LOCUS       sequence-451464-        7054 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Flp and gentamicin to enable excision of antibiotic 
            resistance cassettes; flp-cl-sacB-pL1V-Lv1-gent-pBBR1-ELT3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7054)
  AUTHORS   Jorrin B, Haskett TL, Knights HE, Martyn A, Underwood TJ, Dolliver 
            J, Ledermann R, Poole PS
  TITLE     Stable, fluorescent markers for tracking synthetic communities and 
            assembly dynamics.
  JOURNAL   Microbiome. 2024 May 7;12(1):81. doi: 10.1186/s40168-024-01792-2.
  PUBMED    38715147
REFERENCE   2  (bases 1 to 7054)
  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: 
            "Microbiome."; date: "2024-05-7"; volume: "12(1):81. doi"; pages: " 
            10.1186/s40168-024-01792-2"
COMMENT     Sequence Label: pFlp-Gm
FEATURES             Location/Qualifiers
     source          1..7054
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      9..103
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        join(112..117,118..134,135..140)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             329..862
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     oriT            1056..1164
                     /note="incP origin of transfer"
     CDS             complement(1175..1837)
                     /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      1838..2609
                     /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(2714..2800)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(2861..4195,4196..4282))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        complement(4283..4728)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             complement(4949..6220)
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=FLP
                     /note="FLP is a site-specific recombinase from 
                     Saccharomyces cerevisiae. Recombination occurs at FRT 
                     sequences."
                     /translation="MPQFDILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
                     THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
                     IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
                     SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
                     KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
                     SYNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
                     TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
                     YPAWNGIISQEVLDYLSSYINRRI"
     RBS             6224..6232
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             6321..7034
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
ORIGIN
        1 cgctcttgga ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt
       61 cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatccaggca attgacataa
      121 gcctgttcgg ttcgtaaact gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga
      181 accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac
      241 tgtttttttg tacagcctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt
      301 cgatgtttga tgttatggag cagcaacgat gttacgcagc agcaacgatg ttacgcagca
      361 gggcagtcgc cctaaaacaa agttaggtgg ctcaagtatg ggcatcattc gcacatgtag
      421 gctcggccct gaccaagtca aatccatgcg ggctgctctt gatcttttcg gtcgtgagtt
      481 cggcgacgta gccacctact cccaacatca gccggactcc gattacctcg ggaacttgct
      541 ccgtagtaag acattcatcg cgcttgctgc cttcgaccaa gaagcggttg ttggcgctct
      601 cgcggcttac gttctgccca agtttgagca gccgcgtagt gagatctata tctatgatct
      661 cgcagtctcc ggagagcacc ggaggcaggg cattgccacc gcgctcatca atctcctcaa
      721 gcatgaggcc aacgcgcttg gtgcttatgt gatctacgtg caagcagatt acggtgacga
