LOCUS       sequence-431944-        6679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under induction with the arabinose Marionette 
            Cassette (AraCAM + araE + PBAD-sfGFP) with a BBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6679)
  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 6679)
  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: pMVV104
FEATURES             Location/Qualifiers
     source          1..6679
                     /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"
     CDS             964..1842
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDFFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MLAINGSLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGLE
                     EKVNDVAVKLS"
     primer_bind     complement(3494..3513)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(3878..4540)
                     /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      4541..5312
                     /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(5401..6216)
                     /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(5401..6216)
                     /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     6124..6143
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6208..6227
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        6389..6672
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     6595..6614
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     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 cagggtggtg
      961 aatatggctg aagcgcaaaa tgatcccctg ctgccgggat actcgtttaa tgcccatctg
     1021 gtggcgggtt taacgccgat tgaggccaac ggttatctcg atttttttat cgaccgaccg
     1081 ctgggaatga aaggttatat tctcaatctc accattcgcg gtcagggggt ggtgaaaaat
     1141 cagggacgag aatttgtttg ccgaccgggt gatattttgc tgttcccgcc aggagagatt
     1201 catcactacg gtcgtcatcc ggaggctcgc gaatggtatc accagtgggt ttactttcgt
     1261 ccgcgcgcct actggcatga atggcttaac tggccgtcaa tatttgccaa tacggggttc
     1321 tttcgcccgg atgaagcgca ccagccgcat ttcagcgact tttttgggca aatcattaac
     1381 gccgggcaag gggaagggcg ctattcggag ctgctggcga taaatctgct tgagcaattg
     1441 ttactgcggc gcatgctagc gattaacgga tcgctccatc caccgatgga taatcgggta
     1501 cgcgaggctt gtcagtacat cagcgatcac ctggcagaca gcaattttga tatcgccagc
     1561 gtcgcacagc atgtttgctt gtcgccgtcg cgtctgtcac atcttttccg ccagcagtta
     1621 gggattagcg tcttaagctg gcgcgaggac caacgtatca gccaggcgaa gctgcttttg
     1681 agcaccaccc ggatgcctat cgccaccgtc ggtcgcaatg ttggttttga cgatcaactc
     1741 tatttctcgc gggtatttaa aaaatgcacc ggggccagcc cgagcgagtt ccgtgccggt
     1801 ttggaagaaa aagtgaatga tgtagccgtc aagttgtcat gataagatcc tattccagcg
     1861 ggattaaaga ggagcgatta agcatggtta ctatcaatac ggaatctgct ttaacgccac
     1921 gttctttgcg ggatacgcgg cgtatgaata tgtttgtttc ggtagctgct gcggtcgcag
     1981 gattgttatt tggtcttgat atcggcgtaa tcgccggagc gttgccgttc attaccgatc
     2041 actttgtgct gaccagtcgt ttgcaggaat gggtggttag tagcatgatg ctcggtgcag
     2101 caattggtgc gctgtttaat ggttggctgt cgttccgcct ggggcgtaaa tacagcctga
     2161 tggcgggggc catcctgttt gtactcggtt ctatagggtc cgcttttgcg accagcgtag
     2221 agatgttaat cgccgctcgt gtggtgctgg gcattgctgt cgggatcgcg tcttacaccg
     2281 ctcctctgta tctttctgaa atggcaagtg aaaacgttcg cggtaagatg atcagtatgt
     2341 accagttgat ggtcacactc ggcatcgtgc tggcgttttt atccgataca gcgttcagtt
     2401 atagcggtaa ctggcgcgca atgttggggg ttcttgcttt accagcagtt ctgctgatta
     2461 ttctggtagt atttctgcca aatagcccgc gctggctggc ggaaaagggg cgtcatattg
     2521 aggcggaaga agtattgcgt atgctgcgcg atacgtcgga aaaagcgcga gaagaactca
     2581 acgaaattcg tgaaagcctg aagttaaaac agggcggttg ggcactgttt aagatcaacc
     2641 gtaacgtccg tcgtgctgtg tttctcggta tgttgttgca ggcgatgcag cagtttaccg
     2701 gtatgaacat catcatgtac tacgcgccgc gtatcttcaa aatggcgggc tttacgacca
     2761 cagaacaaca gatgattgcg actctggtcg tagggctgac ctttatgttc gccaccttta
     2821 ttgcggtgtt tacggtagat aaagcagggc gtaaaccggc tctgaaaatt ggtttcagcg
     2881 tgatggcgtt aggcactctg gtgctgggct attgcctgat gcagtttgat aacggtacgg
     2941 cttccagtgg cttgtcctgg ctctctgttg gcatgacgat gatgtgtatt gccggttatg
     3001 cgatgagcgc cgcgccagtg gtgtggatcc tgtgctctga aattcagccg ctgaaatgcc
     3061 gcgatttcgg tattacctgt tcgaccacca cgaactgggt gtcgaatatg attatcggcg
