LOCUS       sequence-375746-        8294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RSF1010-based broad host-range plasmid engineered from pAM5409, 
            kanamycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8294)
  AUTHORS   Dong X, Guthrie BGH, Alexander M, Noecker C, Ramirez L, Glasser NR, 
            Turnbaugh PJ, Balskus EP
  TITLE     Genetic manipulation of the human gut bacterium Eggerthella lenta 
            reveals a widespread family of transcriptional regulators.
  JOURNAL   Nat Commun. 2022 Dec 9;13(1):7624. doi: 10.1038/s41467-022-33576-3.
  PUBMED    36494336
REFERENCE   2  (bases 1 to 8294)
  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"; journalName: "Nat 
            Commun."; date: "2022-12-9"; volume: "13(1):7624. doi"; pages: " 
            10.1038/s41467-022-33576-3"
COMMENT     Sequence Label: pXD68Kan2-AphA
FEATURES             Location/Qualifiers
     source          1..8294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            127..236
                     /note="incP origin of transfer"
     terminator      332..375
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     rep_origin      538..932
                     /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)"
     oriT            1274..1361
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     CDS             2600..3571
                     /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
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELSERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
     CDS             4082..4921
                     /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             4908..5759
                     /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"
     terminator      complement(6191..6218)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    6228..6252
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(6268..6981,6982..6984,6985..6987))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVRFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6313..6334)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6662..6681
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(join(7018..7024,7025..7040,7041..7046))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             7482..8276
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
ORIGIN
        1 taatgagcgg gctttttttt ggcgcgccgc tagcatccgc gcgcgcgcgc gcgatgtcgc
       61 acgatataca ggattttgcc aaagggttcg tgtagacttt ccttggtgta tccaacggcg
      121 tcagccgggc aggataggtg aagtaggccc acccgcgagc gggtgttcct tcttcactgt
      181 cccttattcg cacctggcgg tgctcaacgg gaatcctgct ctgcgaggct ggccggctac
      241 cgccggcgta acagatgagg gcaagcggat ggctgatgaa accaagccaa ccagtctacg
      301 gtcagcgaac caatcagcag ggtcatcgct agaaatcatc cttagcgaaa gctaaggatt
      361 ttttttatct gaattaattc tcgcgagggc cgccacatga ggcatgttcc gcgtccttgc
      421 aatactgtgt ttacatacag tctatcgctt agcggaaagt tcttttaccc tcagccgaaa
      481 tgcctgccgt tgctagacat tgccagccag tgcccgtcac tcccgtacta actgtcacga
      541 acccctgcaa taactgtcac gcccccctgc aataactgtc acgaacccct gcaataactg
      601 tcacgccccc aaacctgcaa acccagcagg ggcgggggct ggcggggtgt tggaaaaatc
