LOCUS       sequence-440376-        4788 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the Acrylic Acid Marionette 
            Cassette (acuRAM + PAcu-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4788)
  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 4788)
  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: pMVV110
FEATURES             Location/Qualifiers
     source          1..4788
                     /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(1818..1837)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2202..2864)
                     /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      2865..3636
                     /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(3725..4540)
                     /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(3725..4540)
                     /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     4448..4467
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4532..4551
                     /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 aatatgccgc tgaccgacac cccgccgtct gttccgcaga aaccgcgtcg tggtcgtccg
     1021 cgtggtgctc cggacgcttc tcttgctcac cagtctctga tccgtgctgg tctggaacac
     1081 ctgaccgaaa aaggttactc ttcggttggt gttgacgaaa tcctgaaagc tgctcgtgtt
     1141 ccgaaaggtt ctttctacca ctacttccgt aacaaagctg acttcggtct ggctctgatc
     1201 aaagcttacg acacctactt cgctcgtctc ctcgaccagg cgttcctgga cggttcgctg
     1261 gctccgctgg ctcgtctgcg tctgttcacc cgtatggctg aagaaggtat ggctcgtcac
     1321 ggtttccgtc gtggttgcct ggttggtaac ctgggtcagg aaatgggcgc tctgccggac
     1381 gacttccgtg ctgctctgat cggtgttctg gaaacctggc aacatcgtac cgctcagctg
     1441 ttccgtgaag ctcaggcttg cggtgaactg tctgctgacc atgacccgga cgctctggct
     1501 gaagctttct ggatcggatg ggaaggtgct atcctgcgtg ctaaactgga actgcgtccg
     1561 gacccgatgc actctttcac ccgtaccttc ggtcgtcact tcgttacccg tacccaggaa
     1621 taatgataag gatcctaatt ggtaacgaat cagacaattg acggctcgag ggagtagcat
     1681 agggtttgca gaatccctgc ttcgtccatt tgacaggcac attatgcatc gatgataagc
     1741 tgtcaaacat gagcagatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac
     1801 aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca
     1861 cttcgggctc atgagcaaat attttatctg aggtgcttcc tcgctcactg actcgctgca
     1921 cgaggcagac ctcagcgcta gcggagtgta tactggctta ctatgttggc actgatgagg
     1981 gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct gcaccggtgc gtcagcagaa
     2041 tatgtgatac aggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg
     2101 actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa
     2161 gatacttaac agggaagtga gagggccgcg gcaaagccgt tctaccggcg cggcagcgtg
     2221 acccgtgtcg gcggctccaa cggctcgcca tcgtccagaa aacacggctc atcgggcatc
     2281 ggcaggcgct gctgcccgcg ccgttcccat tcctccgttt cggtcaaggc tggcaggtct
     2341 ggttccatgc ccggaatgcc gggctggctg ggcggctcct cgccggggcc ggtcggtagt
     2401 tgctgctcgc ccggatacag ggtcgggatg cggcgcaggt cgccatgccc caacagcgat
     2461 tcgtcctggt cgtcgtgatc aaccaccacg gcggcactga acaccgacag gcgcaactgg
     2521 tcgcggggct ggccccacgc cacgcggtca ttgaccacgt aggccgacac ggtgccgggg
     2581 ccgttgagct tcacgacgga gatccagcgc tcggccacca agtccttgac tgcgtattgg
     2641 accgtccgca aagaacgtcc gatgagcttg gaaagtgtct tctggctgac caccacggcg
     2701 ttctggtggc ccatctgcgc cacgaggtga tgcagcagca ttgccgccgt gggtttcctc
     2761 gcaataagcc cggcccacgc ctcatgcgct ttgcgttccg tttgcaccca gtgaccgggc
     2821 ttgttcttgg cttgaatgcc gatttctctg gactgcgtgg ccatgcttat ctccatgcgg
     2881 taggggtgcc gcacggttgc ggcaccatgc gcaatcagct gcaacttttc ggcagcgcga
     2941 caacaattat gcgttgcgta aaagtggcag tcaattacag attttcttta acctacgcaa
     3001 tgagctattg cggggggtgc cgcaatgagc tgttgcgtac cccccttttt taagttgttg
     3061 atttttaagt ctttcgcatt tcgccctata tctagttctt tggtgcccaa agaagggcac
     3121 ccctgcgggg ttcccccacg ccttcggcgc ggctccccct ccggcaaaaa gtggcccctc
     3181 cggggcttgt tgatcgactg cgcggccttc ggccttgccc aaggtggcgc tgcccccttg
     3241 gaacccccgc actcgccgcc gtgaggctcg gggggcaggc gggcgggctt cgcccttcga
     3301 ctgcccccac tcgcataggc ttgggtcgtt ccaggcgcgt caaggccaag ccgctgcgcg
     3361 gtcgctgcgc gagccttgac ccgccttcca cttggtgtcc aaccggcaag cgaagcgcgc
     3421 aggccgcagg ccggaggctt ttccccagag aaaattaaaa aaattgatgg ggcaaggccg
     3481 caggccgcgc agttggagcc ggtgggtatg tggtcgaagg ctgggtagcc ggtgggcaat
     3541 ccctgtggtc aagctcgtgg gcaggcgcag cctgtccatc agcttgtcca gcagggttgt
     3601 ccacgggccg agcgaagcga gccagccggt ggccgcgaag atcatcttat taaggggtct
     3661 gacgctcagt ggaacgaaaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3721 taccttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     3781 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     3841 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     3901 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     3961 cgactgaatc cggtgagaat ggcaaaagct tatgcatttc tttccagact tgttcaacag
     4021 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     4081 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     4141 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     4201 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     4261 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     4321 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     4381 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     4441 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     4501 gcggcctgga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     4561 ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaca
     4621 tcagagattt tgagacacaa ccaattattg aaggcctccc taacgggggg cctttttttg
     4681 tttctggtct cccgcttaac gatcgttggc tgcgctagca agtaaggccg acgcttcaca
     4741 accgcacttg atttaataga ccataccgtc tattatttct ggccatag
//
