LOCUS       sequence-440370-        5532 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under rhamnose induction (RhaS + RhaR + pRhaB-sfGFP)
            with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5532)
  AUTHORS   Venkataraman M, Ynigez-Gutierrez A, Infante V, MacIntyre A, 
            Fernandes-Junior PI, Ane JM, Pfleger B
  TITLE     Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes.
  JOURNAL   ACS Synth Biol. 2023 Dec 15;12(12):3623-3634. doi: 
            10.1021/acssynbio.3c00414. Epub 2023 Nov 21.
  PUBMED    37988619
REFERENCE   2  (bases 1 to 5532)
  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"; doi: 
            "10.1021/acssynbio.3c00414"; journalName: "ACS Synth Biol"; date: 
            "2023-12-15- 15"; volume: "12"; issue: "12"; pages: "3623-3634"
COMMENT     Sequence Label: pMVV052
FEATURES             Location/Qualifiers
     source          1..5532
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      1..72
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      88..115
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(182..844)
                     /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      845..1616
                     /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(1650..1744)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1775..2569)
                     /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(1886..1905)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2496..2515
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(2712..3560)
                     /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
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     CDS             complement(3634..4470)
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     promoter        4616..4734
                     /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)"
     CDS             join(4784..4786,4787..5500)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             4784..5497
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4814..4842)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5437..5457
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
ORIGIN
        1 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
       61 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
      121 acctaggcta cagccgatag tctggaacag cgcacttacg ggttgctgcg caacccaagt
      181 gctaccggcg cggcagcgtg acccgtgtcg gcggctccaa cggctcgcca tcgtccagaa
      241 aacacggctc atcgggcatc ggcaggcgct gctgcccgcg ccgttcccat tcctccgttt
      301 cggtcaaggc tggcaggtct ggttccatgc ccggaatgcc gggctggctg ggcggctcct
      361 cgccggggcc ggtcggtagt tgctgctcgc ccggatacag ggtcgggatg cggcgcaggt
      421 cgccatgccc caacagcgat tcgtcctggt cgtcgtgatc aaccaccacg gcggcactga
      481 acaccgacag gcgcaactgg tcgcggggct ggccccacgc cacgcggtca ttgaccacgt
      541 aggccgacac ggtgccgggg ccgttgagct tcacgacgga gatccagcgc tcggccacca
      601 agtccttgac tgcgtattgg accgtccgca aagaacgtcc gatgagcttg gaaagtgtct
      661 tctggctgac caccacggcg ttctggtggc ccatctgcgc cacgaggtga tgcagcagca
      721 ttgccgccgt gggtttcctc gcaataagcc cggcccacgc ctcatgcgct ttgcgttccg
      781 tttgcaccca gtgaccgggc ttgttcttgg cttgaatgcc gatttctctg gactgcgtgg
      841 ccatgcttat ctccatgcgg taggggtgcc gcacggttgc ggcaccatgc gcaatcagct
      901 gcaacttttc ggcagcgcga caacaattat gcgttgcgta aaagtggcag tcaattacag
      961 attttcttta acctacgcaa tgagctattg cggggggtgc cgcaatgagc tgttgcgtac
     1021 cccccttttt taagttgttg atttttaagt ctttcgcatt tcgccctata tctagttctt
     1081 tggtgcccaa agaagggcac ccctgcgggg ttcccccacg ccttcggcgc ggctccccct
     1141 ccggcaaaaa gtggcccctc cggggcttgt tgatcgactg cgcggccttc ggccttgccc
     1201 aaggtggcgc tgcccccttg gaacccccgc actcgccgcc gtgaggctcg gggggcaggc
     1261 gggcgggctt cgcccttcga ctgcccccac tcgcataggc ttgggtcgtt ccaggcgcgt
     1321 caaggccaag ccgctgcgcg gtcgctgcgc gagccttgac ccgccttcca cttggtgtcc
     1381 aaccggcaag cgaagcgcgc aggccgcagg ccggaggctt ttccccagag aaaattaaaa
     1441 aaattgatgg ggcaaggccg caggccgcgc agttggagcc ggtgggtatg tggtcgaagg
     1501 ctgggtagcc ggtgggcaat ccctgtggtc aagctcgtgg gcaggcgcag cctgtccatc
     1561 agcttgtcca gcagggttgt ccacgggccg agcgaagcga gccagccggt ggccgctcgc
     1621 ggccatcgtc cacatatcct agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa
     1681 ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg
     1741 agtccaagcg agctctcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat
     1801 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag
     1861 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc
     1921 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca
     1981 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc
     2041 gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat
     2101 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg
     2161 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag
     2221 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca
     2281 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc
     2341 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca
     2401 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga
     2461 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct
     2521 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc
     2581 ctcatcctgt ctcttgatca gatcatgatc ccctgcgcca tcagatcctt ggcggcaaga
     2641 aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca
     2701 attccgacgt cttaatcttt ctgcgaattg agatgacgcc actggctggg cgtcatcccg
     2761 gtttcccggg taaacaccac cgaaaaatag ttactatctt caaagccaca ttcggtcgaa
     2821 atatcactga ttaacaggcg gctatgctgg agaagatatt gcgcatgaca cactctgacc
     2881 tgtcgcagat attgattgat ggtcattcca gtctgctggc gaaattgctg acgcaaaacg
     2941 cgctcactgc acgatgcctc atcacaaaat ttatccagcg caaagggact tttcaggcta
     3001 gccgccagcc gggtaatcag cttatccagc aacgtttcgc tggatgttgg cggcaacgaa
     3061 tcactggtgt aacgatggcg attcagcaac atcaccaact gcccgaacag caactcagcc
     3121 atttcgttag caaacggcac atgctgacta ctttcatgct caagctgacc gataacctgc
     3181 cgcgcctgcg ccatccccat gctacctaag cgccagtgtg gttgccctgc gctggcgtta
     3241 aatcccggaa tcgccccctg ccagtcaaga ttcagcttca gacgctccgg gcaataaata
     3301 atattctgca aaaccagatc gttaacggaa gcgtaggagt gtttatcgtc agcatgaatg
     3361 taaaagagat cgccacgggt aatgcgataa gggcgatcgt tgagtacatg caggccatta
     3421 ccgcgccaga caatcaccag ctcacaaaaa tcatgtgtat gttcagcaaa gacatcttgc
     3481 ggataacggt cagccacagc gactgcctgc tggtcgctgg caaaaaaatc atctttgaga
     3541 agttttaact gatgcgccac cgtggctacc tcggccagag aacgaagttg attattcgca
     3601 atatggcgta caaatacgtt gagaagattc gcgttattgc agaaagccat cccgtccctg
     3661 gcgaatatca cgcggtgacc agttaaactc tcggcgaaaa agcgtcgaaa agtggttact
     3721 gtcgctgaat ccacagcgat aggcgatgtc agtaacgctg gcctcgctgt ggcgtagcag
     3781 atgtcgggct ttcatcagtc gcaggcggtt caggtatcgc tgaggcgtca gtcccgtttg
     3841 ctgcttaagc tgccgatgta gcgtacgcag tgaaagagaa aattgatccg ccacggcatc
     3901 ccaattcacc tcatcggcaa aatggtcctc cagccaggcc agaagcaagt tgagacgtga
     3961 tgcgctgttt tccaggttct cctgcaaact gcttttacgc agcaagagca gtaattgcat
     4021 aaacaagatc tcgcgactgg cggtcgaggg taaatcattt tccccttcct gctgttccat
     4081 ctgtgcaacc agctgtcgca cctgctgcaa tacgctgtgg ttaacgcgcc agtgagacgg
     4141 atactgccca tccagctctt gtggcagcaa ctgattcagc ccggcgagaa actgaaatcg
     4201 atccggcgag cgatacagca cattggtcag acacagatta tcggtatgtt catacagatg
     4261 ccgatcatga tcgcgtacga aacagaccgt gccaccggtg atggtatagg gctgcccatt
     4321 aaacacatga atacccgtgc catgttcgac aatcacaatt tcatgaaaat catgatgatg
     4381 ttcaggaaaa tccgcctgcg ggagccgggg ttctatcgcc acggacgcgt taccagacgg
     4441 aaaaaaatcc acactatgta atacggtcat actggcctcc tgatgtcgtc aacacggcga
     4501 aatagtaatc acgaggtcag gttcttacct taaattttcg acggaaaacc acgtaaaaaa
     4561 cgtcgatttt tcaagataca gcgtgaattt tcaggaaatg cggtgagcat cacatcacca
     4621 caattcagca aattgtgaac atcatcacgt tcatctttcc ctggttgcca atggcccatt
     4681 ttcctgtcag taacgagaag gtcgcgaatt caggcgctct ttagactggt cgtaacccgt
     4741 ttttttggga attcaaaaga tcttttaaga aggagatata catatgagca aaggagaaga
     4801 acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa
     4861 attttctgtc cgtggagagg gtgaaggtga tgctacaaac ggaaaactca cccttaaatt
     4921 tatttgcact actggaaaac tacctgttcc gtggccaaca cttgtcacta ctctgaccta
     4981 tggtgttcaa tgcttttccc gttatccgga tcacatgaaa cggcatgact ttttcaagag
     5041 tgccatgccc gaaggttatg tacaggaacg cactatatct ttcaaagatg acgggaccta
     5101 caagacgcgt gctgaagtca agtttgaagg tgataccctt gttaatcgta tcgagttaaa
     5161 gggtattgat tttaaagaag atggaaacat tcttggacac aaacttgagt acaactttaa
     5221 ctcacacaat gtatacatca cggcagacaa acaaaagaat ggaatcaaag ctaacttcaa
     5281 aattcgccac aacgttgaag atggttccgt tcaactagca gaccattatc aacaaaatac
     5341 tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcga cacaatctgt
     5401 cctttcgaaa gatcccaacg aaaagcgtga ccacatggtc cttcttgagt ttgtaactgc
     5461 tgctgggatt acacatggca tggatgagct ctacaaataa ggatccaaac tcgagtaagg
     5521 atctccaggc at
//
