LOCUS       sequence-434104-        4753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the Cuminic Acid Marionette 
            Cassette (CymRAM + PCymRC-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4753)
  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 4753)
  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: pMVV099
FEATURES             Location/Qualifiers
     source          1..4753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        1..50
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(100..102,103..816)
                     /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             100..813
                     /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(130..158)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     753..773
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1767..1786)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2151..2813)
                     /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      2814..3585
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             complement(3674..4489)
                     /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             complement(3674..4489)
                     /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     4397..4416
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4481..4500
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4662..4689
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     protein_bind    4720..4747
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
ORIGIN
        1 ctgtcaccgg atgtgctttc cggtctgatg agtccgtgag gacgaaacag cctctacaaa
       61 taattttgtt taatactaga gaaagagggg aaatactaga tgagcaaagg agaagaactt
      121 ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt
      181 tctgtccgtg gagagggtga aggtgatgct acaaacggaa aactcaccct taaatttatt
      241 tgcactactg gaaaactacc tgttccgtgg ccaacacttg tcactactct gacctatggt
      301 gttcaatgct tttcccgtta tccggatcac atgaaacggc atgacttttt caagagtgcc
      361 atgcccgaag gttatgtaca ggaacgcact atatctttca aagatgacgg gacctacaag
      421 acgcgtgctg aagtcaagtt tgaaggtgat acccttgtta atcgtatcga gttaaagggt
      481 attgatttta aagaagatgg aaacattctt ggacacaaac ttgagtacaa ctttaactca
      541 cacaatgtat acatcacggc agacaaacaa aagaatggaa tcaaagctaa cttcaaaatt
      601 cgccacaacg ttgaagatgg ttccgttcaa ctagcagacc attatcaaca aaatactcca
      661 attggcgatg gccctgtcct tttaccagac aaccattacc tgtcgacaca atctgtcctt
      721 tcgaaagatc ccaacgaaaa gcgtgaccac atggtccttc ttgagtttgt aactgctgct
      781 gggattacac atggcatgga tgagctctac aaataactcg gtaccaaatt ccagaaaaga
      841 ggcctcccga aaggggggcc ttttttcgtt ttggtccaat ggcggcgcgc catcgaatgg
      901 tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
      961 aatatgagcc cgaaacgtcg tacccaggca gaacgtgcaa tggaaaccca gggtaaactg
     1021 attgcagcag cactgggtgt tctgcgtgaa aaaggttatg caggttttcg tattgcagat
     1081 gttccgggtg cagccggtgt tagccgtggt gcacagagcc atcattttcc gaccaaactg
     1141 gaactgctgc tggcaacctt tgaatggctg tatgagcaga ttaccgaacg tagccgtgca
     1201 cgtctggcaa aactgaaacc ggaagatgat gttattcagc agatgctgga tgatgcagca
     1261 gaattttttc tggatgatga ttttagcatc ggcctggatc tgattgttgc agcagatcgt
     1321 gatccggcac tgcgtgaagg tattcagcgt accgttgaac gtaatcgttt tgttgttgaa
     1381 gatatgtggc tgggtgtgct ggtgagccgt ggtctgagcc gtgatgatgc cgaagatatt
     1441 ctgtggctga tttttaacag cgttcgtggt ctggtagttc gtagcctgtg gcagaaagat
     1501 aaagaacgtt ttgaacgtgt gcgtaatagc accctggaaa ttgcacgtga acgttatgca
     1561 aaattcaaac gttgataagg atcctaattg gtaacgaatc agacaattga cggctcgagg
     1621 gagtagcata gggtttgcag aatccctgct tcgtccattt gacaggcaca ttatgcatcg
