LOCUS       sequence-431951-        4749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the aTc Marionette Cassette 
            (TetR + PTet-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4749)
  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 4749)
  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: pMVV103
FEATURES             Location/Qualifiers
     source          1..4749
                     /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"
     CDS             964..1587
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(1037..1056)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(1777..1796)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2161..2823)
                     /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      2824..3595
                     /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(3684..4499)
                     /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(3684..4499)
                     /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     4407..4426
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4491..4510
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4694..4712
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4719..4737
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
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 aatatgtcca gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag
     1021 gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct
     1081 acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg
     1141 ttagataggc accatactca cttttgccct ttagaagggg aaagctggca agatttttta
     1201 cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta
     1261 catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt
     1321 ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat
     1381 tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa
     1441 acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac
     1501 caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa
     1561 caacttaaat gtgaaagtgg gtcctgataa ggatcctaat tggtaacgaa tcagaattga
     1621 cggctcgagg gagtagcata gggtttgcag aatccctgct tcgtccattt gacaggcaca
     1681 ttatgcatcg atgataagct gtcaaacatg agcagatcct ctacgccgga cgcatcgtgg
     1741 ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg
     1801 gggaagatcg ggctcgccac ttcgggctca tgagcaaata ttttatctga ggtgcttcct
     1861 cgctcactga ctcgctgcac gaggcagacc tcagcgctag cggagtgtat actggcttac
     1921 tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg
     1981 caccggtgcg tcagcagaat atgtgataca ggatatattc cgcttcctcg ctcactgact
     2041 cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt
     2101 tcctggaaga tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt
     2161 ctaccggcgc ggcagcgtga cccgtgtcgg cggctccaac ggctcgccat cgtccagaaa
     2221 acacggctca tcgggcatcg gcaggcgctg ctgcccgcgc cgttcccatt cctccgtttc
     2281 ggtcaaggct ggcaggtctg gttccatgcc cggaatgccg ggctggctgg gcggctcctc
     2341 gccggggccg gtcggtagtt gctgctcgcc cggatacagg gtcgggatgc ggcgcaggtc
     2401 gccatgcccc aacagcgatt cgtcctggtc gtcgtgatca accaccacgg cggcactgaa
     2461 caccgacagg cgcaactggt cgcggggctg gccccacgcc acgcggtcat tgaccacgta
     2521 ggccgacacg gtgccggggc cgttgagctt cacgacggag atccagcgct cggccaccaa
     2581 gtccttgact gcgtattgga ccgtccgcaa agaacgtccg atgagcttgg aaagtgtctt
     2641 ctggctgacc accacggcgt tctggtggcc catctgcgcc acgaggtgat gcagcagcat
     2701 tgccgccgtg ggtttcctcg caataagccc ggcccacgcc tcatgcgctt tgcgttccgt
     2761 ttgcacccag tgaccgggct tgttcttggc ttgaatgccg atttctctgg actgcgtggc
     2821 catgcttatc tccatgcggt aggggtgccg cacggttgcg gcaccatgcg caatcagctg
     2881 caacttttcg gcagcgcgac aacaattatg cgttgcgtaa aagtggcagt caattacaga
     2941 ttttctttaa cctacgcaat gagctattgc ggggggtgcc gcaatgagct gttgcgtacc
     3001 cccctttttt aagttgttga tttttaagtc tttcgcattt cgccctatat ctagttcttt
     3061 ggtgcccaaa gaagggcacc cctgcggggt tcccccacgc cttcggcgcg gctccccctc
     3121 cggcaaaaag tggcccctcc ggggcttgtt gatcgactgc gcggccttcg gccttgccca
     3181 aggtggcgct gcccccttgg aacccccgca ctcgccgccg tgaggctcgg ggggcaggcg
     3241 ggcgggcttc gcccttcgac tgcccccact cgcataggct tgggtcgttc caggcgcgtc
     3301 aaggccaagc cgctgcgcgg tcgctgcgcg agccttgacc cgccttccac ttggtgtcca
     3361 accggcaagc gaagcgcgca ggccgcaggc cggaggcttt tccccagaga aaattaaaaa
     3421 aattgatggg gcaaggccgc aggccgcgca gttggagccg gtgggtatgt ggtcgaaggc
     3481 tgggtagccg gtgggcaatc cctgtggtca agctcgtggg caggcgcagc ctgtccatca
     3541 gcttgtccag cagggttgtc cacgggccga gcgaagcgag ccagccggtg gccgcgaaga
     3601 tcatcttatt aaggggtctg acgctcagtg gaacgaaaaa tcaatctaaa gtatatatga
     3661 gtaaacttgg tctgacagtt accttagaaa aactcatcga gcatcaaatg aaactgcaat
     3721 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     3781 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     3841 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     3901 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct
     3961 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     4021 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     4081 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     4141 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     4201 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     4261 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     4321 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     4381 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     4441 tccatgttgg aatttaatcg cggcctggag caagacgttt cccgttgaat atggctcata
     4501 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     4561 ttatcttgtg caatgtacat cagagatttt gagacacaac caattattga aggcctccct
     4621 aacggggggc ctttttttgt ttctggtctc ccgcttaacg atcgttggct gttttcagca
     4681 ggacgcactg acctccctat cagtgataga gattgacatc cctatcagtg atagagatac
     4741 tgagcacag
//
