LOCUS       sequence-313276-        7862 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broad-Host range expression vector, pBBR origin, RhaR-RhaS 
            activator, PrhaBAD promoter, GFPmut3b, Kanamycin Resistance Marker. 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7862)
  AUTHORS   Schuster LA, Reisch CR
  TITLE     A plasmid toolbox for controlled gene expression across the 
            Proteobacteria.
  JOURNAL   Nucleic Acids Res. 2021 Jun 14. pii: 6298615. doi: 
            10.1093/nar/gkab496.
  PUBMED    34125913
REFERENCE   2  (bases 1 to 7862)
  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: "Nucleic 
            Acids Res. 2021 Jun 14. pii: 6298615. doi: 10.1093/nar/gkab496."
COMMENT     Sequence Label: pBRG2
FEATURES             Location/Qualifiers
     source          1..7862
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..772
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             773..1435
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     primer_bind     2129..2151
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2143..2160
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2144..2160
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2163..3011)
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQSIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     CDS             complement(3085..3921)
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHGVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFLREFNWSPRDIRQGRDGFLQ"
     promoter        4067..4185
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     misc_RNA        4200..4249
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             4294..4305
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4312..5025
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4342..4370)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4965..4985
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     misc_feature    5032..5052
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      5053..5110
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(5134..5151)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(5589..6383)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5700..5719)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6310..6329
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gcggccaccg gctggctcgc ttcgctcggc ccgtggacaa ccctgctgga caagctgatg
       61 gacaggctgc gcctgcccac gagcttgacc acagggattg cccaccggct acccagcctt
      121 cgaccacata cccaccggct ccaactgcgc ggcctgcggc cttgccccat caattttttt
      181 aattttctct ggggaaaagc ctccggcctg cggcctgcgc gcttcgcttg ccggttggac
      241 accaagtgga aggcgggtca aggctcgcgc agcgaccgcg cagcggcttg gccttgacgc
      301 gcctggaacg acccaagcct atgcgagtgg gggcagtcga agggcgaagc ccgcccgcct
      361 gccccccgag cctcacggcg gcgagtgcgg gggttccaag ggggcagcgc caccttgggc
      421 aaggccgaag gccgcgcagt cgatcaacaa gccccggagg ggccactttt tgccggaggg
      481 ggagccgcgc cgaaggcgtg ggggaacccc gcaggggtgc ccttctttgg gcaccaaaga
      541 actagatata gggcgaaatg cgaaagactt aaaaatcaac aacttaaaaa aggggggtac
      601 gcaacagctc attgcggcac cccccgcaat agctcattgc gtaggttaaa gaaaatctgt
      661 aattgactgc cacttttacg caacgcataa ttgttgtcgc gctgccgaaa agttgcagct
      721 gattgcgcat ggtgccgcaa ccgtgcggca cccctaccgc atggagataa gcatggccac
      781 gcagtccaga gaaatcggca ttcaagccaa gaacaagccc ggtcactggg tgcaaacgga
      841 acgcaaagcg catgaggcgt gggccgggct tattgcgagg aaacccacgg cggcaatgct
      901 gctgcatcac ctcgtggcgc agatgggcca ccagaacgcc gtggtggtca gccagaagac
      961 actttccaag ctcatcggac gttctttgcg gacggtccaa tacgcagtca aggacttggt
     1021 ggccgagcgc tggatctccg tcgtgaagct caacggcccc ggcaccgtgt cggcctacgt
     1081 ggtcaatgac cgcgtggcgt ggggccagcc ccgcgaccag ttgcgcctgt cggtgttcag
     1141 tgccgccgtg gtggttgatc acgacgacca ggacgaatcg ctgttggggc atggcgacct
     1201 gcgccgcatc ccgaccctgt atccgggcga gcagcaacta ccgaccggcc ccggcgagga
     1261 gccgcccagc cagcccggca ttccgggcat ggaaccagac ctgccagcct tgaccgaaac
