LOCUS       sequence-440371-        4359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under the Anderson J23104 promoter with an RK2 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4359)
  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 4359)
  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: pWN165
FEATURES             Location/Qualifiers
     source          1..4359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        88..122
                     /label=BBa_J23104
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.72"
     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(1120..2268)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      2661..3192
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(3304..3398)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3429..4223)
                     /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(3540..3559)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4150..4169
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gacgtcctcg gtaccaaatt ccagaaaaga ggcctcccga aaggggggcc ttttttcgtt
       61 ttggtccatc ttgaactagc atacatattg acagctagct cagtcctagg tattgtgcta
      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 caacctaggg cgatgcaggt
     1081 ggctgctgaa cccccagccg gaactgaccc cacaaggccc tagcgtttgc aatgcaccag
     1141 gtcatcattg acccaggcgt gttccaccag gccgctgcct cgcaactctt cgcaggcttc
     1201 gccgacctgc tcgcgccact tcttcacgcg ggtggaatcc gatccgcaca tgaggcggaa
     1261 ggtttccagc ttgagcgggt acggctcccg gtgcgagctg aaatagtcga acatccgtcg
     1321 ggccgtcggc gacagcttgc ggtacttctc ccatatgaat ttcgtgtagt ggtcgccagc
     1381 aaacagcacg acgatttcct cgtcgatcag gacctggcaa cgggacgttt tcttgccacg
     1441 gtccaggacg cggaagcggt gcagcagcga caccgattcc aggtgcccaa cgcggtcgga
     1501 cgtgaagccc atcgccgtcg cctgtaggcg cgacaggcat tcctcggcct tcgtgtaata
     1561 ccggccattg atcgaccagc ccaggtcctg gcaaagctcg tagaacgtga aggtgatcgg
     1621 ctcgccgata ggggtgcgct tcgcgtactc caacacctgc tgccacacca gttcgtcatc
     1681 gtcggcccgc agctcgacgc cggtgtaggt gatcttcacg tccttgttga cgtggaaaat
     1741 gaccttgttt tgcagcgcct cgcgcgggat tttcttgttg cgcgtggtga acagggcaga
     1801 gcgggccgtg tcgtttggca tcgctcgcat cgtgtccggc cacggcgcaa tatcgaacaa
     1861 ggaaagctgc atttccttga tctgctgctt cgtgtgtttc agcaacgcgg cctgcttggc
     1921 ttcgctgacc tgttttgcca ggtcctcgcc ggcggttttt cgcttcttgg tcgtcatagt
     1981 tcctcgcgtg tcgatggtca tcgacttcgc caaacctgcc gcctcctgtt cgagacgacg
     2041 cgaacgctcc acggcggccg atggcgcggg cagggcaggg ggagccagtt gcacgctgtc
     2101 gcgctcgatc ttggccgtag cttgctggac tatcgagccg acggactgga aggtttcgcg
     2161 gggcgcacgc atgacggtgc ggcttgcgat ggtttcggca tcctcggcgg aaaaccccgc
     2221 gtcgatcagt tcttgcctgt atgccttccg gtcaaacgtc cgattcattc accctccttg
     2281 cgggattgcc ccggaattaa ttccccggat cgatccgtcg atcttgatcc cctgcgccat
     2341 cagatccttg gcggcaagaa agccatccag tttactttgc agggcttccc aaccttacca
     2401 gagggcgccc cagctggcaa ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc
     2461 tatcgccatg taagcccact gcaagctacc tgctttctct ttgcgcttgc gttttccctt
     2521 gtccagatag cccagtagct gacattcatc cggggtcagc accgtttctg cggactggct
     2581 ttctacgtgg ctgccatttt tggggtgagg ccgttcgcgg ccgaggggcg cagcccctgg
     2641 ggggatggga ggcccgcgtt agcgggccgg gagggttcga gaaggggggg cacccccctt
     2701 cggcgtgcgc ggtcacgcgc acagggcgca gccctggtta aaaacaaggt ttataaatat
     2761 tggtttaaaa gcaggttaaa agacaggtta gcggtggccg aaaaacgggc ggaaaccctt
     2821 gcaaatgctg gattttctgc ctgtggacag cccctcaaat gtcaataggt gcgcccctca
     2881 tctgtcagca ctctgcccct caagtgtcaa ggatcgcgcc cctcatctgt cagtagtcgc
     2941 gcccctcaag tgtcaatacc gcagggcact tatccccagg cttgtccaca tcatctgtgg
     3001 gaaactcgcg taaaatcagg cgttttcgcc gatttgcgag gctggccagc tccacgtcgc
     3061 cggccgaaat cgagcctgcc cctcatctgt caacgccgcg ccgggtgagt cggcccctca
     3121 agtgtcaacg tccgcccctc atctgtcagt gagggccaag ttttccgcga ggtatccaca
     3181 acgccggcgg ccctacatgg ctctgctgta gtgagtgggt tgcgctccgg cagcggtcct
     3241 gatcccccgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cactagtgct
     3301 tggattctca ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg
     3361 gagttctgag gtcattactg gatctatcaa caggagtcca agcgagctct cgaaccccag
     3421 agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga
     3481 gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca
     3541 atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag
     3601 tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca
     3661 tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg
     3721 gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc
     3781 atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc
     3841 ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga
     3901 gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt
     3961 cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac
     4021 gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc ggtcttgaca
     4081 aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt
     4141 gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg
     4201 tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagatcat
     4261 gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc
     4321 ttcccaacct taccagaggg cgccccagct ggcaattcc
//