      661 catccatgat tatctaagaa taatccacta ggcgcggtta tcagcgccct tgtggggcgc
      721 tgctgccctt gcccaatatg cccggccaga ggccggatag ctggtctatt cgctgcgcta
      781 ggctacacac cgccccaccg ctgcgcggca gggggaaagg cgggcaaagc ccgctaaacc
      841 ccacaccaaa ccccgcagaa atacgctgga gcgcttttag ccgctttagc ggcctttccc
      901 cctacccgaa gggtgggggc gcgtgtgcag ccccgcaggg cctgtctcgg tcgatcattc
      961 agcccggctc atccttctgg cgtggcggca gaccgaacaa ggcgcggtcg tggtcgcgtt
     1021 caaggtacgc atccattgcc gccatgagcc gatcctccgg ccactcgctg ctgttcacct
     1081 tggccaaaat catggccccc accagcacct tgcgccttgt ttcgttcttg cgctcttgct
     1141 gctgttccct tgcccgcacc cgctgaattt cggcattgat tcgcgctcgt tgttcttcga
     1201 gcttggccag ccgatccgcc gccttgttgc tccccttaac catcttgaca ccccattgtt
     1261 aatgtgctgt ctcgtaggct atcatggagg cacagcggcg gcaatcccga ccctactttg
     1321 taggggaggg cgcacttacc ggtttctctt cgagaaactg gcctaacggc cacccttcgg
     1381 gcggtgcgct ctccgagggc cattgcatgg agccgaaaag caaaagcaac agcgaggcag
     1441 catggcgatt tatcacctta cggcgaaaac cggcagcagg tcgggcggcc aatcggccag
     1501 ggccaaggcc gacttcatcc agcgcgaagg caagtatgcc cgcgacatgg atgaagtctt
     1561 gcacgccgaa tccgggcaca tgccggagtt cgtcgagcgg cccgccgact actgggatgc
     1621 tgccgacctg tatgaacgcg ccaatgggcg gctgttcaag gaggtcgaat ttgccctgcc
     1681 ggtcgagctg accctcgacc agcagaaggc gctggcgtcc gagttcgccc agcacctgac
     1741 cggtgccgag cgcctgccgt atacgctggc catccatgcc ggtggcggcg agaacccgca
     1801 ctgccacctg atgatctccg agcggatcaa tgacggcatc gagcggcccg ccgctcagtg
     1861 gttcaagcgg tacaacggca agaccccgga gaagggcggg gcacagaaga ccgaagcgct
     1921 caagcccaag gcatggcttg agcagacccg cgaggcatgg gccgaccatg ccaaccgggc
     1981 attagagcgg gcaggccacg acgcccgcat tgaccacaga acacttgagg cgcagggcat
     2041 cgagcgcctg cccggtgttc acctggggcc gaacgtggtg gagatggaag gccggggcat
     2101 ccgcaccgac cgggcagacg tggccctgaa catcgacacc gccaacgccc agatcatcga
     2161 cttacaggaa taccgggagg caatagacca tgaacgcaat cgacagagtg aagaaatcca
     2221 gaggcatcaa cgagttagcg gagcagatcg aaccgctggc ccagagcatg gcgacactgg
     2281 ccgacgaagc ccggcaggtc atgagccaga cccagcaggc cagcgaggcg caggcggcgg
     2341 agtggctgaa agcccagcgc cagacagggg cggcatgggt ggagctggcc aaagagttgc
     2401 gggaggtagc cgccgaggtg agcagcgccg cgcagagcgc ccggagcgcg tcgcgggggt
     2461 ggcactggaa gctatggcta accgtgatgc tggcttccat gatgcctacg gtggtgctgc
     2521 tgatcgcatc gttgctcttg ctcgacctga cgccactgac aaccgaggac ggctcgatct
     2581 ggctgcgctt ggtggcccga tgaagaacga caggactttg caggccatag gccgacagct
     2641 caaggccatg ggctgtgagc gcttcgatat cggcgtcagg gacgccacca ccggccagat
     2701 gatgaaccgg gaatggtcag ccgccgaagt gctccagaac acgccatggc tcaagcggat
     2761 gaatgcccag ggcaatgacg tgtatatcag gcccgccgag caggagcggc atggtctggt
     2821 gctggtggac gacctcagcg agtttgacct ggatgacatg aaagccgagg gccgggagcc
     2881 tgccctggta gtggaaacca gcccgaagaa ctatcaggca tgggtcaagg tggccgacgc
     2941 cgcaggcggt gaacttcggg ggcagattgc ccggacgctg gccagcgagt acgacgccga
     3001 cccggccagc gccgacagcc gccactatgg ccgcttggcg ggcttcacca accgcaagga
     3061 caagcacacc acccgcgccg gttatcagcc gtgggtgctg ctgcgtgaat ccaagggcaa
     3121 gaccgccacc gctggcccgg cgctggtgca gcaggctggc cagcagatcg agcaggccca
