LOCUS       sequence-434114-        5070 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the Salicylic Acid Marionette 
            Cassette (NahRAM + PSalTTC-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5070)
  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 5070)
  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: pMVV108
FEATURES             Location/Qualifiers
     source          1..5070
                     /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(2058..2077)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2442..3104)
                     /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      3105..3876
                     /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(3965..4780)
                     /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(3965..4780)
                     /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     4688..4707
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4772..4791
                     /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 tttttcgttt tggtccaatg gcggcgcgcc atcgaatggt
      901 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc atgggggttg
      961 aatatggaac tgcgtgacct ggatttaaac ctgctggtgg tgttcaacca gttgctggtc
     1021 gacagacgcg tctctgtcac tgcggagaac ctgggcctga cccagcctgc cgtgagcaat
     1081 gcgctgaaac gcctgcgcac ctcgctacag gacccactct tcgtgcgcac acatcaggga
     1141 atggaaccca caccctatgc cgcgcatctg gccgagcacg tcacttcggc catgcacgca
     1201 ctgcgcaacg ccctacagca ccatgaaagc ttcgatccgc tgaccagcga gcgtaccttc
     1261 accctggcca tgaccgacat tggcgagatc tacttcatgc cgcggctgat ggatgcgctg
     1321 gctcaccagg cccccaattg cgtgatcagt acggtgcgcg acagttcgat gagcctgatg
     1381 caggccttgc agaacggaac cgtggacttg gccgtgggcc tgcttcccaa tctgcaaact
     1441 ggcttctttc agcgccggct gctccagaat cactacgtgt gcctatgtcg caaggaccat
     1501 ccagtcaccc gcgaacccct gactctggag cgcttctgtt cctacggcca cgtgcgtgtc
     1561 atcgccgctg gcaccggcca cggcgaggtg gacacgtaca tgacacgggt cggcatccgg
     1621 cgcgacatcc gtctggaagt gccgcacttc gccgccgttg gccacatcct ccagcgcacc
     1681 gatctgctcg ccactgtgcc gatatgttta gccgactgct gcgtagagcc cttcggccta
     1741 agcgccttgc cgcacccagt cgtcttgcct gaaatagcca tcaacatgtt ctggcatgcg
     1801 aagtaccaca aggacctagc caatatttgg ttgcggcaac tgatgtttga cctgtttacg
     1861 gattgataag gatcctaatt ggtaacgaat cagacaattg acggctcgag ggagtagcat
     1921 agggtttgca gaatccctgc ttcgtccatt tgacaggcac attatgcatc gatgataagc
     1981 tgtcaaacat gagcagatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac
     2041 aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca
     2101 cttcgggctc atgagcaaat attttatctg aggtgcttcc tcgctcactg actcgctgca
     2161 cgaggcagac ctcagcgcta gcggagtgta tactggctta ctatgttggc actgatgagg
     2221 gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct gcaccggtgc gtcagcagaa
     2281 tatgtgatac aggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg
     2341 actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa
     2401 gatacttaac agggaagtga gagggccgcg gcaaagccgt tctaccggcg cggcagcgtg
     2461 acccgtgtcg gcggctccaa cggctcgcca tcgtccagaa aacacggctc atcgggcatc
     2521 ggcaggcgct gctgcccgcg ccgttcccat tcctccgttt cggtcaaggc tggcaggtct
     2581 ggttccatgc ccggaatgcc gggctggctg ggcggctcct cgccggggcc ggtcggtagt
     2641 tgctgctcgc ccggatacag ggtcgggatg cggcgcaggt cgccatgccc caacagcgat
     2701 tcgtcctggt cgtcgtgatc aaccaccacg gcggcactga acaccgacag gcgcaactgg
     2761 tcgcggggct ggccccacgc cacgcggtca ttgaccacgt aggccgacac ggtgccgggg
     2821 ccgttgagct tcacgacgga gatccagcgc tcggccacca agtccttgac tgcgtattgg
     2881 accgtccgca aagaacgtcc gatgagcttg gaaagtgtct tctggctgac caccacggcg
     2941 ttctggtggc ccatctgcgc cacgaggtga tgcagcagca ttgccgccgt gggtttcctc
     3001 gcaataagcc cggcccacgc ctcatgcgct ttgcgttccg tttgcaccca gtgaccgggc
     3061 ttgttcttgg cttgaatgcc gatttctctg gactgcgtgg ccatgcttat ctccatgcgg
     3121 taggggtgcc gcacggttgc ggcaccatgc gcaatcagct gcaacttttc ggcagcgcga
     3181 caacaattat gcgttgcgta aaagtggcag tcaattacag attttcttta acctacgcaa
     3241 tgagctattg cggggggtgc cgcaatgagc tgttgcgtac cccccttttt taagttgttg
     3301 atttttaagt ctttcgcatt tcgccctata tctagttctt tggtgcccaa agaagggcac
     3361 ccctgcgggg ttcccccacg ccttcggcgc ggctccccct ccggcaaaaa gtggcccctc
     3421 cggggcttgt tgatcgactg cgcggccttc ggccttgccc aaggtggcgc tgcccccttg
     3481 gaacccccgc actcgccgcc gtgaggctcg gggggcaggc gggcgggctt cgcccttcga
     3541 ctgcccccac tcgcataggc ttgggtcgtt ccaggcgcgt caaggccaag ccgctgcgcg
     3601 gtcgctgcgc gagccttgac ccgccttcca cttggtgtcc aaccggcaag cgaagcgcgc
     3661 aggccgcagg ccggaggctt ttccccagag aaaattaaaa aaattgatgg ggcaaggccg
     3721 caggccgcgc agttggagcc ggtgggtatg tggtcgaagg ctgggtagcc ggtgggcaat
     3781 ccctgtggtc aagctcgtgg gcaggcgcag cctgtccatc agcttgtcca gcagggttgt
     3841 ccacgggccg agcgaagcga gccagccggt ggccgcgaag atcatcttat taaggggtct
     3901 gacgctcagt ggaacgaaaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3961 taccttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     4021 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     4081 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     4141 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     4201 cgactgaatc cggtgagaat ggcaaaagct tatgcatttc tttccagact tgttcaacag
     4261 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     4321 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     4381 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     4441 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     4501 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     4561 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     4621 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     4681 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     4741 gcggcctgga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     4801 ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaca
     4861 tcagagattt tgagacacaa ccaattattg aaggcctccc taacgggggg cctttttttg
     4921 tttctggtct cccgcttaac gatcgttggc tgggggcctc gcttgggtta ttgctggtgc
     4981 ccggccgggc gcaatattca tgttgatgat ttattatata tcgagtggtg tatttattta
     5041 tattgtttgc tccgttaccg ttattaacag
//