     1321 ggaggaatgg gaacggcgcg ggcagcagcg cctgccgatg cccgatgagc cgtgttttct
     1381 ggacgatggc gagccgttgg agccgccgac acgggtcacg ctgccgcgcc ggtagcactt
     1441 gggttgcgca gcaacccgta agtgcgctgt tccagactat cggctgtagc cgcctcgccg
     1501 ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga
     1561 cctgaatgga agccggcggc acctcgctaa cggattcacc gtttttatca ggctctggga
     1621 ggcagaataa atgatcatat cgtcaattat tacctccacg gggagagcct gagcaaactg
     1681 gcctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa
     1741 ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcgaa
     1801 tttgctttcg aatttctgcc attcatccgc ttattatcac ttattcaggc gtagcaacca
     1861 ggcgtttaag ggcaccaata actgccttaa aaaaattacg ccccgccctg ccactcatcg
     1921 cagtacggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     1981 caaaatatta acgcttacaa tttccattcg ccattcaggc tgcgcaactg ttgggaaggg
     2041 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     2101 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     2161 gattaatctt tctgcgaatt gagatgacgc cactggctgg gcgtcatccc ggtttcccgg
     2221 gtaaacacca ccgaaaaata gttactatct tcaaagccac attcggtcga aatatcactg
     2281 attaacaggc ggctatgctg gagaagatat tgcgcatgac acactctgac ctgtcgcaga
     2341 tattgattga tggtcattcc agtctgctgg cgaaattgct gacgcaaaac gcgctcactg
     2401 cacgatgcct catcacaaaa tttatccagc gcaaagggac ttttcaggct agccgccagc
     2461 cgggtaatca gcttatccag caacgtttcg ctggatgttg gcggcaacga atcactggtg
     2521 taacgatggc gattcagcaa catcaccaac tgcccgaaca gcaactcagc catttcgtta
     2581 gcaaacggca catgctgact actttcatgc tcaagctgac cgataacctg ccgcgcctgc
     2641 gccatcccca tgctacctaa gcgccagtgt ggttgccctg cgctggcgtt aaatcccgga
     2701 atcgccccct gccagtcaag attcagcttc agacgctccg ggcaataaat aatactctgc
     2761 aaaaccagat cgttaacgga agcgtaggag tgtttatcgt cagcatgaat gtaaaagaga
     2821 tcgccacggg taatgcgata agggcgatcg ttgagtacat gcaggccatt accgcgccag
     2881 acaatcacca gctcacaaaa atcatgtgta tgttcagcaa agacatcttg cggataacgg
     2941 tcagccacag cgactgcctg ctggtcgctg gcaaaaaaat catctttgag aagttttaac
     3001 tgatgcgcca ccgtggctac ctcggccaga gaacgaagtt gattattcgc aatatggcgt
     3061 acaaatacgt tgagaagatt cgcgttattg cagaaagcca tcccgtccct ggcgaatatc
     3121 acgcggtgac cagttaaact ctcggagaaa aagcgtcgaa aagtggttac tgtcgctgaa
     3181 tccacagcga taggcgatgt cagtaacgct ggcctcgctg tggcgtagca gatgtcgggc
     3241 tttcatcagt cgcaggcggt tcaggtatcg ctgaggcgtc agtcccgttt gctgcttaag
     3301 ctgccgatgt agcgtacgca gtgaaagaga aaattgatcc gccacggcat cccaattcac
     3361 ctcatcggca aaatggtcct ccagccaggc cagaagcaag ttgagacgtg atgcgctgtt
     3421 ttccaggttc tcctgcaaac tgcttttacg cagcaagagc agtaattgca taaacaggat
     3481 ctcgcgactg gcggtcgagg gtaaatcatt ttccccttcc tgctgttcca tctgtgcaac
     3541 cagctgtcgc acctgctgca atacgccgtg gttaacgcgc cagtgagacg gatactgccc
     3601 atccagctct tgtggcagca actgattcag cccggcgaga aactgaaatc gatccggcga
     3661 gcgatacagc acattggtca gacacagatt atcggtatgt tcatacagat gccgatcatg
     3721 atcgcgtacg aaacagaccg tgccaccggt gatggtatag ggctgcccat taaacacatg
     3781 aatacccgtg ccatgttcga caatcacaat ttcatgaaaa tcatgatgat gttcaggaaa
     3841 atccgcctgc gggagccggg gttctatcgc cacggacgcg ttaccagacg gaaaaaaatc
     3901 cacactatgt aatacggtca tactggcttc ctgatgtcgt caacacggcg aaatagtaat
     3961 cacgaggtca ggttcttacc ttaaattttc gacggaaaac cacgtaaaaa acgtcgattt
     4021 ttcaagatac agcgtgaatt ttcaggaaat gcggtgagca tcacatcacc acaattcagc
     4081 aaattgtgaa catcatcacg ttcatctttc cctggttgcc aatggcccat tttcctgtca
     4141 gtaacgagaa ggtcgcgaat tcaggcgctt tttagactgg tcgtaatgaa attcagcagc
     4201 tgtcaccgga tgtgctttcc ggtctgatga gtccgtgagg acgaaacagc ctctacaaat
     4261 aattttgttt aagaactggg aagagcgcag attaaagagg agaaatacta gatgcgtaaa
     4321 ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat
     4381 gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc
     4441 cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact tgtcactact
     4501 ttcggttatg gtgttcaatg ctttgcgaga tacccagatc atatgaaaca gcatgacttt