      781 tcccgcagtg gctctctata caaagttggg catacgggaa gaagtgatgc actttgatat
      841 cgacccaagt accgccacct aacaattcgt tcaagccgag atcggcttcc cggccgcgga
      901 gttgttcggt aaattgacta cttttccgct gcataaccct gcttcggggt cattatagcg
      961 attttttcgg tatatccatc ctttttcgca cgatatacag gattttgcca aagggttcgt
     1021 gtagactttc cttggtgtat ccaacggcgt cagccgggca ggataggtga agtaggccca
     1081 cccgcgagcg ggtgttcctt cttcactgtc ccttattcgc acctggcggt gctcaacggg
     1141 aatcctgctc tgcgaggctg gccgtaggcc ggccctaccg gcgcggcagc gttacccgtg
     1201 tcggcggctc caacggctcg ccatcgtcca gaaaacacgg ctcatcgggc atcggcaggc
     1261 gctgctgccc gcgccgttcc cattcctccg tttcggtcaa ggctggcagg tctggttcca
     1321 tgcccggaat gccgggctgg ctgggcggct cctcgccggg gccggtcggt agttgctgct
     1381 cgcccggata cagggtcggg atgcggcgca ggtcgccatg ccccaacagc gattcgtcct
     1441 ggtcgtcgtg atcaaccacc acggcggcac tgaacaccga caggcgcaac tggtcgcggg
     1501 gctggcccca cgccacgcgg tcattgacca cgtaggccga cacggtgccg gggccgttga
     1561 gcttcacgac ggagatccag cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc
     1621 gcaaagaacg tccgatgagc ttggaaagag tcttttggct gaccaccacg gcgttctggt
     1681 ggcccatctg cgcgacgagg tgatgcagca gcattgccgc cgtgggtttc ctcgcaataa
     1741 gcccggccca cgcctcatgc gctttgcgtt ccgtttggac ccagtgaccg ggcttgttct
     1801 tggcttgaat gccgatttct ctggactgcg tggccatgct tatctccatg cggtaggggt
     1861 gccgcacggt tgcggcacca tgcgcaatca gctgcaactt ttcggcagcg cgacaacaat
     1921 tatgcgttgc gtaaaagtgg cagtcaatta cagattttct ttaacctacg caatgagcta
     1981 ttgcgggggg tgccgcaatg agctgttgcg tacccccctt ttttaagttg ttgattttta
     2041 agtctttcgc atttcgccct atatctagtt ctttggtgcc caaagaaggg cacccctgcg
     2101 gggttccccc acgccttcgg cgcggctccc cctccggcaa aaagtggccc ctccggggct
     2161 tgttgatcga ctgcgcggcc ttcggccttg cccaaggtgg cgctgccccc ttggaacccc
     2221 cgcactcgcc gccgtgaggc tcggggggca ggcgggcggg cttcgccctt cgactgcccc
     2281 cactcgcata ggcttgggtc gttccaggcg cgtcaaggcc aagccgctgc gcggtcgctg
     2341 cgcgagcctt gacccgcctt cctcttggtg tccaaccggc aagcgaagcg cgcaggccgc
     2401 aggccggagg cttttcccca gagaaaatta aaaaaattga tggggcaagg ccgcaggccg
     2461 cgcagttgga gccggtgggt atgtggtcga aggctgggta gccggtgggc aatccctgtg
     2521 gtcaagctcg tgggcaggcg cagcctgtcc atcagcttgt ccagcagggt tgtccacggg
     2581 ccgagcgaag cgagccagcc ggtggccgct cgcggccatc gtccacatat ccacgggctg
     2641 gcaagggagc gcagcgaccg cgcagggcga agcccggaga gcaagcccgt agggggttac
     2701 tctagggcgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac
     2761 gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctgccg aattcggatc
     2821 cggagatgcc aataggatat cggcattttc ttttgcgttt ttatttgtta actgttaatt
     2881 gtccttgttc aaggatgctg tctttgacaa cagatgtttt cttgcctttg atgttcagca
     2941 ggaagctagg cgcaaacgtt gattgtttgt ctgcgtagaa tcctctgttt gtcatatagc
     3001 ttgtaatcac gacattgttt cctttcgctt gaggtacagc gaagtgtgag taagtaaagg
     3061 ttacatcgtt aggatcaaga tccattttta acacaaggcc agttttgttc agcggcttgt
     3121 atgggccagt taaagaatta gaaacataac caagcatgta aatatcgtta gacgtaatgc
     3181 cgtcaatcgt catttttgat ccgcgggagt cagtgaacag ataccatttg ccgttcattt
     3241 taaagacgtt cgcgcgttca atttcatctg ttactgtgtt agatgcaatc agcggtttca
     3301 tcactttttt cagtgtgtaa tcatcgttta gctcaatcat accgagagcg ccgtttgcta
     3361 actcagccgt gcgtttttta tcgctttgca gaagtttttg actttcttga cggaagaatg
     3421 atgtgctttt gccatagtat gctttgttaa ataaagattc ttcgccttgg tagccatctt
     3481 cagttccagt gtttgcttca aatactaagt atttgtggcc tttatcttct acgtagtgag
     3541 gatctctcag cgtatggttg tcgcctgagc tgtagttgcc ttcatcgatg aactgctgta
     3601 cattttgata cgtttttccg tcaccgtcaa agattgattt ataatcctct acaccgttga
     3661 tgttcaaaga gctgtctgat gctgatacgt taacttgtgc agttgtcagt gtttgtttgc