     3181 gcggcagcag gagaaggccc gcaggctggc cagcctcgaa ctgtccgagc ggcagcttag
     3241 ccgccaccgg cgcacggcgc tggacgagta ccgcagcgag atggccgggc tggtcaagcg
     3301 cttcggtgat gacctcagca agtgcgactt tatcgccgcg cagaagctgg ccagccgggg
     3361 ccgcagtgcc gaggaaatcg gcaaggccat ggccgaggcc agcccagcgc tggcagagcg
     3421 caagcccggc cacgaagcgg attacatcga gcgcaccgtc agcaaggtca tgggtctgcc
     3481 cagcgtccag cttgcgcggg ccgagctggc acgggcaccg gcaccccgcc agcgaggcat
     3541 ggacaggggc gggccagatt tcagcatgta gtgcttgcgt tggtactcac gcctgttata
     3601 ctatgagtac tcacgcacag aagggggttt tatggaatac gaaaaaagcg cttcagggtc
     3661 ggtctacctg atcaaaagtg acaagggcta ttggttgccc ggtggctttg gttatacgtc
     3721 aaacaaggcc gaggctggcc gcttttcagt cgctgatatg gccagcctta accttgacgg
     3781 ctgcaccttg tccttgttcc gcgaagacaa gcctttcggc cccggcaagt ttctcggtga
     3841 ctgatatgaa agaccaaaag gacaagcaga ccggcgacct gctggccagc cctgacgctg
     3901 tacgccaagc gcgatatgcc gagcgcatga aggccaaagg gatgcgtcag cgcaagttct
     3961 ggctgaccga cgacgaatac gaggcgctgc gcgagtgcct ggaagaactc agagcggcgc
     4021 agggcggggg tagtgacccc gccagcgcct aaccaccaac tgcctgcaaa ggaggcaatc
     4081 aatggctacc cataagccta tcaatattct ggaggcgttc gcagcagcgc cgccaccgct
     4141 ggactacgtt ttgcccaaca tggtggccgg tacggtcggg gcgctggtgt cgcccggtgg
     4201 tgccggtaaa tccatgctgg ccctgcaact ggccgcacag attgcaggcg ggccggatct
     4261 gctggaggtg ggcgaactgc ccaccggccc ggtgatctac ctgcccgccg aagacccgcc
     4321 caccgccatt catcaccgcc tgcacgccct tggggcgcac ctcagcgccg aggaacggca
     4381 agccgtggct gacggcctgc tgatccagcc gctgatcggc agcctgccca acatcatggc
     4441 cccggagtgg ttcgacggcc tcaagcgcgc cgccgagggc cgccgcctga tggtgctgga
     4501 cacgctgcgc cggttccaca tcgaggaaga aaacgccagc ggccccatgg cccaggtcat
     4561 cggtcgcatg gaggccatcg ccgccgatac cgggtgctct atcgtgttcc tgcaccatgc
     4621 cagcaagggc gcggccatga tgggcgcagg cgaccagcag caggccagcc ggggcagctc
     4681 ggtactggtc gataacatcc gctggcagtc ctacctgtcg agcatgacca gcgccgaggc
     4741 cgaggaatgg ggtgtggacg acgaccagcg ccggttcttc gtccgcttcg gtgtgagcaa
     4801 ggccaactat ggcgcaccgt tcgctgatcg gtggttcagg cggcatgacg gcggggtgct
     4861 caagcccgcc gtgctggaga ggcagcgcaa gagcaagggg gtgccccgtg gtgaagccta
     4921 agaacaagca cagcctcagc cacgtccgac acgacccggc gcactgtctg gcccccggcc
     4981 tgttccgtgc cctcaagcgg ggcgagcgca agcgcagcaa gctggacgtg acgtatgact
     5041 acggcgacgg caagcggatc gagttcagcg gcccggagcc gctgggcgct gatgatctgc
     5101 gcatcctgca agggctggtg gccatggctg ggcctaatgg cctagtgctt ggcccggaac
     5161 ccaagaccga aggcggacgg cagctccggc tgttcctgga acccaagtgg gaggccgtca
     5221 ccgctgatgc catggtggtc aaaggtagct atcgggcgct ggcaaaggaa atcggggcag
     5281 aggtcgatag tggtggggcg ctcaagcaca tacaggactg catcgagcgc ctttggaagg
     5341 tatccatcat cgcccagaat ggccgcaagc ggcaggggtt tcggctgctg tcggagtacg
     5401 ccagcgacga ggcggacggg cgcctgtacg tggccctgaa ccccttgatc gcgcaggccg
     5461 tcatgggtgg cggccagcat gtgcgcatca gcatggacga ggtgcgggcg ctggacagcg
     5521 aaaccgcccg cctgctgcac cagcggctgt gtggctggat cgaccccggc aaaaccggca
     5581 aggcttccat agataccttg tgcggctatg tctggccgtc agaggccagt ggttcgacca
     5641 tgcgcaagcg ccgccagcgg gtgcgcgagg cgttgccgga gctggtcgcg ctgggctgga
     5701 cggtaaccga gttcgcggcg ggcaagtacg acatcacccg gcccaaggcg gcaggctgac