     4561 ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac
     4621 gggaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc
     4681 gagttaaaag gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac
     4741 aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagtt
     4801 aacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa
     4861 caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca
     4921 caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt
     4981 gtaacagctg ctgggattac acatggcatg gatgaactat acaaataata atactagtag
     5041 cggccgctgc agtccggcaa aaaagggcaa ggtgtcacca ccctgccctt tttctttaaa
     5101 accgaaaaga ttacttcgcg ttatgcaggc ttcctcgctc actgactcgc tgcgctcggt
     5161 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatgcttt ccagtcggga
     5221 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5281 attgggcgca tgcataaaaa ctgttgtaat tcattaagca ttctgccgac atggaagcca
     5341 tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta
     5401 taatatttgc ccatgggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat
     5461 catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt
     5521 ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaaccccag
     5581 agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga
     5641 gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca
     5701 atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag
     5761 tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca
     5821 tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg
     5881 gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc
     5941 atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc
     6001 ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga
     6061 gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt
     6121 cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac
     6181 gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc ggtcttgaca
     6241 aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt
     6301 gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg
     6361 tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagatctt
     6421 gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc
     6481 ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc tgtccataaa
     6541 accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt tctctttgcg
     6601 cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatcccagg tggcactttt
     6661 cgtggaaatg tgcgcgcccg cgttcctgct ggcgctgggc ctgtttctgg cgctggactt
     6721 cccgctgttc cgtcagcagc ttttcgccca cggccttgat gatcgcggcg gccttggcct
     6781 gcatatcccg attcaacggc cccagggcgt ccagaacggt cttcaggcgc tcccgaaggt
     6841 ctcgggccgt ctcttgggct tgatcggcct tcttgcgcat ctcacgcgct cctgcggcgg
     6901 cctgtagggc aggctcatac ccctgccgaa ccgcttttgt cagccggtcg gccacggctt
     6961 ccggcgtctc aacgcgcttt gagattccca gcttttcggc caatccctgc ggtgcatagg
     7021 cgcgtggctc gaccgcttgc gggctgatgg tgacgtggcc cactggtggc cgctccaggg
     7081 cctcgtagaa cgcctgaatg cgcgtgtgac gtgccttgct gccctcgatg ccccgttgca
     7141 gccctagatc ggccacagcg gccgcaaacg tggtctggtc gcgggtcatc tgcgctttgt
     7201 tgccgatgaa ctccttggcc gacagcctgc cgtcctgcgt cagcggcacc acgaacgcgg
     7261 tcatgtgcgg gctggtttcg tcacggtgga tgctggccgt cacgatgcga tccgccccgt
     7321 acttgtccgc cagccacttg tgcgccttct cgaagaacgc cgcctgctgt tcttggctgg
     7381 ccgacttcca ccattccggg ctggccgtca tgacgtactc gaccgccaac acagcgtcct
     7441 tgcgccgctt ctctggcagc aactcgcgca gtcggcccat cgcttcatcg gtgctgctgg
     7501 ccgcccagtg ctcgttctct ggcgtcctgc tggcgtcagc gttgggcgtc tcgcgctcgc
     7561 ggtaggcgtg cttgagactg gccgccacgt tgcccatttt cgccagcttc ttgcatcgca
     7621 tgatcgcgta tgccgccatg cctgcccctc ccttttggtg tccaaccggc tcgacggggg
     7681 cagcgcaagg cggtgcctcc ggcgggccac tcaatgcttg agtatactca ctagactttg
     7741 cttcgcaaag tagtgaccgc ctacggcggc tgcggcgccc tacgggcttg ctctcagggc
     7801 ttcgccctgc gcggtcgctg cgctcccttg ccagcccgtg gatatgtgga cgatggccgc
     7861 ga
//
