LOCUS       sequence-434099-        4100 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under the Anderson J23119 promoter with a pRO1600 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4100)
  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 4100)
  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: pMVV094
FEATURES             Location/Qualifiers
     source          1..4100
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        88..122
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     CDS             194..217
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             230..241
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             245..958
                     /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="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             245..955
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(272..300)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     895..915
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     rep_origin      1147..1735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1636..1655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1789..2622)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pRO1600 from Pseudomonas aeruginosa "
                     /label=pRO1600 Rep
                     /translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     rep_origin      2636..2987
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     terminator      complement(3045..3139)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3170..3964)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3281..3300)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3891..3910
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gacgtcctcg gtaccaaatt ccagaaaaga ggcctcccga aaggggggcc ttttttcgtt
       61 ttggtccatc ttgaactagc atacatattg acagctagct cagtcctagg tataatgcta
      121 gcacgtcgac ttatctcgag tgagatattg ttgacgaact cataaagtca agtaggagat
      181 taattccatg ggccatcacc atcaccatca ccatcacagc agcggccata tcgaaggtcg
      241 tcatagcaaa ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg
      301 tgatgttaat gggcacaaat tttctgtccg tggagagggt gaaggtgatg ctacaaacgg
      361 aaaactcacc cttaaattta tttgcactac tggaaaacta cctgttccgt ggccaacact
      421 tgtcactact ctgacctatg gtgttcaatg cttttcccgt tatccggatc acatgaaacg
      481 gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaacgca ctatatcttt
      541 caaagatgac gggacctaca agacgcgtgc tgaagtcaag tttgaaggtg atacccttgt
      601 taatcgtatc gagttaaagg gtattgattt taaagaagat ggaaacattc ttggacacaa
      661 acttgagtac aactttaact cacacaatgt atacatcacg gcagacaaac aaaagaatgg
      721 aatcaaagct aacttcaaaa ttcgccacaa cgttgaagat ggttccgttc aactagcaga
      781 ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta
      841 cctgtcgaca caatctgtcc tttcgaaaga tcccaacgaa aagcgtgacc acatggtcct
      901 tcttgagttt gtaactgctg ctgggattac acatggcatg gatgagctct acaaataata
      961 agaattcttc agccaaaaaa cttaagaccg ccggtcttgt ccactacctt gcagtaatgc
     1021 ggtggacagg atcggcggtt ttcttttctc ttctcaacct agggataatc tcatgaccaa
     1081 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     1141 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     1201 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     1261 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     1321 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     1381 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     1441 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     1501 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     1561 cgaagggaga aaggcggaca ggcatccggt aagcggcagg gtcggaacag gagagcgcac
     1621 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     1681 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     1741 cagcaacgcg gccgtgaaag gcaggccggt ccgtggtggc cacggcctct aggccagatc
     1801 cagcggcatc tgggttagtc gagcgcgggc cgcttcccat gtctcaccag ggcgagcctg
     1861 tttcgcgatc tcagcatctg aaatcttccc ggccttgcgc ttcgctgggg ccttacccac
     1921 cgccttggcg ggcttcttcg gtccaaaact gaacaacaga tgtgtgacct tgcgcccggt
     1981 ctttcgctgc gcccactcca cctgtagcgg gctgtgctcg ttgatctgcg tcacggctgg
     2041 atcaagcact cgcaacttga agtccttgat cgagggatac cggccttcca gttgaaacca
     2101 ctttcgcagc tggtcaattt ctatttcgcg ctggccgatg ctgtcccatt gcatgagcag
     2161 ctcgtaaagc ctgatcgcgt gggtgctgtc catcttggcc acgtcagcca aggcgtattt
     2221 ggtgaactgt ttggtgagtt ccgtcaggta cggcagcatg tctttggtga acctgagttc
     2281 tacacggccc tcaccctccc ggtagatgat tgtttgcacc cagccggtaa tcatcacact
     2341 cggtcttttc cccttgccat tgggctcttg ggttaaccgg acttcccgcc gtttcaggcg
     2401 cagggccgct tctttgagct ggttgtagga agattcgata gggacacccg ccatcgtcgc
     2461 tatgtcctcc gccgtcactg aatacatcac ttcatcggtg acaggctcgc tcctcttcac
     2521 ctggctaata caggccagaa cgatccgctg ttcctgaaca ctgaggcgat acgcggcctc
     2581 gaccagggca ttgcttttgt aaaccattgg gggtgaggcc acgttcgaca ttccttgtgt
     2641 ataaggggac actgtatctg cgtcccacaa tacaacaaat ccgtcccttt acaacaacaa
     2701 atccgtccct tcttaacaac aaatccgtcc cttaatggca acaaatccgt ccctttttaa
     2761 actctacagg ccacggatta cgtggcctgt agacgtccta aaaggtttaa aagggaaaag
     2821 gaagaaaagg gtggaaacgc aaaaaacgca ccactacgtg gccccgttgg ggccgcattt
     2881 gtgcccctga aggggcgggg gaggcgtctg ggcaatcccc gttttaccag tcccctatcg
     2941 ccgcctgaga gggcgcagga agcgagtaat cagggtatcg aggcggattc acccttggcg
     3001 tccaaccagc ggcaccagcg gcgcctgaga ggactagtgc ttggattctc accaataaaa
     3061 aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact
     3121 ggatctatca acaggagtcc aagcgagctc tcgaacccca gagtcccgct cagaagaact
     3181 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     3241 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     3301 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     3361 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct
     3421 cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     3481 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
     3541 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     3601 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     3661 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     3721 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
     3781 cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
     3841 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
     3901 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
     3961 tcatgcgaaa cgatcctcat cctgtctctt gatcagatca tgatcccctg cgccatcaga
     4021 tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg
     4081 gcgccccagc tggcaattcc
//
