LOCUS       sequence-431949-        3647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sfGFP under the Anderson J23107 promoter with a BBR1 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3647)
  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 3647)
  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: pWN212
FEATURES             Location/Qualifiers
     source          1..3647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        88..122
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     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(1124..1786)
                     /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      1787..2558
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     terminator      complement(2592..2686)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2717..3511)
                     /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(2828..2847)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3438..3457
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gacgtcctcg gtaccaaatt ccagaaaaga ggcctcccga aaggggggcc ttttttcgtt
       61 ttggtccatc ttgaactagc atacatattt acggctagct cagccctagg tattatgcta
      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 caacctaggc tacagccgat
     1081 agtctggaac agcgcactta cgggttgctg cgcaacccaa gtgctaccgg cgcggcagcg
     1141 tgacccgtgt cggcggctcc aacggctcgc catcgtccag aaaacacggc tcatcgggca
     1201 tcggcaggcg ctgctgcccg cgccgttccc attcctccgt ttcggtcaag gctggcaggt
     1261 ctggttccat gcccggaatg ccgggctggc tgggcggctc ctcgccgggg ccggtcggta
     1321 gttgctgctc gcccggatac agggtcggga tgcggcgcag gtcgccatgc cccaacagcg
     1381 attcgtcctg gtcgtcgtga tcaaccacca cggcggcact gaacaccgac aggcgcaact
     1441 ggtcgcgggg ctggccccac gccacgcggt cattgaccac gtaggccgac acggtgccgg
     1501 ggccgttgag cttcacgacg gagatccagc gctcggccac caagtccttg actgcgtatt
     1561 ggaccgtccg caaagaacgt ccgatgagct tggaaagtgt cttctggctg accaccacgg
     1621 cgttctggtg gcccatctgc gccacgaggt gatgcagcag cattgccgcc gtgggtttcc
     1681 tcgcaataag cccggcccac gcctcatgcg ctttgcgttc cgtttgcacc cagtgaccgg
     1741 gcttgttctt ggcttgaatg ccgatttctc tggactgcgt ggccatgctt atctccatgc
     1801 ggtaggggtg ccgcacggtt gcggcaccat gcgcaatcag ctgcaacttt tcggcagcgc
     1861 gacaacaatt atgcgttgcg taaaagtggc agtcaattac agattttctt taacctacgc
     1921 aatgagctat tgcggggggt gccgcaatga gctgttgcgt accccccttt tttaagttgt
     1981 tgatttttaa gtctttcgca tttcgcccta tatctagttc tttggtgccc aaagaagggc
     2041 acccctgcgg ggttccccca cgccttcggc gcggctcccc ctccggcaaa aagtggcccc
     2101 tccggggctt gttgatcgac tgcgcggcct tcggccttgc ccaaggtggc gctgccccct
     2161 tggaaccccc gcactcgccg ccgtgaggct cggggggcag gcgggcgggc ttcgcccttc
     2221 gactgccccc actcgcatag gcttgggtcg ttccaggcgc gtcaaggcca agccgctgcg
     2281 cggtcgctgc gcgagccttg acccgccttc cacttggtgt ccaaccggca agcgaagcgc
     2341 gcaggccgca ggccggaggc ttttccccag agaaaattaa aaaaattgat ggggcaaggc
     2401 cgcaggccgc gcagttggag ccggtgggta tgtggtcgaa ggctgggtag ccggtgggca
     2461 atccctgtgg tcaagctcgt gggcaggcgc agcctgtcca tcagcttgtc cagcagggtt
     2521 gtccacgggc cgagcgaagc gagccagccg gtggccgctc gcggccatcg tccacatatc
     2581 ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa
     2641 tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag cgagctctcg
     2701 aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
     2761 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
     2821 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
     2881 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
     2941 catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga
     3001 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
     3061 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
     3121 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
     3181 cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc
     3241 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
     3301 ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg
     3361 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
     3421 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
     3481 aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat
     3541 cagatcatga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt
     3601 tgcagggctt cccaacctta ccagagggcg ccccagctgg caattcc
//
