LOCUS       sequence-138904-        6766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mCherry-encoding vector for markerless deletion mutagenesis in 
            Bradyrhizobium diazoefficiens and other Alphaproteobacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6766)
  AUTHORS   Ledermann R, Strebel S, Kampik C, Fischer HM
  TITLE     Versatile vectors for efficient mutagenesis of Bradyrhizobium 
            diazoefficiens and other Alphaproteobacteria.
  JOURNAL   Appl Environ Microbiol. 2016 Feb 26. pii: AEM.04085-15.
  PUBMED    26921431
REFERENCE   2  (bases 1 to 6766)
  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. 2016 Feb 26. pii: AEM.04085-15."
COMMENT     Sequence Label: pTETSIX
FEATURES             Location/Qualifiers
     source          1..6766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(73..90)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(244..832)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(324..343)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(1091..1096,1097..1113,1114..1119)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             1167..2357
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     CDS             complement(3175..3546)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            3579..3688
                     /note="incP origin of transfer"
     CDS             complement(join(4790..5581,5582..5650))
                     /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     5413..5432
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5651..5755)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6038..6745
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6183..6201)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6451..6470
                     /label=mCherry-F
                     /note="mCherry, forward primer"
ORIGIN
        1 ctgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
       61 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
      121 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      181 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      241 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      301 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      361 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      421 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      481 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      541 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      601 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      661 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      721 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      781 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      841 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      901 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      961 taaatcaatc taaagtatat atgagtaaac ttggtctgac aggtttaaac tctagacaat
     1021 tgcctaggat gcatgctagc cttaagattt aaatgttaca tcgatgattc actagtgata
     1081 attctcatgt ttgacagctt atcatcgata agctttaatg cggtagttta tcacagttaa
     1141 attgctaacg cagtcaggca ccgtgtatga aatctaacaa tgcgctcatc gtcatcctcg
     1201 gcaccgtcac cctggatgct gtaggcatag gcttggttat gccggtactg ccgggcctct
     1261 tgcgggatat cgtccattcc gacagcatcg ccagtcacta tggcgtgctg ctagcgctat
     1321 atgcgttgat gcaatttcta tgcgcacccg ttctcggagc actgtccgac cgctttggcc
     1381 gccgcccagt cctgctcgct tcgctacttg gagccactat cgactacgcg atcatggcga
     1441 ccacacccgt cctgtggatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca
     1501 caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc
     1561 acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg
     1621 gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc
     1681 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgaccga
     1741 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     1801 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     1861 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     1921 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     1981 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccgacgcg ctgggctacg
     2041 tcttgctggc gttcgcgacg cgaggctgga tggccttccc cattatgatt cttctcgctt
     2101 ccggcggcat cgggatgccc gcgttgcagg ccatgctgtc caggcaggta gatgacgacc
     2161 atcagggaca gcttcaagga tcgctcgcgg ctcttaccag cctaacttcg atcattggac
     2221 cgctgatcgt cacggcgatt tatgccgcct cggcgagcac atggaacggg ttggcatgga
     2281 ttgtaggcgc cgccctatac cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc
     2341 gggccacctc gacctgaatg gaagccggcg gcacctcgct aacggattca ccactccaag
     2401 aattggagcc aatcaattct tgcggagaac tgtgaatgcg cacctagatc ttcgaacgat
     2461 actagtcata tgaattcgta atccggttta ttgactaccg gaagcagtgt gaccgtgtgc
     2521 ttctcaaatg cctgaggcca gtttgctcag gctctccccg tggaggtaat aattgacgat