     3121 cgaccttcct gacactgctt gatagcattg gcgctgccgg tacgttctgg ctctacactg
     3181 cgctgaacat tgcgtttgtg ggcattactt tctggctcat tccggaaacc aaaaatgtca
     3241 cgctggaaca tatcgaacgc aaactgatgg caggcgagaa gttgagaaat atcggcgtct
     3301 gataaggatc ctaattggta acgaatcaga caattgacgg ctcgagggag tagcataggg
     3361 tttgcagaat ccctgcttcg tccatttgac aggcacatta tgcatcgatg ataagctgtc
     3421 aaacatgagc agatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
     3481 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc
     3541 gggctcatga gcaaatattt tatctgaggt gcttcctcgc tcactgactc gctgcacgag
     3601 gcagacctca gcgctagcgg agtgtatact ggcttactat gttggcactg atgagggtgt
     3661 cagtgaagtg cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca gcagaatatg
     3721 tgatacagga tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg
     3781 cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata
     3841 cttaacaggg aagtgagagg gccgcggcaa agccgttcta ccggcgcggc agcgtgaccc
     3901 gtgtcggcgg ctccaacggc tcgccatcgt ccagaaaaca cggctcatcg ggcatcggca
     3961 ggcgctgctg cccgcgccgt tcccattcct ccgtttcggt caaggctggc aggtctggtt
     4021 ccatgcccgg aatgccgggc tggctgggcg gctcctcgcc ggggccggtc ggtagttgct
     4081 gctcgcccgg atacagggtc gggatgcggc gcaggtcgcc atgccccaac agcgattcgt
     4141 cctggtcgtc gtgatcaacc accacggcgg cactgaacac cgacaggcgc aactggtcgc
     4201 ggggctggcc ccacgccacg cggtcattga ccacgtaggc cgacacggtg ccggggccgt
     4261 tgagcttcac gacggagatc cagcgctcgg ccaccaagtc cttgactgcg tattggaccg
     4321 tccgcaaaga acgtccgatg agcttggaaa gtgtcttctg gctgaccacc acggcgttct
     4381 ggtggcccat ctgcgccacg aggtgatgca gcagcattgc cgccgtgggt ttcctcgcaa
     4441 taagcccggc ccacgcctca tgcgctttgc gttccgtttg cacccagtga ccgggcttgt
     4501 tcttggcttg aatgccgatt tctctggact gcgtggccat gcttatctcc atgcggtagg
     4561 ggtgccgcac ggttgcggca ccatgcgcaa tcagctgcaa cttttcggca gcgcgacaac
     4621 aattatgcgt tgcgtaaaag tggcagtcaa ttacagattt tctttaacct acgcaatgag
     4681 ctattgcggg gggtgccgca atgagctgtt gcgtaccccc cttttttaag ttgttgattt
     4741 ttaagtcttt cgcatttcgc cctatatcta gttctttggt gcccaaagaa gggcacccct
     4801 gcggggttcc cccacgcctt cggcgcggct ccccctccgg caaaaagtgg cccctccggg
     4861 gcttgttgat cgactgcgcg gccttcggcc ttgcccaagg tggcgctgcc cccttggaac
     4921 ccccgcactc gccgccgtga ggctcggggg gcaggcgggc gggcttcgcc cttcgactgc
     4981 ccccactcgc ataggcttgg gtcgttccag gcgcgtcaag gccaagccgc tgcgcggtcg
     5041 ctgcgcgagc cttgacccgc cttccacttg gtgtccaacc ggcaagcgaa gcgcgcaggc
     5101 cgcaggccgg aggcttttcc ccagagaaaa ttaaaaaaat tgatggggca aggccgcagg
     5161 ccgcgcagtt ggagccggtg ggtatgtggt cgaaggctgg gtagccggtg ggcaatccct
     5221 gtggtcaagc tcgtgggcag gcgcagcctg tccatcagct tgtccagcag ggttgtccac
     5281 gggccgagcg aagcgagcca gccggtggcc gcgaagatca tcttattaag gggtctgacg
     5341 ctcagtggaa cgaaaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5401 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     5461 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     5521 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     5581 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     5641 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
     5701 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     5761 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     5821 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     5881 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     5941 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     6001 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     6061 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     6121 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     6181 cctggagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     6241 gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtacatcag
     6301 agattttgag acacaaccaa ttattgaagg cctccctaac ggggggcctt tttttgtttc
     6361 tggtctcccg cttaacgatc gttggctgag aaaccaattg tccatattgc atcagacatt
     6421 gccgtcactg cgtcttttac tggctcttct cgctaaccaa accggtaacc ccgcttatta
     6481 aaagcattct gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac aaaagtgtct
     6541 ataatcacgg cagaaaagtc cacattgatt atttgcacgg cgtcacactt tgctatgcca
     6601 tagcattttt atccataaga ttagcggatc ctacctgacg ctttttatcg caactctcta
     6661 ctgtttctcc atacccgag
//
