LOCUS       sequence-334678-        6474 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broad-host-range allelic exchange vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6474)
  TITLE     Broad-host-range allelic exchange vector
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6474)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pPS04
FEATURES             Location/Qualifiers
     source          1..6474
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(815..1186)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            1219..1328
                     /note="incP origin of transfer"
     promoter        join(1965..1970,1971..1987,1988..1993)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             complement(join(2165..3499,3500..3586))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        complement(3587..4032)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     primer_bind     complement(4273..4290)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4444..5032)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4524..4543)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5583..5602)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5594..6409
                     /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"
     CDS             5594..6409
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5667..5686)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
       61 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
      121 agatctgaat tcagctgtac aattggtacc atggatgcat atgctgcagc tcgagcggcc
      181 gcgggcccta cgtacgcgtg ttaaccggtg agctcactag aggatccagc cgaccaggct
      241 ttccacgccc gcgtgccgct ccatgtcgtt cgcgcggttc tcggaaacgc gctgccgcgt
      301 ttcgtgattg tcacgctcaa gcccgtagtc ccgttcgagc gtcgcgcaga ggtcagcgag
      361 ggcgcggtag gcccgatacg gctcatggat ggtgtttcgg gtcgggtgaa tcttgttgat
      421 ggcgatatgg atgtgcaggt tgtcggtgtc gtgatgcacg gcactgacgc gctgatgctc
      481 ggcgaagcca agcccagcgc agatgcggtc ctcaatcgcg cgcaacgtct ccgcgtcggg
      541 cttctctccc gcgcggaagc taaccagcag gtgataggtc ttgtcggcct cggaacgggt
      601 gttgccgtgc tgggtcgcca tcacctcggc catgacagcg ggcagggtgt ttgcctcgca
      661 gttcgtgacg cgcacgtgac ccaggcgctc ggtcttgcct tgctcgtcgg tgatgtactt
      721 caccagctcc gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac
      781 gcgcaccccc cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca
      841 cgcccatcat gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga
      901 ggatcgtggc atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca
      961 ggccgcccag gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt
     1021 ccacgacgcc cgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca
     1081 tgccggccgc cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct
     1141 tgataggtgg gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat
     1201 ctgttacgcc ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca
     1261 ggtgcgaata agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta
     1321 tcctgcccgg ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa
     1381 atcctgtata tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga
     1441 agcagggtta tgcagcggaa aagcgctgct tccctgctgt tttgtggaat atctaccgac
     1501 tggaaacagg caaatgcagg aaattactga actgagggga caggcgagag acgatgccaa
     1561 agagctacac cgacgagctg gccgagtggg ttgaatcccg cgcggccaag aagcgccggc
     1621 gtgatgaggc tgcggttgcg ttcctggcgg tgagggcgga tgtcgaggcg gcgttagcgt
     1681 ccggctatgc gctcgtcacc atttgggagc acatgcggga aacggggaag gtcaagttct
     1741 cctacgagac gttccgctcg cacgccaggc ggcacatcaa ggccaagccc gccgatgtgc
     1801 ccgcaccgca ggccaaggct gcggaacccg cgccggcacc caagacgccg gagccacggc
     1861 ggccgaagca ggggggcaag gctgaaaagc cggcccccgc tgcggccccg accggcttca
     1921 ccttcaaccc aacaccggac aaaaaggatc cccaattctc atgtttgaca gcttatcatc
     1981 gataagcttt aatgcggtag tttatcacag ttaaattgct aacgcagtca ggcaccgtgt
     2041 atgaaatcta acaatgcgct catcgtcatc ctcggcaccg tcaccctgga tgctgtaggc
     2101 ataggcttgg ttatgccggt actgccgggc ctcttgcggg atatcggcat tttcttttgc
     2161 gtttttattt gttaactgtt aattgtcctt gttcaaggat gctgtctttg acaacagatg
     2221 ttttcttgcc tttgatgttc agcaggaagc ttggcgcaaa cgttgattgt ttgtctgcgt
     2281 agaatcctct gtttgtcata tagcttgtaa tcacgacatt gtttcctttc gcttgaggta
     2341 cagcgaagtg tgagtaagta aaggttacat cgttaggatc aagatccatt tttaacacaa
     2401 ggccagtttt gttcagcggc ttgtatgggc cagttaaaga attagaaaca taaccaagca
