LOCUS       sequence-434095-        5808 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under the Anderson J23113 promoter with an RSF1010 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5808)
  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 5808)
  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: pWN172
FEATURES             Location/Qualifiers
     source          1..5808
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        88..122
                     /label=BBa_J23113
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.01"
     CDS             194..223
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             236..247
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             251..964
                     /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             251..961
                     /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(278..306)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     901..921
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             complement(1070..1921)
                     /codon_start=1
                     /gene="repC"
                     /product="replication protein C of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepC
                     /translation="MVKPKNKHSLSHVRHDPAHCLAPGLFRALKRGERKRSKLDVTYDY
                     GDGKRIEFSGPEPLGADDLRILQGLVAMAGPNGLVLGPEPKTEGGRQLRLFLEPKWEAV
                     TADAMVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
                     YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
                     TGKASIDTLCGYVWPSEASGSTMRKRRQRVREALPELVALGWTVTEFAAGKYDITRPKA
                     AG"
     CDS             complement(1908..2747)
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein A of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepA
                     /translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
                     LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
                     GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
                     EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
                     WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
     CDS             complement(3258..4229)
                     /codon_start=1
                     /gene="repB"
                     /product="replication protein B of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepB
                     /translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
                     QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
                     YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
     rep_origin      4336..4730
                     /label=RSF1010 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     RSF1010; requires the RSF1010 RepA/B/C proteins for 
                     replication (Scholz et al., 1989)"
     terminator      complement(4753..4847)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4878..5672)
                     /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(4989..5008)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5599..5618
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gacgtcctcg gtaccaaatt ccagaaaaga ggcctcccga aaggggggcc ttttttcgtt
       61 ttggtccatc ttgaactagc atacatactg atggctagct cagtcctagg gattatgcta
      121 gcacgtcgac ttatctcgag tgagatattg ttgacgaact cataaagtca agtaggagat
      181 taattccatg ggccatcacc atcaccatca ccatcaccat cacagcagcg gccatatcga
      241 aggtcgtcat agcaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt
      301 agatggtgat gttaatgggc acaaattttc tgtccgtgga gagggtgaag gtgatgctac
      361 aaacggaaaa ctcaccctta aatttatttg cactactgga aaactacctg ttccgtggcc
      421 aacacttgtc actactctga cctatggtgt tcaatgcttt tcccgttatc cggatcacat
      481 gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg aacgcactat
      541 atctttcaaa gatgacggga cctacaagac gcgtgctgaa gtcaagtttg aaggtgatac
      601 ccttgttaat cgtatcgagt taaagggtat tgattttaaa gaagatggaa acattcttgg
      661 acacaaactt gagtacaact ttaactcaca caatgtatac atcacggcag acaaacaaaa
      721 gaatggaatc aaagctaact tcaaaattcg ccacaacgtt gaagatggtt ccgttcaact
      781 agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa
      841 ccattacctg tcgacacaat ctgtcctttc gaaagatccc aacgaaaagc gtgaccacat
      901 ggtccttctt gagtttgtaa ctgctgctgg gattacacat ggcatggatg agctctacaa
      961 ataataagaa ttcttcagcc aaaaaactta agaccgccgg tcttgtccac taccttgcag
     1021 taatgcggtg gacaggatcg gcggttttct tttctcttct caacctaggt cagcctgccg
     1081 ccttgggccg ggtgatgtcg tacttgcccg ccgcgaactc ggttaccgtc cagcccagcg
     1141 cgaccagctc cggcaacgcc tcgcgcaccc gctggcggcg cttgcgcatg gtcgaaccac
     1201 tggcctctga cggccagaca tagccgcaca aggtatctat ggaagccttg ccggttttgc
     1261 cggggtcgat ccagccacac agccgctggt gcagcaggcg ggcggtttcg ctgtccagcg
     1321 cccgcacctc gtccatgctg atgcgcacat gctggccgcc acccatgacg gcctgcgcga
     1381 tcaaggggtt cagggccacg tacaggcgcc cgtccgcctc gtcgctggcg tactccgaca
     1441 gcagccgaaa cccctgccgc ttgcggccat tctgggcgat gatggatacc ttccaaaggc
     1501 gctcgatgca gtcctgtatg tgcttgagcg ccccaccact atcgacctct gccccgattt