     1681 atgataagct gtcaaacatg agcagatcct ctacgccgga cgcatcgtgg ccggcatcac
     1741 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
     1801 ggctcgccac ttcgggctca tgagcaaata ttttatctga ggtgcttcct cgctcactga
     1861 ctcgctgcac gaggcagacc tcagcgctag cggagtgtat actggcttac tatgttggca
     1921 ctgatgaggg tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg caccggtgcg
     1981 tcagcagaat atgtgataca ggatatattc cgcttcctcg ctcactgact cgctacgctc
     2041 ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga
     2101 tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt ctaccggcgc
     2161 ggcagcgtga cccgtgtcgg cggctccaac ggctcgccat cgtccagaaa acacggctca
     2221 tcgggcatcg gcaggcgctg ctgcccgcgc cgttcccatt cctccgtttc ggtcaaggct
     2281 ggcaggtctg gttccatgcc cggaatgccg ggctggctgg gcggctcctc gccggggccg
     2341 gtcggtagtt gctgctcgcc cggatacagg gtcgggatgc ggcgcaggtc gccatgcccc
     2401 aacagcgatt cgtcctggtc gtcgtgatca accaccacgg cggcactgaa caccgacagg
     2461 cgcaactggt cgcggggctg gccccacgcc acgcggtcat tgaccacgta ggccgacacg
     2521 gtgccggggc cgttgagctt cacgacggag atccagcgct cggccaccaa gtccttgact
     2581 gcgtattgga ccgtccgcaa agaacgtccg atgagcttgg aaagtgtctt ctggctgacc
     2641 accacggcgt tctggtggcc catctgcgcc acgaggtgat gcagcagcat tgccgccgtg
     2701 ggtttcctcg caataagccc ggcccacgcc tcatgcgctt tgcgttccgt ttgcacccag
     2761 tgaccgggct tgttcttggc ttgaatgccg atttctctgg actgcgtggc catgcttatc
     2821 tccatgcggt aggggtgccg cacggttgcg gcaccatgcg caatcagctg caacttttcg
     2881 gcagcgcgac aacaattatg cgttgcgtaa aagtggcagt caattacaga ttttctttaa
     2941 cctacgcaat gagctattgc ggggggtgcc gcaatgagct gttgcgtacc cccctttttt
     3001 aagttgttga tttttaagtc tttcgcattt cgccctatat ctagttcttt ggtgcccaaa
     3061 gaagggcacc cctgcggggt tcccccacgc cttcggcgcg gctccccctc cggcaaaaag
     3121 tggcccctcc ggggcttgtt gatcgactgc gcggccttcg gccttgccca aggtggcgct
     3181 gcccccttgg aacccccgca ctcgccgccg tgaggctcgg ggggcaggcg ggcgggcttc
     3241 gcccttcgac tgcccccact cgcataggct tgggtcgttc caggcgcgtc aaggccaagc
     3301 cgctgcgcgg tcgctgcgcg agccttgacc cgccttccac ttggtgtcca accggcaagc
     3361 gaagcgcgca ggccgcaggc cggaggcttt tccccagaga aaattaaaaa aattgatggg
     3421 gcaaggccgc aggccgcgca gttggagccg gtgggtatgt ggtcgaaggc tgggtagccg
     3481 gtgggcaatc cctgtggtca agctcgtggg caggcgcagc ctgtccatca gcttgtccag
     3541 cagggttgtc cacgggccga gcgaagcgag ccagccggtg gccgcgaaga tcatcttatt
     3601 aaggggtctg acgctcagtg gaacgaaaaa tcaatctaaa gtatatatga gtaaacttgg
     3661 tctgacagtt accttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     3721 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     3781 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     3841 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     3901 catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt
     3961 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     4021 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     4081 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     4141 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     4201 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     4261 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     4321 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     4381 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     4441 aatttaatcg cggcctggag caagacgttt cccgttgaat atggctcata acaccccttg
     4501 tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg
     4561 caatgtacat cagagatttt gagacacaac caattattga aggcctccct aacggggggc
     4621 ctttttttgt ttctggtctc ccgcttaacg atcgttggct gaacaaacag acaatctggt
     4681 ctgtttgtat tatggaaaat ttttctgtat aatagattca acaaacagac aatctggtct
     4741 gtttgtatta tag
//