     5761 cccccccact ctattgtaaa caagacattt ttatctttta tattcaatgg cttattttcc
     5821 tgctaattgg taataccatg aaaaatacca tgctcagaaa aggcttaaca atattttgaa
     5881 aaattgccta ctgagcgctg ccgcacagct ccataggccg ctttcctggc tttgcttcca
     5941 gatgtatgct cttctgctcc cgaacgccag caagacgtag cccagcgcgt cggccagctt
     6001 gcaattcgcg ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     6061 acctgtcgtg ccagctcgcg agaattaatt caaaaaaagc ccgctcatta agcgggctaa
     6121 ttcgagggcg agacgaggac ttccattgtt ctagaatccc cccgggggcc cccgattcta
     6181 gaaagcttca aaaaggccat ccgtcaggat ggccttctcc tgcagggccg agtttgtaca
     6241 agaaagctgg gtcggcgcgc ccacccttta cttgtacagc tcgtccatgc cgagagtgat
     6301 cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt
     6361 gctcagggcg gactggtagc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc
     6421 gatgggggtg ttctgctggt agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg
     6481 gcggatcttg aagttcacct tgatgccgtt cttctgcttg tcggccatga tatagacgtt
     6541 gtggctgttg tagttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc
     6601 gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacctca cctcggcgcg
     6661 ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt agccttcggg
     6721 catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggg cgaagcactg
     6781 caggccgtag ccgaaggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt
     6841 gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac
     6901 gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt
     6961 gaacagctcc tcgcccttgc tcaccatctt aagacctcct ttatttaaat agggtaccat
     7021 tatacgagcc gatgattaat tgtcaatctc aattcgaaac cggtatccgg cggcggcggc
     7081 gggataccgg tttcgaatgc gtggagaccg aaaccttgcg ctcgttcgcc agccaggaca
     7141 gaaatgcctc gacttcgctg ctgcccaagg ttgccgggtg acgcacaccg tggaaacgga
     7201 tgaaggcacg aacccagtgg acataagcct gttcggttcg taagctgtaa tgcaagtagc
     7261 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
     7321 tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg cctcgggcat
     7381 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
     7441 ttacgcagca gggcagtcgc cctaaaacaa agttaaacat catggctaaa atgagaatat
     7501 caccggaatt gaaaaaactg atcgaaaaat accgctgcgt aaaagatacg gaaggaatgt
     7561 ctcctgctaa ggtatataag ctggtgggag aaaatgaaaa cctatattta aaaatgacgg
     7621 acagccggta taaagggacc acctatgatg tggaacggga aaaggacatg atgctatggc
     7681 tggaaggaaa gctgcctgtt ccaaaggtcc tgcactttga acggcatgat ggctggagca
     7741 atctgctcat gagtgaggcc gatggcgtcc tttgctcgga agagtatgaa gatgaacaaa
     7801 gccctgaaaa gattatcgag ctgtatgcgg agtgcatcag gctctttcac tccatcgaca
     7861 tatcggattg tccctatacg aatagcttag acagccgctt agccgaattg gattacttac
     7921 tgaataacga tctggccgat gtggattgcg aaaactggga agaagacact ccatttaaag
     7981 atccgcgcga gctgtatgat tttttaaaga cggaaaagcc cgaagaggaa cttgtctttt
     8041 cccacggcga cctgggagac agcaacatct ttgtgaaaga tggcaaagta agtggcttta
     8101 ttgatcttgg gagaagcggc agggcggaca agtggtatga cattgccttc tgcgtccggt
     8161 cgatcaggga ggatatcggg gaagaacagt atgtcgagct attttttgac ttactgggga
     8221 tcaagcctga ttgggagaaa ataaaatatt atattttact ggatgaattg ttttagaagg
     8281 ccggccagcc cgcc
//