     2461 tgtaaatatc gttagacgta atgccgtcaa tcgtcatttt tgatccgcgg gagtcagtga
     2521 acaggtacca tttgccgttc attttaaaga cgttcgcgcg ttcaatttca tctgttactg
     2581 tgttagatgc aatcagcggt ttcatcactt ttttcagtgt gtaatcatcg tttagctcaa
     2641 tcataccgag agcgccgttt gctaactcag ccgtgcgttt tttatcgctt tgcagaagtt
     2701 tttgactttc ttgacggaag aatgatgtgc ttttgccata gtatgctttg ttaaataaag
     2761 attcttcgcc ttggtagcca tcttcagttc cagtgtttgc ttcaaatact aagtatttgt
     2821 ggcctttatc ttctacgtag tgaggatctc tcagcgtatg gttgtcgcct gagctgtagt
     2881 tgccttcatc gatgaactgc tgtacatttt gatacgtttt tccgtcaccg tcaaagattg
     2941 atttataatc ctctacaccg ttgatgttca aagagctgtc tgatgctgat acgttaactt
     3001 gtgcagttgt cagtgtttgt ttgccgtaat gtttaccgga gaaatcagtg tagaataaac
     3061 ggatttttcc gtcagatgta aatgtggctg aacctgacca ttcttgtgtt tggtctttta
     3121 ggatagaatc atttgcatcg aatttgtcgc tgtctttaaa gacgcggcca gcgtttttcc
     3181 agctgtcaat agaagtttcg ccgacttttt gatagaacat gtaaatcgat gtgtcatccg
     3241 catttttagg atctccggct aatgcaaaga cgatgtggta gccgtgatag tttgcgacag
     3301 tgccgtcagc gttttgtaat ggccagctgt cccaaacgtc caggcctttt gcagaagaga
     3361 tatttttaat tgtggacgaa tcgaattcag gaacttgata tttttcattt ttttgctgtt
     3421 cagggatttg cagcatatca tggcgtgtaa tatgggaaat gccgtatgtt tccttatatg
     3481 gcttttggtt cgtttctttc gcaaacgctt gagttgcgcc tcctgccagc agtgcggtag
     3541 taaaggttaa tactgttgct tgttttgcaa actttttgat gttcatcgtt catgtctcct
     3601 tttttatgta ctgtgttagc ggtctgcttc ttccagccct cctgtttgaa gatggcaagt
     3661 tagttacgca caataaaaaa agacctaaaa tatgtaaggg gtgacgccaa agtatacact
     3721 ttgcccttta cacattttag gtcttgcctg ctttatcagt aacaaacccg cgcgatttac
     3781 ttttcgacct cattctatta gactctcgtt tggattgcaa ctggtctatt ttcctctttt
     3841 gtttgataga aaatcataaa aggatttgca gactacgggc ctaaagaact aaaaaatcta
     3901 tctgtttctt ttcattctct gtatttttta tagtttctgt tgcatgggca taaagttgcc
     3961 tttttaatca caattcagaa aatatcataa tatctcattt cactaaataa tagtgaacgg
     4021 caggtatatg tgatgggtta aaaaggatcg atcctctagc cacgggtgcg catgatcgtg
     4081 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     4141 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     4201 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     4261 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4321 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4381 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4441 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4501 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4561 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4621 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4681 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4741 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4801 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4861 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4921 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4981 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5041 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5101 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5161 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5221 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5281 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5341 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5401 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta atatccccgt
     5461 gtcggacctg cagggggggg gggaaagcca cgttgtgtct caaaatctct gatgttacat
     5521 tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa
     5581 tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctcgaggc cgcgattaaa
     5641 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     5701 aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     5761 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     5821 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     5881 actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc
     5941 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     6001 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     6061 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     6121 acaagtctgg aaagaaatgc ataagctttt gccattctca ccggattcag tcgtcactca
     6181 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     6241 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     6301 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     6361 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt
     6421 taattggttg taacactggc agagcattac gctgacttga cgggacggaa tatt
//
