LOCUS       sequence-442628-        4859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the 3OC6-HSL Marionette 
            Cassette (LuxR + PLuxB-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4859)
  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 4859)
  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: pMVV100
FEATURES             Location/Qualifiers
     source          1..4859
                     /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(1908..1927)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2292..2954)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHRNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      2955..3726
                     /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(3815..4630)
                     /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(3815..4630)
                     /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     4538..4557
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4622..4641
                     /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 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
      961 aatatgaaaa acataaatgc cgacgacaca tacagaataa ttaataaaat taaagcttgt
     1021 agaagcaata atgatattaa tcaatgctta tctgatatga ctaaaatggt acattgtgaa
     1081 tattatttac tcgcgatcat ttatcctcat tctatggtta aatctgatat ttcaatccta
     1141 gataattacc ctaaaaaatg gaggcaatat tatgatgacg ctaatttaat aaaatatgat
     1201 cctatagtag attattctaa ctccaatcat tcaccaatta attggaatat atttgaaaac
     1261 aatgctgtaa ataaaaaatc tccaaatgta attaaagaag cgaaaacatc aggtcttatc
     1321 actgggttta gtttccctat tcatacggct aacaatggct tcggaatgct tagttttgca
     1381 cattcagaaa aagacaacta tatagatagt ttatttttac atgcgtgtat gaacatacca
     1441 ttaattgttc cttctctagt tgataattat cgaaaaataa atatagcaaa taataaatca
     1501 aacaacgatt taaccaaaag agaaaaagaa tgtttagcgt gggcatgcga aggaaaaagc
     1561 tcttgggata tttcaaaaat attaggttgc agtgagcgta ctgtcacttt ccatttaacc
     1621 aatgcgcaaa tgaaactcaa tacaacaaac cgctgccaaa gtatttctaa agcaatttta
     1681 acaggagcaa ttgattgccc atactttaaa aattgataag gatcctaatt ggtaacgaat
     1741 cagacaattg acggctcgag ggagtagcat agggtttgca gaatccctgc ttcgtccatt
     1801 tgacaggcac attatgcatc gatgataagc tgtcaaacat gagcagatcc tctacgccgg
     1861 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
     1921 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcaaat attttatctg
     1981 aggtgcttcc tcgctcactg actcgctgca cgaggcagac ctcagcgcta gcggagtgta
     2041 tactggctta ctatgttggc actgatgagg gtgtcagtga agtgcttcat gtggcaggag
     2101 aaaaaaggct gcaccggtgc gtcagcagaa tatgtgatac aggatatatt ccgcttcctc
     2161 gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg
     2221 gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg
     2281 gcaaagccgt tctaccggcg cggcagcgtg acccgtgtcg gcggctccaa cggctcgcca
     2341 tcgtccagaa aacacggctc atcgggcatc ggcaggcgct gctgcccgcg ccgttcccat
     2401 tcctccgttt cggtcaaggc tggcaggtct ggttccatgc ccggaatgcc gggctggctg
     2461 ggcggctcct cgccggggcc ggtcggtagt tgctgctcgc ccggatacag ggtcgggatg
     2521 cggcgcaggt cgccatgccc caacagcgat tcgtcctggt cgtcgtgatc aaccaccacg
     2581 gcggcactga acaccgacag gcgcaactgg tcgcggggct ggccccacgc cacgcggtca
     2641 ttgaccacgt aggccgacac ggtgccgggg ccgttgagct tcacgacgga gatccagcgc
     2701 tcggccacca agtccttgac tgcgtattgg accgtccgca aagaacgtcc gatgagcttg
     2761 gaaagtgtct tctggctgac caccacggcg ttccggtggc ccatctgcgc cacgaggtga
     2821 tgcagcagca ttgccgccgt gggtttcctc gcaataagcc cggcccacgc ctcatgcgct
     2881 ttgcgttccg tttgcaccca gtgaccgggc ttgttcttgg cttgaatgcc gatttctctg
     2941 gactgcgtgg ccatgcttat ctccatgcgg taggggtgcc gcacggttgc ggcaccatgc
     3001 gcaatcagct gcaacttttc ggcagcgcga caacaattat gcgttgcgta aaagtggcag
     3061 tcaattacag attttcttta acctacgcaa tgagctattg cggggggtgc cgcaatgagc
     3121 tgttgcgtac cccccttttt taagttgttg atttttaagt ctttcgcatt tcgccctata
     3181 tctagttctt tggtgcccaa agaagggcac ccctgcgggg ttcccccacg ccttcggcgc
     3241 ggctccccct ccggcaaaaa gtggcccctc cggggcttgt tgatcgactg cgcggccttc
     3301 ggccttgccc aaggtggcgc tgcccccttg gaacccccgc actcgccgcc gtgaggctcg
     3361 gggggcaggc gggcgggctt cgcccttcga ctgcccccac tcgcataggc ttgggtcgtt
     3421 ccaggcgcgt caaggccaag ccgctgcgcg gtcgctgcgc gagccttgac ccgccttcca
     3481 cttggtgtcc aaccggcaag cgaagcgcgc aggccgcagg ccggaggctt ttccccagag
     3541 aaaattaaaa aaattgatgg ggcaaggccg caggccgcgc agttggagcc ggtgggtatg
     3601 tggtcgaagg ctgggtagcc ggtgggcaat ccctgtggtc aagctcgtgg gcaggcgcag
     3661 cctgtccatc agcttgtcca gcagggttgt ccacgggccg agcgaagcga gccagccggt
     3721 ggccgcgaag atcatcttat taaggggtct gacgctcagt ggaacgaaaa atcaatctaa
     3781 agtatatatg agtaaacttg gtctgacagt taccttagaa aaactcatcg agcatcaaat
     3841 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     3901 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     3961 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     4021 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct
     4081 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     4141 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     4201 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     4261 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     4321 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     4381 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     4441 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     4501 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     4561 ataaatcagc atccatgttg gaatttaatc gcggcctgga gcaagacgtt tcccgttgaa
     4621 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     4681 atgatatatt tttatcttgt gcaatgtaca tcagagattt tgagacacaa ccaattattg
     4741 aaggcctccc taacgggggg cctttttttg tttctggtct cccgcttaac gatcgttggc
     4801 tgacctgtag gatcgtacag gtttacgcaa gaaaatggtt tgttacagtc gaataaaag
//
