LOCUS       sequence-434110-        4912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the Vanillic Acid Marionette 
            Cassette (VanRAM + PVanCC-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4912)
  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 4912)
  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: pMVV101
FEATURES             Location/Qualifiers
     source          1..4912
                     /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(1891..1910)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2350..3012)
                     /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      3013..3784
                     /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(3873..4688)
                     /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(3873..4688)
                     /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     4596..4615
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4680..4699
                     /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 caggggggtg
      961 aataatggac atgcctcgta ttaaaccggg tcagcgtgtt atgatggcac tgcgtaaaat
     1021 gattgcaagc ggtgaaatca aaagtggtga acgtattgca gaaattccga ccgcagcagc
     1081 actgggtgtt agccgtatgc cggttcgtat cgcactgcgt tcactggaac aagaaggtct
     1141 ggttgttcgt ctgggtgcac gtggttatgc agcccgtggt gttagcagcg atcagattcg
     1201 tgatgcaatt gaagttcgtg gtgttctgga aggttttgca gcacgtcgtc tggcagaacg
     1261 tggtatgacc gcagaaaccc atgcacgttt tgttgtactg attgcagaag gtgaagcact
     1321 gtttgcagcc ggtcgcctga atggtgaaga tctggatcgt tatgccgcat ataatcaggc
     1381 atttcatgat accctggtta gcgcagcagg taatggtgca gttgaaagcg cactggcacg
     1441 taatggtttt gaaccgtttg cagcagccgg tgcactggcc ctggatctga tggacctgtc
     1501 tgccgaatat gaacatctgc tggcagcaca tcgtcagcat caggcagttc tggatgcagt
     1561 tagctgtggt gatgccgaag gtgcagaacg tattatgcgt gatcatgcac tggcagcaat
     1621 tcgtaatgca aaagtttttg aagcagcagc aagcgcaggc gcaccgctgg gtgcagcatg
     1681 gtcaattcgt gcagattgat aaggatccta attggtaacg aatcagacaa ttgacggctc
     1741 gagggagtag catagggttt gcagaatccc tgcttcgtcc atttgacagg cacattatgc
     1801 atcgatgata agctgtcaaa catgagcaga tcctctacgc cagacgcatc gtggccggca
     1861 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
     1921 atcgggctcg ccacttcggg ctcatgagca aatattttat ctgaggtgct tcctcgctca
     1981 ctgactcgct gcacgaggca gacctcagcg ctagcggagt gtatactggc ttactatgtt
     2041 ggcactgatg agggtgtcag tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg
     2101 tgcgtcagca gaatatgtga tacaggatat attccgcttc ctcgctcact gactcgctac
     2161 gctcggtcgt tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg
     2221 aagatgccag gaagatactt aacagggaag tgagagggcc gcggcaaagc cgttctaccg
     2281 gcgccgcggc aaagccgttc taccggcgcc gcggcaaagc cgttctaccg gcgccgcggc
     2341 aaagccgttc taccggcgcg gcagcgtgac ccgtgtcggc ggctccaacg gctcgccatc
     2401 gtccagaaaa cacggctcat cgggcatcgg caggcgctgc tgcccgcgcc gttcccattc
     2461 ctccgtttcg gtcaaggctg gcaggtctgg ttccatgccc ggaatgccgg gctggctggg
     2521 cggctcctcg ccggggccgg tcggtagttg ctgctcgccc ggatacaggg tcgggatgcg
     2581 gcgcaggtcg ccatgcccca acagcgattc gtcctggtcg tcgtgatcaa ccaccacggc
     2641 ggcactgaac accgacaggc gcaactggtc gcggggctgg ccccacgcca cgcggtcatt
     2701 gaccacgtag gccgacacgg tgccggggcc gttgagcttc acgacggaga tccagcgctc
     2761 ggccaccaag tccttgactg cgtattggac cgtccgcaaa gaacgtccga tgagcttgga
     2821 aagtgtcttc tggctgacca ccacggcgtt ctggtggccc atctgcgcca cgaggtgatg
     2881 cagcagcatt gccgccgtgg gtttcctcgc aataagcccg gcccacgcct catgcgcttt
     2941 gcgttccgtt tgcacccagt gaccgggctt gttcttggct tgaatgccga tttctctgga
     3001 ctgcgtggcc atgcttatct ccatgcggta ggggtgccgc acggttgcgg caccatgcgc
     3061 aatcagctgc aacttttcgg cagcgcgaca acaattatgc gttgcgtaaa agtggcagtc
     3121 aattacagat tttctttaac ctacgcaatg agctattgcg gggggtgccg caatgagctg
     3181 ttgcgtaccc ccctttttta agttgttgat ttttaagtct ttcgcatttc gccctatatc
     3241 tagttctttg gtgcccaaag aagggcaccc ctgcggggtt cccccacgcc ttcggcgcgg
     3301 ctccccctcc ggcaaaaagt ggcccctccg gggcttgttg atcgactgcg cggccttcgg
     3361 ccttgcccaa ggtggcgctg cccccttgga acccccgcac tcgccgccgt gaggctcggg
     3421 gggcaggcgg gcgggcttcg cccttcgact gcccccactc gcataggctt gggtcgttcc
     3481 aggcgcgtca aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc gccttccact
     3541 tggtgtccaa ccggcaagcg aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa
     3601 aattaaaaaa attgatgggg caaggccgca ggccgcgcag ttggagccgg tgggtatgtg
     3661 gtcgaaggct gggtagccgg tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc
     3721 tgtccatcag cttgtccagc agggttgtcc acgggccgag cgaagcgagc cagccggtgg
     3781 ccgcgaagat catcttatta aggggtctga cgctcagtgg aacgaaaaat caatctaaag
     3841 tatatatgag taaacttggt ctgacagtta ccttagaaaa actcatcgag catcaaatga
     3901 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     3961 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     4021 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     4081 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta
     4141 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     4201 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     4261 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     4321 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     4381 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     4441 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     4501 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     4561 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     4621 aaatcagcat ccatgttgga atttaatcgc ggcctggagc aagacgtttc ccgttgaata
     4681 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat
     4741 gatatatttt tatcttgtgc aatgtacatc agagattttg agacacaacc aattattgaa
     4801 ggcctcccta acggggggcc tttttttgtt tctggtctcc cgcttaacga tcgttggctg
     4861 attggatcca attgacagct agctcagtcc taggtaccat tggatccaat ag
//
