LOCUS       sequence-440372-        4720 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the DAPG Marionette Cassette 
            (PhIFAM + PPhIF-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4720)
  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 4720)
  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: pMVV098
FEATURES             Location/Qualifiers
     source          1..4720
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(39..854)
                     /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(39..854)
                     /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     762..781
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     846..865
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     misc_RNA        1095..1144
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(1194..1196,1197..1910)
                     /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             1194..1907
                     /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(1224..1252)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1847..1867
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2852..2871)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(3236..3898)
                     /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      3899..4670
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
ORIGIN
        1 ctaaagtata tatgagtaaa cttggtctga cagttacctt agaaaaactc atcgagcatc
       61 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
      121 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
      181 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
      241 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
      301 agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
      361 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
      421 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
      481 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
      541 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
      601 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
      661 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
      721 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
      781 ccatataaat cagcatccat gttggaattt aatcgcggcc tggagcaaga cgtttcccgt
      841 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
      901 catgatgata tatttttatc ttgtgcaatg tacatcagag attttgagac acaaccaatt
      961 attgaaggcc tccctaacgg ggggcctttt tttgtttctg gtctcccgct taacgatcgt
     1021 tggctgcgac gtacggtgga atctgattcg ttaccaattg acatgatacg aaacgtaccg
     1081 tatcgttaag gtagctgtca ccggatgtgc tttccggtct gatgagtccg tgaggacgaa
     1141 acagcctcta caaataattt tgtttaatac tagagaaaga ggggaaatac tagatgagca
     1201 aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta
     1261 atgggcacaa attttctgtc cgtggagagg gtgaaggtga tgctacaaac ggaaaactca
     1321 cccttaaatt tatttgcact actggaaaac tacctgttcc gtggccaaca cttgtcacta
     1381 ctctgaccta tggtgttcaa tgcttttccc gttatccgga tcacatgaaa cggcatgact
     1441 ttttcaagag tgccatgccc gaaggttatg tacaggaacg cactatatct ttcaaagatg
     1501 acgggaccta caagacgcgt gctgaagtca agtttgaagg tgataccctt gttaatcgta
     1561 tcgagttaaa gggtattgat tttaaagaag atggaaacat tcttggacac aaacttgagt
     1621 acaactttaa ctcacacaat gtatacatca cggcagacaa acaaaagaat ggaatcaaag
     1681 ctaacttcaa aattcgccac aacgttgaag atggttccgt tcaactagca gaccattatc
     1741 aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcga
     1801 cacaatctgt cctttcgaaa gatcccaacg aaaagcgtga ccacatggtc cttcttgagt
     1861 ttgtaactgc tgctgggatt acacatggca tggatgagct ctacaaataa ctcggtacca
     1921 aattccagaa aagaggcctc ccgaaagggg ggcctttttt cgttttggtc caatggcggc
     1981 gcgccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     2041 aattcatggg ggtgaatatg gcacgtaccc cgagccgtag cagcattggt agcctgcgta
     2101 gtccgcatac ccataaagca attctgacca gcaccattga aatcctgaaa gaatgtggtt
     2161 atagcggtct gagcattgaa agcgtggcac gtcgcgccgg tgcaggcaaa ccgaccattt
     2221 atcgttggtg gaccaacaaa gcagcactga ttgccgaagt gtatgaaaat gaaatcgaac
     2281 aggtacgtaa atttccggat ttgggtagct ttaaagccga tctggatttt ctgctgcata
     2341 atctgtggaa agtttggcgt gaaaccattt gtggtgaagc atttcgttgt gttattgcag
     2401 aagcacagtt ggaccctgta accctgaccc aactgaaaga tcagtttatg gaacgtcgtc
     2461 gtgagatacc gaaaaaactg gttgaagatg ccattagcaa tggtgaactg ccgaaagata
     2521 tcaatcgtga actgctgctg gatatgattt ttggtttttg ttggtatcgc ctgctgaccg
     2581 aacagttgac cgttgaacag gatattgaag aatttacctt cctgctgatt aatggtgttt
     2641 gtccgggtac acagtgttga taaggatcct aattggtaac gaatcagaca attgacggct
     2701 cgagggagta gcatagggtt tgcagaatcc ctgcttcgtc catttgacag gcacattatg
     2761 catcgatgat aagctgtcaa acatgagcag atcctctacg ccggacgcat cgtggccggc
     2821 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
     2881 gatcgggctc gccacttcgg gctcatgagc aaatatttta tctgaggtgc ttcctcgctc
     2941 actgactcgc tgcacgaggc agacctcagc gctagcggag tgtatactgg cttactatgt
     3001 tggcactgat gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa ggctgcaccg
     3061 gtgcgtcagc agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta
     3121 cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg
     3181 gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgttctacc
     3241 ggcgcggcag cgtgacccgt gtcggcggct ccaacggctc gccatcgtcc agaaaacacg
     3301 gctcatcggg catcggcagg cgctgctgcc cgcgccgttc ccattcctcc gtttcggtca
     3361 aggctggcag gtctggttcc atgcccggaa tgccgggctg gctgggcggc tcctcgccgg
     3421 ggccggtcgg tagttgctgc tcgcccggat acagggtcgg gatgcggcgc aggtcgccat
     3481 gccccaacag cgattcgtcc tggtcgtcgt gatcaaccac cacggcggca ctgaacaccg
     3541 acaggcgcaa ctggtcgcgg ggctggcccc acgccacgcg gtcattgacc acgtaggccg
     3601 acacggtgcc ggggccgttg agcttcacga cggagatcca gcgctcggcc accaagtcct
     3661 tgactgcgta ttggaccgtc cgcaaagaac gtccgatgag cttggaaagt gtcttctggc
     3721 tgaccaccac ggcgttctgg tggcccatct gcgccacgag gtgatgcagc agcattgccg
     3781 ccgtgggttt cctcgcaata agcccggccc acgcctcatg cgctttgcgt tccgtttgca
     3841 cccagtgacc gggcttgttc ttggcttgaa tgccgatttc tctggactgc gtggccatgc
     3901 ttatctccat gcggtagggg tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact
     3961 tttcggcagc gcgacaacaa ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc
     4021 tttaacctac gcaatgagct attgcggggg gtgccgcaat gagctgttgc gtacccccct
     4081 tttttaagtt gttgattttt aagtctttcg catttcgccc tatatctagt tctttggtgc
     4141 ccaaagaagg gcacccctgc ggggttcccc cacgccttcg gcgcggctcc ccctccggca
     4201 aaaagtggcc cctccggggc ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg
     4261 gcgctgcccc cttggaaccc ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg
     4321 gcttcgccct tcgactgccc ccactcgcat aggcttgggt cgttccaggc gcgtcaaggc
     4381 caagccgctg cgcggtcgct gcgcgagcct tgacccgcct tccacttggt gtccaaccgg
     4441 caagcgaagc gcgcaggccg caggccggag gcttttcccc agagaaaatt aaaaaaattg
     4501 atggggcaag gccgcaggcc gcgcagttgg agccggtggg tatgtggtcg aaggctgggt
     4561 agccggtggg caatccctgt ggtcaagctc gtgggcaggc gcagcctgtc catcagcttg
     4621 tccagcaggg ttgtccacgg gccgagcgaa gcgagccagc cggtggccgc gaagatcatc
     4681 ttattaaggg gtctgacgct cagtggaacg aaaaatcaat
//