     2581 atgatccagc cgaccaggct ttccacgccc gcgtgccgct ccatgtcgtt cgcgcggttc
     2641 tcggaaacgc gctgccgcgt ttcgtgattg tcacgctcaa gcccgtagtc ccgttcgagc
     2701 gtcgcgcaga ggtcagcgag ggcgcggtag gcccgatacg gctcatggat ggtgtttcgg
     2761 gtcgggtgaa tcttgttgat ggcgatatgg atgtgcaggt tgtcggtgtc gtgatgcacg
     2821 gcactgacgc gctgatgctc ggcgaagcca agcccagcgc agatgcggtc ctcaatcgcg
     2881 cgcaacgtct ccgcgtcggg cttctctccc gcgcggaagc taaccagcag gtgataggtc
     2941 ttgtcggcct cggaacgggt gttgccgtgc tgggtcgcca tcacctcggc catgacagcg
     3001 ggcagggtgt ttgcctcgca gttcgtgacg cgcacgtgac ccaggcgctc ggtcttgcct
     3061 tgctcgtcgg tgatgtactt caccagctcc gcgaagtcgc tcttcttgat ggagcgcatg
     3121 gggacgtgct tggcaatcac gcgcaccccc cggccgtttt agcggctaaa aaagtcatgg
     3181 ctctgccctc gggcggacca cgcccatcat gaccttgcca agctcgtcct gcttctcttc
     3241 gatcttcgcc agcagggcga ggatcgtggc atcaccgaac cgcgccgtgc gcgggtcgtc
     3301 ggtgagccag agtttcagca ggccgcccag gcggcccagg tcgccattga tgcgggccag
     3361 ctcgcggacg tgctcatagt ccacgacgcc cgtgattttg tagccctggc cgacggccag
     3421 caggtaggcc gacaggctca tgccggccgc cgccgccttt tcctcaatcg ctcttcgttc
     3481 gtctggaagg cagtacacct tgataggtgg gctgcccttc ctggttggct tggtttcatc
     3541 agccatccgc ttgccctcat ctgttacgcc ggcggtagcc ggccagcctc gcagagcagg
     3601 attcccgttg agcaccgcca ggtgcgaata agggacagtg aagaaggaac acccgctcgc
     3661 gggtgggcct acttcaccta tcctgcccgg ctgacgccgt tggatacacc aaggaaagtc
     3721 tacacgaacc ctttggcaaa atcctgtata tcgtgcgaaa aaggatggat ataccgaaaa
     3781 aatcgctata atgaccccga agcagggtta tgcagcggaa aagcgctgct tccctgctgt
     3841 tttgtggaat atctaccgac tggaaacagg caaatgcagg aaattactga actgagggga
     3901 caggcgagag acgatgccaa agagctacac cgacgagctg gccgagtggg ttgaatcccg
     3961 cgcggccaag aagcgccggc gtgatgaggc tgcggttgcg ttcctggcgg tgagggcgga
     4021 tgtcgaggcg gcgttagcgt ccggctatgc gctcgtcacc atttgggagc acatgcggga
     4081 aacggggaag gtcaagttct cctacgagac gttccgctcg cacgccaggc ggcacatcaa
     4141 ggccaagccc gccgatgtgc ccgcaccgca ggccaaggct gcggaacccg cgccggcacc
     4201 caagacgccg gagccacggc ggccgaagca ggggggcaag gctgaaaagc cggcccccgc
     4261 tgcggccccg accggcttca ccttcaaccc aacaccggac aaaaaggatc tactgtaatg
     4321 gcgaaaattc acatggtttt gcagggcaag ggcggggtcg gcaagtcggc catcgccgcg
     4381 atcattgcgc agtacaagat ggacaagggg cagacaccct tgtgcatcga caccgacccg
     4441 gtgaacgcga cgttcgaggg ctacaaggcc ctgaacgtcc gccggctgaa catcatggcc
     4501 ggcgacgaaa ttaactcgcg caacttcgac accctggtcg agctgattgc gccgaccaag
     4561 gatgacgtgg tgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
     4621 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatccttg aagctgtccc
     4681 tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg
     4741 ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgt taccaatgct
     4801 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4861 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4921 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4981 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5041 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5101 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5161 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5221 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5281 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5341 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5401 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5461 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5521 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5581 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5641 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5701 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5761 ttccaattcg cccttagtga agacaaaggt aagcttgctc gatctcggcg agcgccgcgg
     5821 cggcgccccg gaggcgcagc ggctctacga gcaggtcggc gaggccgcgt cctgacccct
     5881 cattcggcgc gatccgccct gccggcaaac ctgccgcttg atcatcgtcg cacaagcctc
     5941 taatcggtcc gcgcctttac gcccggctta tggccggtcc cgcggtagga ttgcacgaag
     6001 cttagatctt gactagaaat aagaaggaga tataattatg gtgagcaagg gcgaggagga
     6061 taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa
     6121 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac
     6181 cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc
     6241 tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt
     6301 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg
     6361 cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa
     6421 gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg
     6481 ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca
     6541 gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc
     6601 caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc
     6661 ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac
     6721 cggcggcatg gacgagctgt acaaggaatt gaagcttaga tcttga
//
