LOCUS       sequence-440373-        4984 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the DHBA Marionette Cassette 
            (PcaUAM + P3B5B-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4984)
  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 4984)
  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: pMVV107
FEATURES             Location/Qualifiers
     source          1..4984
                     /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(1995..2014)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2379..3041)
                     /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      3042..3813
                     /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(3902..4717)
                     /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(3902..4717)
                     /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     4625..4644
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4709..4728
                     /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 cagggaggtg
      961 aatatgtggt cgaacatgga tgacaagaaa gtgaaagagg agaatattct gcacaattcc
     1021 accaacaaga agatcatccg ccacgaagat tttgtagccg gcattagcaa agggatggcg
     1081 attctggatt cgtttggtac agatcgtcat cgcctcaata tcaccatggc cgcagagaaa
     1141 accggtatga cacgtgcagc agctcgtcgc cacctgctta ctctggagta tctgggctat
     1201 ctggaaagtg acggccacta cttctactta actcccaaaa tcctgaaatt cagtggttca
     1261 tatttgggtg gtgctcaatt gccgaaaatt tcccaaccac tgttgaactt gcttacgacc
     1321 cagaccagcc tgatttacag cgtgatggtg ttggatggct atgaagccat taccattgcg
     1381 cgttctgccg ctcatcagca aaccgaccgc gttaacccgt atggtttaca tctcgggaat
     1441 cgcttaccag cgcatacaac gtcagcgggc aaaatcctgt tagcgtattt ggatgaccat
     1501 gcccagcaag agtggctcaa tcagtaccct ctgcaacggc tcacgaaata cacgtatacc
     1561 aaccacatcg actttctgcg ccttttgagt gaaatcaagg aacagggttg gtgctatagt
     1621 tcggaagaac acgaactggg agtacacgcc cttgcggttc cgatttacgg acagcagtct
     1681 cgcgtcgtag cggcactgaa cattgtcagc ccgacaatgc ggaccacgaa agaatacctg
     1741 attcagcata ttctgccgtt actgcaagaa actgcgcgtg aattgcgcaa tatcctgtaa
     1801 tgataaggat cctaattggt aacgaatcag acaattgacg gctcgaggga gtagcatagg
     1861 gtttgcagaa tccctgcttc gtccatttga caggcacatt atgcatcgat gataagctgt
     1921 caaacatgag cagatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg
     1981 tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt
     2041 cgggctcatg agcaaatatt ttatctgagg tgcttcctcg ctcactgact cgctgcacga
     2101 ggcagacctc agcgctagcg gagtgtatac tggcttacta tgttggcact gatgagggtg
     2161 tcagtgaagt gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat
     2221 gtgatacagg atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact
     2281 gcggcgagcg gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat
     2341 acttaacagg gaagtgagag ggccgcggca aagccgttct accggcgcgg cagcgtgacc
     2401 cgtgtcggcg gctccaacgg ctcgccatcg tccagaaaac acggctcatc gggcatcggc
     2461 aggcgctgct gcccgcgccg ttcccattcc tccgtttcgg tcaaggctgg caggtctggt
     2521 tccatgcccg gaatgccggg ctggctgggc ggctcctcgc cggggccggt cggtagttgc
     2581 tgctcgcccg gatacagggt cgggatgcgg cgcaggtcgc catgccccaa cagcgattcg
     2641 tcctggtcgt cgtgatcaac caccacggcg gcactgaaca ccgacaggcg caactggtcg
     2701 cggggctggc cccacgccac gcggtcattg accacgtagg ccgacacggt gccggggccg
     2761 ttgagcttca cgacggagat ccagcgctcg gccaccaagt ccttgactgc gtattggacc
     2821 gtccgcaaag aacgtccgat gagcttggaa agtgtcttct ggctgaccac cacggcgttc
     2881 tggtggccca tctgcgccac gaggtgatgc agcagcattg ccgccgtggg tttcctcgca
     2941 ataagcccgg cccacgcctc atgcgctttg cgttccgttt gcacccagtg accgggcttg
     3001 ttcttggctt gaatgccgat ttctctggac tgcgtggcca tgcttatctc catgcggtag
     3061 gggtgccgca cggttgcggc accatgcgca atcagctgca acttttcggc agcgcgacaa
     3121 caattatgcg ttgcgtaaaa gtggcagtca attacagatt ttctttaacc tacgcaatga
     3181 gctattgcgg ggggtgccgc aatgagctgt tgcgtacccc ccttttttaa gttgttgatt
     3241 tttaagtctt tcgcatttcg ccctatatct agttctttgg tgcccaaaga agggcacccc
     3301 tgcggggttc ccccacgcct tcggcgcggc tccccctccg gcaaaaagtg gcccctccgg
     3361 ggcttgttga tcgactgcgc ggccttcggc cttgcccaag gtggcgctgc ccccttggaa
     3421 cccccgcact cgccgccgtg aggctcgggg ggcaggcggg cgggcttcgc ccttcgactg
     3481 cccccactcg cataggcttg ggtcgttcca ggcgcgtcaa ggccaagccg ctgcgcggtc
     3541 gctgcgcgag ccttgacccg ccttccactt ggtgtccaac cggcaagcga agcgcgcagg
     3601 ccgcaggccg gaggcttttc cccagagaaa attaaaaaaa ttgatggggc aaggccgcag
     3661 gccgcgcagt tggagccggt gggtatgtgg tcgaaggctg ggtagccggt gggcaatccc
     3721 tgtggtcaag ctcgtgggca ggcgcagcct gtccatcagc ttgtccagca gggttgtcca
     3781 cgggccgagc gaagcgagcc agccggtggc cgcgaagatc atcttattaa ggggtctgac
     3841 gctcagtgga acgaaaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3901 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     3961 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     4021 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     4081 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     4141 ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc
     4201 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     4261 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     4321 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     4381 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     4441 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     4501 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     4561 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     4621 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     4681 gcctggagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     4741 tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca atgtacatca
     4801 gagattttga gacacaacca attattgaag gcctccctaa cggggggcct ttttttgttt
     4861 ctggtctccc gcttaacgat cgttggctgt tttgttcgat tatcgaacaa attattgaaa
     4921 tatcgaacaa aacctctaaa ctactgtggc actgaatcaa aaaattataa accatgatca
     4981 gaag
//
