761 gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat
     2821 ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac
     2881 catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc
     2941 tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac
     3001 caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     3061 gctggcacga caggtttccc gactggaaag cgggcagtaa catcaccacc atcattagga
     3121 tggtggtgat gataatgctg atcatcccgc tggtggtgat cggcgccttc gccgcccgcg
     3181 ccgcgatcgg agcggcgctg ggcgccggca tcgagctggg gatgcagacc ggcaagcagg
     3241 tgctggggca gatgaaggac aactgggaca gcgatcgcga cctcaccgat atcaagtgga
     3301 agtgcatcga catcaactgg aaacacgtcg gcgcctcggc ggccatcggc accgtggcgc
     3361 cgggcatgct cagcaccggc aagaccgtgg tgcagtccgc caaggcgatc aggacccttt
     3421 ccggacaggc ggccaacacc gccaaccgcg cggccaagct ggccgcgcgc aaggccgccc
     3481 atgccgatac catcaagaag gccgtggcca cccaggccgc ctggcagacg ggcaagcaga
     3541 tcgtcaaatg cccactgaag gacgaagaag aggagtgtcc gccgcaatga ctcgagcacc
     3601 accaccacca ccactgaggt acccggggat cctctagagt cgacctgcag gcatgcaagc
     3661 ttgcggccgc gtcgtgactg ggaaaaccct ggcgcaaagc ccgccgaaag gcgggctttt
     3721 ctgttataaa cgcagaaagg cccacccgaa ggtgagccag tgtgactcta gtagagagcg
     3781 ttcaccgaca aaaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta
     3841 tttgatgcct ggctcgagct cgagaaaaaa agcccggacg actgttcggg cttgtctttt
     3901 tatatgttgg aaaatcagtg gcaatgcaat ggcccaacag aaaatggtga aaccagtaac
     3961 gttatactga atctaagtat caatgtgagc gctcacaagt caacactctt tttgataaaa
     4021 ctagtcttgg actcctgttg atagatccag taatgacctc agaactccat ctggatttgt
     4081 tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaggg gtccccaata
     4141 attacgattt aaatttgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     4201 atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     4261 ccatattcag cgtgaaacga gctgtagccg tccgcgtctg aacagcaaca tggatgcgga
     4321 tctgtatggc tataaatggg cgcgtgataa cgtgggtcag agcggcgcga ccatttatcg
     4381 tctgtatggc aaaccggatg cgccggaact gtttctgaaa catggcaaag gcagcgtggc
     4441 gaacgatgtg accgatgaaa tggtgcgtct gaactggctg accgaattta tgccgctgcc
     4501 gaccattaaa cattttattc gcaccccgga tgatgcgtgg ctgctgacca ccgcgattcc
     4561 gggcaaaacc gcgtttcagg tgctggaaga atatccggat agcggcgaaa acattgtgga
     4621 tgcgctggcc gtgtttctgc gtcgtctgca tagcattccg gtgtgcaact gcccgtttaa
     4681 cagcgatcgt gtgtttcgtc tggcccaggc gcagagccgt atgaacaacg gcctggtgga
     4741 tgcgagcgat tttgatgatg aacgtaacgg ctggccggtg gaacaggtgt ggaaagaaat
     4801 gcataaactg ctgccgttta gcccggatag cgtggtgacc cacggcgatt ttagcctgga
     4861 taacctgatt ttcgatgaag gcaaactgat tggctgcatt gatgtgggcc gtgtgggcat
     4921 tgcggatcgt tatcaggatc tggccattct gtggaactgc ctgggcgaat ttagcccgag
     4981 cctgcaaaaa cgtctgtttc agaaatatgg cattgataat ccggatatga acaaactgca
     5041 atttcatctg atgctggatg aatttttcta ataattaatt ggaccgcggt ccgcgcgttg
     5101 tccttttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc ggtatatcca
     5161 tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt tccttggtgt
     5221 atccaacggc gtcagccggg caggataggt gaagtaggcc cacccgcgag cgggtgttcc
     5281 ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc
     5341 tggccgtagg ccggccctac cggcgcggca gcgttacccg tgtcggcggc tccaacggct
     5401 cgccatcgtc cagaaaacac ggctcatcgg gcatcggcag gcgctgctgc ccgcgccgtt
     5461 cccattcctc cgtttcggtc aaggctggca ggtctggttc catgcccgga atgccgggct
     5521 ggctgggcgg ctcctcgccg gggccggtcg gtagttgctg ctcgcccgga tacagggtcg
     5581 ggatgcggcg caggtcgcca tgccccaaca gcgattcgtc ctggtcgtcg tgatcaacca
     5641 ccacggcggc actgaacacc gacaggcgca actggtcgcg gggctggccc cacgccacgc
     5701 ggtcattgac cacgtaggcc gacacggtgc cggggccgtt gagcttcacg acggagatcc
     5761 agcgctcggc caccaagtcc ttgactgcgt attggaccgt ccgcaaagaa cgtccgatga
     5821 gcttggaaag tgtcttctgg ctgaccacca cggcgttctg gtggcccatc tgcgccacga
     5881 ggtgatgcag cagcattgcc gccgtgggtt tcctcgcaat aagcccggcc cacgcctcat
     5941 gcgctttgcg ttccgtttgc acccagtgac cgggcttgtt cttggcttga atgccgattt
     6001 ctctggactg cgtggccatg cttatctcca tgcggtaggg gtgccgcacg gttgcggcac
     6061 catgcgcaat cagctgcaac ttttcggcag cgcgacaaca attatgcgtt gcgtaaaagt
     6121 ggcagtcaat tacagatttt ctttaaccta cgcaatgagc tattgcgggg ggtgccgcaa
     6181 tgagctgttg cgtacccccc ttttttaagt tgttgatttt taagtctttc gcatttcgcc
     6241 ctatatctag ttctttggtg cccaaagaag ggcacccctg cggggttccc ccacgccttc
     6301 ggcgcggctc cccctccggc aaaaagtggc ccctccgggg cttgttgatc gactgcgcgg
     6361 ccttcggcct tgcccaaggt ggcgctgccc ccttggaacc cccgcactcg ccgccgtgag
     6421 gctcgggggg caggcgggcg ggcttcgccc ttcgactgcc cccactcgca taggcttggg
     6481 tcgttccagg cgcgtcaagg ccaagccgct gcgcggtcgc tgcgcgagcc ttgacccgcc
     6541 ttccacttgg tgtccaaccg gcaagcgaag cgcgcaggcc gcaggccgga ggcttttccc
     6601 cagagaaaat taaaaaaatt gatggggcaa ggccgcaggc cgcgcagttg gagccggtgg
     6661 gtatgtggtc gaaggctggg tagccggtgg gcaatccctg tggtcaagct cgtgggcagg
     6721 cgcagcctgt ccatcagctt gtccagcagg gttgtccacg ggccgagcga agcgagccag
     6781 ccggtggccg ctcgcggcca tcgtccacat atccacgggc tggcaaggga gcgcagcgac
     6841 cgcgcagggc gaagcccgga gagcaagccc gtaggggggg cgcgcccagc tgtctagggc
     6901 ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc
     6961 cagtctttcg actgagcctt tcgttttatt tgatgcct
//
