LOCUS       sequence-434113-        4702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the choline Marionette Cassette
            (BetIAM + PBetI-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4702)
  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 4702)
  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: pMVV105
FEATURES             Location/Qualifiers
     source          1..4702
                     /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(1743..1762)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2127..2789)
                     /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      2790..3561
                     /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(3650..4465)
                     /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(3650..4465)
                     /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     4373..4392
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4457..4476
                     /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 cacgggggtg
      961 aatatgccga aactgggtat gcagagcatt cgtcgtcgtc agctgattga tgcaaccctg
     1021 gaagcaatta atgaagttgg tatgcatgat gcaaccattg cacagattgc acgtcgtgcc
     1081 ggtgttagca ccggtattat tagccattat ttccgcgata aaaacggtct actggaagca
     1141 accatgcgtg atattaccag ccagctgcgt gatgcagttc tgaatcgtct gcatgcactg
     1201 ccgcagggta gcgcagaaca gcgtctgcag gcaattgttg gtggtaattt tgatgaaacc
     1261 caggttagca gcgcagcaat gaaagcatgg ctggcatttt gggcaatcag catgcatcag
     1321 ccgatgctgt atcgtctgca gcaggttagc agtcgtcgtc tgctgagcaa tctggttagc
     1381 gaatttcgtc gtgaactgcc tcgtgaacag gcacaagagg caggttatgg tctggcagca
     1441 ctgattgatg gtctgtggct gcgtgcagca ctgagcggta aaccgctgga taaaacccgt
     1501 gcaaatagcc tgacccgtca ttttatcacc cagcatctgc cgaccgattg ataaggatcc
     1561 taattggtaa cgaatcagac aattgacggc tcgagggagt agcatagggt ttgcagaatc
     1621 cctgcttcgt ccatttgaca ggcacattat gcatcgatga taagctgtca aacatgagca
     1681 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     1741 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     1801 caaatatttt atctgaggtg cttcctcgct cactgactcg ctgcacgagg cagacctcag
     1861 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc
     1921 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat
     1981 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga
     2041 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga
     2101 agtgagaggg ccgcggcaaa gccgttctac cggcgcggca gcgtgacccg tgtcggcggc
     2161 tccaacggct cgccatcgtc cagaaaacac ggctcatcgg gcatcggcag gcgctgctgc
     2221 ccgcgccgtt cccattcctc cgtttcggtc aaggctggca ggtctggttc catgcccgga
     2281 atgccgggct ggctgggcgg ctcctcgccg gggccggtcg gtagttgctg ctcgcccgga
     2341 tacagggtcg ggatgcggcg caggtcgcca tgccccaaca gcgattcgtc ctggtcgtcg
     2401 tgatcaacca ccacggcggc actgaacacc gacaggcgca actggtcgcg gggctggccc
     2461 cacgccacgc ggtcattgac cacgtaggcc gacacggtgc cggggccgtt gagcttcacg
     2521 acggagatcc agcgctcggc caccaagtcc ttgactgcgt attggaccgt ccgcaaagaa
     2581 cgtccgatga gcttggaaag tgtcttctgg ctgaccacca cggcgttctg gtggcccatc
     2641 tgcgccacga ggtgatgcag cagcattgcc gccgtgggtt tcctcgcaat aagcccggcc
     2701 cacgcctcat gcgctttgcg ttccgtttgc acccagtgac cgggcttgtt cttggcttga
     2761 atgccgattt ctctggactg cgtggccatg cttatctcca tgcggtaggg gtgccgcacg
     2821 gttgcggcac catgcgcaat cagctgcaac ttttcggcag cgcgacaaca attatgcgtt
     2881 gcgtaaaagt ggcagtcaat tacagatttt ctttaaccta cgcaatgagc tattgcgggg
     2941 ggtgccgcaa tgagctgttg cgtacccccc ttttttaagt tgttgatttt taagtctttc
     3001 gcatttcgcc ctatatctag ttctttggtg cccaaagaag ggcacccctg cggggttccc
     3061 ccacgccttc ggcgcggctc cccctccggc aaaaagtggc ccctccgggg cttgttgatc
     3121 gactgcgcgg ccttcggcct tgcccaaggt ggcgctgccc ccttggaacc cccgcactcg
     3181 ccgccgtgag gctcgggggg caggcgggcg ggcttcgccc ttcgactgcc cccactcgca
     3241 taggcttggg tcgttccagg cgcgtcaagg ccaagccgct gcgcggtcgc tgcgcgagcc
     3301 ttgacccgcc ttccacttgg tgtccaaccg gcaagcgaag cgcgcaggcc gcaggccgga
     3361 ggcttttccc cagagaaaat taaaaaaatt gatggggcaa ggccgcaggc cgcgcagttg
     3421 gagccggtgg gtatgtggtc gaaggctggg tagccggtgg gcaatccctg tggtcaagct
     3481 cgtgggcagg cgcagcctgt ccatcagctt gtccagcagg gttgtccacg ggccgagcga
     3541 agcgagccag ccggtggccg cgaagatcat cttattaagg ggtctgacgc tcagtggaac
     3601 gaaaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacct tagaaaaact
     3661 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     3721 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     3781 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     3841 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     3901 agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     3961 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     4021 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     4081 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     4141 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     4201 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     4261 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     4321 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     4381 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctggagcaag
     4441 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     4501 gttttattgt tcatgatgat atatttttat cttgtgcaat gtacatcaga gattttgaga
     4561 cacaaccaat tattgaaggc ctccctaacg gggggccttt ttttgtttct ggtctcccgc
     4621 ttaacgatcg ttggctgagc gcgggtgaga gggattcgtt accaatagac aattgattgg
     4681 acgttcaata taatgctagc ag
//
