LOCUS       sequence-431950-        5444 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the Erythromycin Marionette 
            Cassette (MphRAM + EryR + PMph-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5444)
  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 5444)
  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: pMVV111
FEATURES             Location/Qualifiers
     source          1..5444
                     /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(2496..2515)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2880..3542)
                     /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      3543..4314
                     /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(4403..5218)
                     /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(4403..5218)
                     /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     5126..5145
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5210..5229
                     /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 tagggaggtg
      961 aatatgcctc gtccgaaact gaaaagtgat gatgaagttc tggaagcagc aaccgttgtt
     1021 ctgaaacgtt gtggtccgat tgaatttacc ctgagcggtg ttgcaaaaga agttggtctg
     1081 agtcgcgcag cactgattca gcgttttacc aatcgtgata ccctgctggt tcgtatgatg
     1141 gaacgtggtg ttgaacaggt tcgtcattat ctgaatgcaa ttccgattgg tgcaggtccg
     1201 cagggtctgt gggaatttct gcaggttctg gttcgtagca tggatacccg taatgatttc
     1261 agcgtgaact atctgatcag ctggtatgaa ctgcaggttc cggaactgcg taccctggca
     1321 attcagcgta atcgtgcagt tgttgaaggt attcgtaaac gtctgcctcc gggtgcaccg
     1381 gcagcagcag aactgctgct gcatagcgtt attgccggtg caaccatgca gtgggcagtt
     1441 gatccggatg gtgaactggc agatcatgtt ctggcacaga ttgcagcaat tctgtgtctg
     1501 atgtttccgg aacatgatga ttttcagctg ctgcaggcac atgcataata atattgaaaa
     1561 aggaagagta tgaacgagaa aaatataaaa cacagtcaaa actttattac ttcaaaacat
     1621 aatatagata aaataatgac aaatataaga ttaaatgaac atgataatat ctttgaaatc
     1681 ggctcaggaa aaggccattt tacccttgaa ttagtaaaga ggtgtaattt cgtaactgcc
     1741 attgaaatag accataaatt atgcaaaact acagaaaata aacttgttga tcacgataat
     1801 ttccaagttt taaacaagga tatattgcag tttaaatttc ctaaaaacca atcctataaa
     1861 atatatggta atatacctta taacataagt acggatataa tacgcaaaat tgtttttgat
     1921 agtatagcta atgagattta tttaatcgtg gaatacgggt ttgctaaaag attattaaat
     1981 acaaaacgct cattggcatt acttttaatg gcagaagttg atatttctat attaagtatg
     2041 gttccaagag aatattttca tcctaaacct aaagtgaata gctcacttat cagattaagt
     2101 agaaaaaaat caagaatatc acacaaagat aaacaaaagt ataattattt cgttatgaaa
     2161 tgggttaaca aagaatacaa gaaaatattt acaaaaaatc aatttaacaa ttccttaaaa
     2221 catgcaggaa ttgacgattt aaacaatatt agctttgaac aattcttatc tcttttcaat
     2281 agctataaat tatttaataa gtgataagga tcctaattgg taacgaatca gacaattgac
     2341 ggctcgaggg agtagcatag ggtttgcaga atccctgctt cgtccatttg acaggcacat
     2401 tatgcatcga tgataagctg tcaaacatga gcagatcctc tacgccggac gcatcgtggc
     2461 cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg
     2521 ggaagatcgg gctcgccact tcgggctcat gagcaaatat tttatctgag gtgcttcctc
     2581 gctcactgac tcgctgcacg aggcagacct cagcgctagc ggagtgtata ctggcttact
     2641 atgttggcac tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa aaaaggctgc
     2701 accggtgcgt cagcagaata tgtgatacag gatatattcc gcttcctcgc tcactgactc
     2761 gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg gcggagattt
     2821 cctggaagat gccaggaaga tacttaacag ggaagtgaga gggccgcggc aaagccgttc
     2881 taccggcgcg gcagcgtgac ccgtgtcggc ggctccaacg gctcgccatc gtccagaaaa
     2941 cacggctcat cgggcatcgg caggcgctgc tgcccgcgcc gttcccattc ctccgtttcg
     3001 gtcaaggctg gcaggtctgg ttccatgccc ggaatgccgg gctggctggg cggctcctcg
     3061 ccggggccgg tcggtagttg ctgctcgccc ggatacaggg tcgggatgcg gcgcaggtcg
     3121 ccatgcccca acagcgattc gtcctggtcg tcgtgatcaa ccaccacggc ggcactgaac
     3181 accgacaggc gcaactggtc gcggggctgg ccccacgcca cgcggtcatt gaccacgtag
     3241 gccgacacgg tgccggggcc gttgagcttc acgacggaga tccagcgctc ggccaccaag
     3301 tccttgactg cgtattggac cgtccgcaaa gaacgtccga tgagcttgga aagtgtcttc
     3361 tggctgacca ccacggcgtt ctggtggccc atctgcgcca cgaggtgatg cagcagcatt
     3421 gccgccgtgg gtttcctcgc aataagcccg gcccacgcct catgcgcttt gcgttccgtt
     3481 tgcacccagt gaccgggctt gttcttggct tgaatgccga tttctctgga ctgcgtggcc
     3541 atgcttatct ccatgcggta ggggtgccgc acggttgcgg caccatgcgc aatcagctgc
     3601 aacttttcgg cagcgcgaca acaattatgc gttgcgtaaa agtggcagtc aattacagat
     3661 tttctttaac ctacgcaatg agctattgcg gggggtgccg caatgagctg ttgcgtaccc
     3721 ccctttttta agttgttgat ttttaagtct ttcgcatttc gccctatatc tagttctttg
     3781 gtgcccaaag aagggcaccc ctgcggggtt cccccacgcc ttcggcgcgg ctccccctcc
     3841 ggcaaaaagt ggcccctccg gggcttgttg atcgactgcg cggccttcgg ccttgcccaa
     3901 ggtggcgctg cccccttgga acccccgcac tcgccgccgt gaggctcggg gggcaggcgg
     3961 gcgggcttcg cccttcgact gcccccactc gcataggctt gggtcgttcc aggcgcgtca
     4021 aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc gccttccact tggtgtccaa
     4081 ccggcaagcg aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa aattaaaaaa
     4141 attgatgggg caaggccgca ggccgcgcag ttggagccgg tgggtatgtg gtcgaaggct
     4201 gggtagccgg tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc tgtccatcag
     4261 cttgtccagc agggttgtcc acgggccgag cgaagcgagc cagccggtgg ccgcgaagat
     4321 catcttatta aggggtctga cgctcagtgg aacgaaaaat caatctaaag tatatatgag
     4381 taaacttggt ctgacagtta ccttagaaaa actcatcgag catcaaatga aactgcaatt
     4441 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     4501 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     4561 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     4621 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta tgcatttctt
     4681 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     4741 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     4801 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     4861 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     4921 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     4981 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     5041 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     5101 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     5161 ccatgttgga atttaatcgc ggcctggagc aagacgtttc ccgttgaata tggctcataa
     5221 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt
     5281 tatcttgtgc aatgtacatc agagattttg agacacaacc aattattgaa ggcctcccta
     5341 acggggggcc tttttttgtt tctggtctcc cgcttaacga tcgttggctg ggattgaata
     5401 taaccgacgt gactgttaca tttaggtggc taaacccgtc aaag
//