     1561 cctttgccag cgcccgatag ctacctttga ccaccatggc atcagcggtg acggcctccc
     1621 acttgggttc caggaacagc cggagctgcc gtccgccttc ggtcttgggt tccgggccaa
     1681 gcactaggcc attaggccca gccatggcca ccagcccttg caggatgcgc agatcatcag
     1741 cgcccagcgg ctccgggccg ctgaactcga tccgcttgcc gtcgccgtag tcatacgtca
     1801 cgtccagctt gctgcgcttg cgctcgcccc gcttgagggc acggaacagg ccgggggcca
     1861 gacagtgcgc cgggtcgtgc cggacgtggc tgaggctgtg cttgttctta ggcttcacca
     1921 cggggcaccc ccttgctctt gcgctgcctc tccagcacgg cgggcttgag caccccgccg
     1981 tcatgccgcc tgaaccaccg atcagcgaac ggtgcgccat agttggcctt gctcacaccg
     2041 aagcggacga agaaccggcg ctggtcgtcg tccacacccc attcctcggc ctcggcgctg
     2101 gtcatgctcg acaggtagga ctgccagcgg atgttatcga ccagtaccga gctgccccgg
     2161 ctggcctgct gctggtcgcc tgcgcccatc atggccgcgc ccttgctggc atggtgcagg
     2221 aacacgatag agcacccggt atcggcggcg atggcctcca tgcgaccgat gacctgggcc
     2281 atggggccgc tggcgttttc ttcctcgatg tggaaccggc gcagcgtgtc cagcaccatc
     2341 aggcggcggc cctcggcggc gcgcttgagg ccgtcgaacc actccggggc catgatgttg
     2401 ggcaggctgc cgatcagcgg ctggatcagc aggccgtcag ccacggcttg ccgttcctcg
     2461 gcgctgaggt gcgccccaag ggcgtgcagg cggtgatgaa tggcggtggg cgggtcttcg
     2521 gcgggcaggt agatcaccgg gccggtgggc agttcgccca cctccagcag atccggcccg
     2581 cctgcaatct gtgcggccag ttgcagggcc agcatggatt taccggcacc accgggcgac
     2641 accagcgccc cgaccgtacc ggccaccatg ttgggcaaaa cgtagtccag cggtggcggc
     2701 gctgctgcga acgcctccag aatattgata ggcttatggg tagccattga ttgcctcctt
     2761 tgcaggcagt tggtggttag gcgctggcgg ggtcactacc cccgccctgc gccgctctga
     2821 gttcttccag gcactcgcgc agcgcctcgt attcgtcgtc ggtcagccag aacttgcgct
     2881 gacgcatccc tttggccttc atgcgctcgg catatcgcgc ttggcgtaca gcgtcagggc
     2941 tggccagcag gtcgccggtc tgcttgtcct tttggtcttt catatcagtc accgagaaac
     3001 ttgccggggc cgaaaggctt gtcttcgcgg aacaaggaca aggtgcagcc gtcaaggtta
     3061 aggctggcca tatcagcgac tgaaaagcgg ccagcctcgg ccttgtttga cgtataacca
     3121 aagccaccgg gcaaccaata gcccttgtca cttttgatca ggtagaccga ccctgaagcg
     3181 cttttttcgt attccataaa acccccttct gtgcgtgagt actcatagta taacaggcgt
     3241 gagtaccaac gcaagcacta catgctgaaa tctggcccgc ccctgtccat gcctcgctgg
     3301 cggggtgccg gtgcccgtgc cagctcggcc cgcgcaagct ggacgctggg cagacccatg
     3361 accttgctga cggtgcgctc gatgtaatcc gcttcgtggc cgggcttgcg ctctgccagc
     3421 gctgggctgg cctcggccat ggccttgccg atttcctcgg cactgcggcc ccggctggcc
     3481 agcttctgcg cggcgataaa gtcgcacttg ctgaggtcat caccgaagcg cttgaccagc
     3541 ccggccatct cgctgcggta ctcgtccagc gccgtgcgcc ggtggcggct aagctgccgc
     3601 tcgggcagtt cgaggctggc cagcctgcgg gccttctcct gctgccgctg ggcctgctcg
     3661 atctgctggc cagcctgctg caccagcgcc gggccagcgg tggcggtctt gcccttggat
     3721 tcacgcagca gcacccacgg ctgataaccg gcgcgggtgg tgtgcttgtc cttgcggttg
     3781 gtgaagcccg ccaagcggcc atagtggcgg ctgtcggcgc tggccgggtc ggcgtcgtac
     3841 tcgctggcca gcgtccgggc aatctgcccc cgaagttcac cgcctgcggc gtcggccacc
     3901 ttgacccatg cctgatagtt cttcgggctg gtttccacta ccagggcagg ctcccggccc
     3961 tcggctttca tgtcatccag gtcaaactcg ctgaggtcgt ccaccagcac cagaccatgc
     4021 cgctcctgct cggcgggcct gatatacacg tcattgccct gggcattcat ccgcttgagc
     4081 catggcgtgt tctggagcac ttcggcggct gaccattccc ggttcatcat ctggccggtg
     4141 gtggcgtccc tgacgccgat atcgaagcgc tcacagccca tggccttgag ctgtcggcct
     4201 atggcctgca aagtcctgtc gttcttcatc gggccaccaa gcgattccca cacattatac
     4261 gagccggaag cataaagtgt agctagatcc gaaggatgag ccgggctgaa tgatcgaccg
     4321 agacaggccc tgcggggctg cacacgcgcc cccacccttc gggtagggga aaaggccgct
     4381 aaagcggcta aaagcgctcc agcgtatttc tgcggggttt ggtgtggggt ttagcgggct
     4441 ttgcccgcct ttccccctgc cgcgcagcgg tggggcggtg tgtagcctag cgcagcgaat
     4501 agaccagcta tccggcctct ggccgggcat attgggcaag ggcagcagcg ccccacaagg
     4561 gcgctgataa ccgcgcctag tggattattc ttagataatc atggatggat ttttccaaca
     4621 ccccgccagc ccccgcccct gctgggtttg caggtttggg ggcgtgacag ttattgcagg
     4681 ggttcgtgac agttattgca ggggggcgtg acagttattg caggggttcg tgacagttag
     4741 actagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa
     4801 atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctctc
     4861 gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc
     4921 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
     4981 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc
     5041 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag
     5101 gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg
     5161 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga
     5221 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg
     5281 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc
     5341 tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc
     5401 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg
     5461 gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg
     5521 gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag
     5581 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga
     5641 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
     5701 tcagatcatg atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact
     5761 ttgcagggct tcccaacctt accagagggc gccccagctg gcaattcc
//
