LOCUS       sequence-434119-        5029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the 3OC14-HSL Marionette 
            Cassette (CinRAM + PCin-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5029)
  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 5029)
  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: pMVV109
FEATURES             Location/Qualifiers
     source          1..5029
                     /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(1907..1926)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2291..2953)
                     /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      2954..3725
                     /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(3814..4629)
                     /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(3814..4629)
                     /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     4537..4556
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4621..4640
                     /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 aatatgattg agaataccta tagcgaaaag ttcgagtccg cgttcgaaca gatcaaagcg
     1021 gcggccaacg tggatgccgc catccgtatt ctccaggcgg aatataacct cgatttcgtc
     1081 acctaccatc tcgcccagac aatcgcgagc aagatcgatt cgcccttcgt gcgcaccacc
     1141 tatccggatg cctgggtttc ccgttacctc ctcaactgct atgtgaaggt cgatccgatc
     1201 atcaagcagg gcttcgaacg ccagctgccc ttcgactgga gcgaggtcga accgacgccg
     1261 gaggcctatg ccatgctggt cgacgcccag aaacacggca tcgatgacaa tggctactcc
     1321 atccccgtcg ccgacaaggc gcagcgccgc gccctgctgt cgctgaatgc ccatataccg
     1381 gccgacgaat ggaccgagct cgtgcgccgc tgccgcaatg agtggatcga gatcgcccat
     1441 ctgatccacc gcaaggccgt atatgagctg catggcgaaa acgatccggt gccggcattg
     1501 tcgccgcgcg agatcgagtg tctgcactgg accgccctcg gcaaggatta caaggatatt
     1561 tcggtcatcc tgggcatatc agagcatacc acacgcgatt acctgaaaac cgcccgcttc
     1621 aggctcggct gcaccacgat ctcggccgcc gcgtcgcggg ctgttcaatt gcgcatcatc
     1681 aatccctata ggatccgcat gacgcgacgt aattggtaat agcctaattg gtaacgaatc
     1741 agacaattga cggctcgagg gagtagcata gggtttgcag aatccctgct tcgtccattt
     1801 gacaggcaca ttatgcatcg atgataagct gtcaaacatg agcagatcct ctacgccgga
     1861 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
     1921 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcaaata ttttatctga
     1981 ggtgcttcct cgctcactga ctcgctgcac gaggcagacc tcagcgctag cggagtgtat
     2041 actggcttac tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg tggcaggaga
     2101 aaaaaggctg caccggtgcg tcagcagaat atgtgataca ggatatattc cgcttcctcg
     2161 ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg
     2221 ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag agggccgcgg
     2281 caaagccgtt ctaccggcgc ggcagcgtga cccgtgtcgg cggctccaac ggctcgccat
     2341 cgtccagaaa acacggctca tcgggcatcg gcaggcgctg ctgcccgcgc cgttcccatt
     2401 cctccgtttc ggtcaaggct ggcaggtctg gttccatgcc cggaatgccg ggctggctgg
     2461 gcggctcctc gccggggccg gtcggtagtt gctgctcgcc cggatacagg gtcgggatgc
     2521 ggcgcaggtc gccatgcccc aacagcgatt cgtcctggtc gtcgtgatca accaccacgg
     2581 cggcactgaa caccgacagg cgcaactggt cgcggggctg gccccacgcc acgcggtcat
     2641 tgaccacgta ggccgacacg gtgccggggc cgttgagctt cacgacggag atccagcgct
     2701 cggccaccaa gtccttgact gcgtattgga ccgtccgcaa agaacgtccg atgagcttgg
     2761 aaagtgtctt ctggctgacc accacggcgt tctggtggcc catctgcgcc acgaggtgat
     2821 gcagcagcat tgccgccgtg ggtttcctcg caataagccc ggcccacgcc tcatgcgctt
     2881 tgcgttccgt ttgcacccag tgaccgggct tgttcttggc ttgaatgccg atttctctgg
     2941 actgcgtggc catgcttatc tccatgcggt aggggtgccg cacggttgcg gcaccatgcg
     3001 caatcagctg caacttttcg gcagcgcgac aacaattatg cgttgcgtaa aagtggcagt
     3061 caattacaga ttttctttaa cctacgcaat gagctattgc ggggggtgcc gcaatgagct
     3121 gttgcgtacc cccctttttt aagttgttga tttttaagtc tttcgcattt cgccctatat
     3181 ctagttcttt ggtgcccaaa gaagggcacc cctgcggggt tcccccacgc cttcggcgcg
     3241 gctccccctc cggcaaaaag tggcccctcc ggggcttgtt gatcgactgc gcggccttcg
     3301 gccttgccca aggtggcgct gcccccttgg aacccccgca ctcgccgccg tgaggctcgg
     3361 ggggcaggcg ggcgggcttc gcccttcgac tgcccccact cgcataggct tgggtcgttc
     3421 caggcgcgtc aaggccaagc cgctgcgcgg tcgctgcgcg agccttgacc cgccttccac
     3481 ttggtgtcca accggcaagc gaagcgcgca ggccgcaggc cggaggcttt tccccagaga
     3541 aaattaaaaa aattgatggg gcaaggccgc aggccgcgca gttggagccg gtgggtatgt
     3601 ggtcgaaggc tgggtagccg gtgggcaatc cctgtggtca agctcgtggg caggcgcagc
     3661 ctgtccatca gcttgtccag cagggttgtc cacgggccga gcgaagcgag ccagccggtg
     3721 gccgcgaaga tcatcttatt aaggggtctg acgctcagtg gaacgaaaaa tcaatctaaa
     3781 gtatatatga gtaaacttgg tctgacagtt accttagaaa aactcatcga gcatcaaatg
     3841 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     3901 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     3961 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     4021 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt
     4081 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     4141 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     4201 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     4261 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     4321 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     4381 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     4441 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     4501 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     4561 taaatcagca tccatgttgg aatttaatcg cggcctggag caagacgttt cccgttgaat
     4621 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     4681 tgatatattt ttatcttgtg caatgtacat cagagatttt gagacacaac caattattga
     4741 aggcctccct aacggggggc ctttttttgt ttctggtctc ccgcttaacg atcgttggct
     4801 gccctttgtg cgtccaaacg gacgcacggc gctctaaagc gggtcgcgat ctttcagatt
     4861 cgctcctcgc gctttcagtc tttgttttgg cgcatgtcgt tatcgcaaaa ccgctgcaca
     4921 cttttgcgcg acatgctctg atccccctca tctggggggg cctatctgag ggaatttccg
     4981 atccggctcg cctgaaccat tctgctttcc acgaacttga aaacgctag
//
