LOCUS       sequence-431948-        4738 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the naringenin Marionette 
            Cassette (TtgRAM + PTtG-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4738)
  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 4738)
  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: pMVV106
FEATURES             Location/Qualifiers
     source          1..4738
                     /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(1791..1810)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2175..2837)
                     /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      2838..3609
                     /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(3698..4513)
                     /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(3698..4513)
                     /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     4421..4440
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4505..4524
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
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 cagggaggtg
      961 aatatggttc gtcgtaccaa agaagaggca caagaaaccc gtgcacagat tattgaagca
     1021 gcagaacgtg cattctataa acgtggtgtt gcacgtacca ccctggcaga tattgcagaa
     1081 ctggcaggcg ttacccgtgg tgcaatttat tggcatttta acaacaaagc cgaactggtt
     1141 caggcactgc tggatagcct gcatgaaacc catgatcatc tggcacgtgc aagcgaaagc
     1201 gaagatgaag ttgatccgct gggttgtatg cgtaaactgc tgctgcaggt ttttaatgaa
     1261 ctggttctgg atgcacgtac ccgtcgtatt aatgaaatcc tgcatcacaa atgcgagttc
     1321 accgatgata tgtgtgaaat tcgtcagcag catcagagcg cagttctgga ttgtcataaa
     1381 ggtattaccc tgacactggc aaatgtagtt cgtcgtggtc agctgcctgg tgaactggat
     1441 gcagaacgtg ccgcagttgc aatgtttgcc tatgttgatg gtctgattcg tcgttggctg
     1501 ctgctgccgg atagcgttga tctgctgggt gatgttgaaa aatgggttga taccggtctg
     1561 gatatgctgc gtctgagtcc ggcactgcgt aaataatgat aaggatccta attggtaacg
     1621 aatcagacaa ttgacggctc gagggagtag catagggttt gcagaatccc tgcttcgtcc
     1681 atttgacagg cacattatgc atcgatgata agctgtcaaa catgagcaga tcctctacgc
     1741 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
     1801 cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagca aatattttat
     1861 ctgaggtgct tcctcgctca ctgactcgct gcacgaggca gacctcagcg ctagcggagt
     1921 gtatactggc ttactatgtt ggcactgatg agggtgtcag tgaagtgctt catgtggcag
     1981 gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga tacaggatat attccgcttc
     2041 ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa tggcttacga
     2101 acggggcgga gatttcctgg aagatgccag gaagatactt aacagggaag tgagagggcc
     2161 gcggcaaagc cgttctaccg gcgcggcagc gtgacccgtg tcggcggctc caacggctcg
     2221 ccatcgtcca gaaaacacgg ctcatcgggc atcggcaggc gctgctgccc gcgccgttcc
     2281 cattcctccg tttcggtcaa ggctggcagg tctggttcca tgcccggaat gccgggctgg
     2341 ctgggcggct cctcgccggg gccggtcggt agttgctgct cgcccggata cagggtcggg
     2401 atgcggcgca ggtcgccatg ccccaacagc gattcgtcct ggtcgtcgtg atcaaccacc
     2461 acggcggcac tgaacaccga caggcgcaac tggtcgcggg gctggcccca cgccacgcgg
     2521 tcattgacca cgtaggccga cacggtgccg gggccgttga gcttcacgac ggagatccag
     2581 cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc gcaaagaacg tccgatgagc
     2641 ttggaaagtg tcttctggct gaccaccacg gcgttctggt ggcccatctg cgccacgagg
     2701 tgatgcagca gcattgccgc cgtgggtttc ctcgcaataa gcccggccca cgcctcatgc
     2761 gctttgcgtt ccgtttgcac ccagtgaccg ggcttgttct tggcttgaat gccgatttct
     2821 ctggactgcg tggccatgct tatctccatg cggtaggggt gccgcacggt tgcggcacca
     2881 tgcgcaatca gctgcaactt ttcggcagcg cgacaacaat tatgcgttgc gtaaaagtgg
     2941 cagtcaatta cagattttct ttaacctacg caatgagcta ttgcgggggg tgccgcaatg
     3001 agctgttgcg tacccccctt ttttaagttg ttgattttta agtctttcgc atttcgccct
     3061 atatctagtt ctttggtgcc caaagaaggg cacccctgcg gggttccccc acgccttcgg
     3121 cgcggctccc cctccggcaa aaagtggccc ctccggggct tgttgatcga ctgcgcggcc
     3181 ttcggccttg cccaaggtgg cgctgccccc ttggaacccc cgcactcgcc gccgtgaggc
     3241 tcggggggca ggcgggcggg cttcgccctt cgactgcccc cactcgcata ggcttgggtc
     3301 gttccaggcg cgtcaaggcc aagccgctgc gcggtcgctg cgcgagcctt gacccgcctt
     3361 ccacttggtg tccaaccggc aagcgaagcg cgcaggccgc aggccggagg cttttcccca
     3421 gagaaaatta aaaaaattga tggggcaagg ccgcaggccg cgcagttgga gccggtgggt
     3481 atgtggtcga aggctgggta gccggtgggc aatccctgtg gtcaagctcg tgggcaggcg
     3541 cagcctgtcc atcagcttgt ccagcagggt tgtccacggg ccgagcgaag cgagccagcc
     3601 ggtggccgcg aagatcatct tattaagggg tctgacgctc agtggaacga aaaatcaatc
     3661 taaagtatat atgagtaaac ttggtctgac agttacctta gaaaaactca tcgagcatca
     3721 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     3781 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     3841 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     3901 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     3961 gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     4021 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     4081 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     4141 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     4201 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     4261 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     4321 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     4381 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     4441 catataaatc agcatccatg ttggaattta atcgcggcct ggagcaagac gtttcccgtt
     4501 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     4561 atgatgatat atttttatct tgtgcaatgt acatcagaga ttttgagaca caaccaatta
     4621 ttgaaggcct ccctaacggg gggccttttt ttgtttctgg tctcccgctt aacgatcgtt
     4681 ggctgcaccc agcagtattt acaaacaacc atgaatgtaa gtatattcct tagcaaag
//