     3721 cgtaatgttt accggagaaa tcagtgtaga ataaacggat ttttccgtca gatgtaaatg
     3781 tggctgaacc tgaccattct tgtgtttggt cttttaggat agaatcattt gcatcgaatt
     3841 tgtcgctgtc tttaaagacg cggccagcgt ttttccagct gtcaatagaa gtttcgccga
     3901 ctttttgata gaacatgtaa atcgatgtgt catccgcatt tttaggatct ccggctaatg
     3961 caaagacgat gtggtagccg tgatagtttg cgacagtgcc gtcagcgttt tgtaatggcc
     4021 agctgtccca aacgtccagg ccttttgcag aagagatatt tttaattgtg gacgaatcga
     4081 actcaggaac ttgatatttt tcattttttt gctgttcagg gatttgcagc atatcatggc
     4141 gtgtaatatg ggaaatgccg tatgtttcct tatatggctt ttggttcgtt tctttcgcaa
     4201 acgcttgagt tgcgcctcct gccagcagtg cggtagtaaa ggttaatact gttgcttgtt
     4261 ttgcaaactt tttgatgttc atcgttcatg tctccttttt tatgtactgt gttagcggtc
     4321 tgcttcttcc agccctcctg tttgaagatg gcaagttagt tacgcacaat aaaaaaagac
     4381 ctaaaatatg taaggggtga cgccaaagta tacactttgc cctttacaca ttttaggtct
     4441 tgcctgcttt atcagtaaca aacccgcgcg atttactttt cgacctcatt ctattagact
     4501 ctcgtttgga ttgcaactgg tctattttcc tcttttgttt gatagaaaat cataaaagga
     4561 tttgcagact acgggcctaa agaactaaaa aatctatctg tttcttttca ttctctgtat
     4621 tttttatagt ttctgttgca tgggcataaa gttgcctttt taatcacaat tcagaaaata
     4681 tcataatatc tcatttcact aaataatagt gaacggcagg tatatgtgat gggttaaaaa
     4741 ggatcgatcc tctagaggat ccccctagag aataggaact tcggaatagg aacttcaaag
     4801 cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc tgcgcacata cagctcactg
     4861 ttcacgtcgc acctatatct gcgtgttgcc tgtatatata tatacatgag aagaacggca
     4921 tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct atttatgtag gatgaaaggt
     4981 agtctagtac ctcctgtgat attatcccat tccatgcggg gtatcgtatg cttccttcag
     5041 cactaccctt tagctgttct atatgctgcc actcctcaat tggattagtc tcatccttca
     5101 atgctatcat ttcctttgat attggatcat atgcatagta ccgagaaact agtgcgaagt
     5161 agtgatcagg tattgctgtt atctgatgag tatacgttgt cctggccacg gcagaagcac
     5221 gcttatcgct ccaatttccc acaacattag tcaactccgt taggcccttc attgaaagaa
     5281 atgaggtcat caaatgtctt ccaatgtgag attttgggcc attttttata gcaaagattg
     5341 aataaggcgc atttttcttc aaagctttat tgtacgatct gactaagtta tcttttaata
     5401 attggtattc ctgtttattg cttgaagaat tgccggtcct atttactcgt tttaggactg
     5461 gttcagaatt cctcaaaaat tcatccaaat atacaagtgg atcgatccta ccccttgcgc
     5521 taaagaagta tatgtgccta ctaacgcttg tctttgtctc tgtcactaaa cactggatta
     5581 ttactcccag atacttattt tggactaatt taaatgattt cggatcaacg ttcttaatat
     5641 cgctgaatct tccacaattg atgaaagtag ctaggaagag gaattggtat aaagtttttg
     5701 tttttgtaaa tctcgaagta tactcaaacg aatttagtat tttctcagtg atctcccaga
     5761 tgctttcacc ctcacttaga agtgctttaa gcattttttt actgtggcta tttcccttat
     5821 ctgcttcttc cgatgattcg aactgtaatt gcaaactact tacaatatca gtgatatcag
     5881 attgatgttt ttgtccatag taaggaataa ttgtaaattc ccaagcagga atcaatttct
     5941 ttaatgaggc ttccagaatt gttgcttttt gcgtcttgta tttaaactgg agtgatttat
     6001 tgacaatatc gaaactcagc gaattgctta tgatagtatt atagctcatg aatgtggctc
     6061 tcttgattgc tgttccgtta tgtgtaatca tccaacataa ataggttagt tcagcagcac
     6121 ataatgctat tttctcacct gaaggtcttt caaacctttc cacaaactga cgaacaagca
     6181 ccttaggtgg tgttttacat aatatatcaa attgtggcat acaacctcct tagtacatgc
     6241 aaccattatc accgccagag gtaaaatagt caacacgcac ggtgttagat atttatccct
     6301 tgcggtgata gatttaacgt atgagcacaa aaaagaaacc attaacacaa gagcagcttg
     6361 aggacgcacg tcgccttaaa gcaatttatg aaaaaaagaa aaatgaactt ggcttatccc
     6421 aggaatctgt cgcagacaag atggggatgg ggcagtcagg cgttggtgct ttatttaatg
     6481 gcatcaatgc attaaatgct tataacgccg cattgcttac aaaaattctc aaagttagcg
     6541 ttgaagaatt tagcccttca atcgccagag aaatctacga gatgtatgaa gcggttagta
     6601 tgcagccgtc acttagaagt gagtatgagt accctgtttt ttctcatgtt caggcaggga
     6661 tgttctcacc taagcttaga acctttacca aaggtgatgc ggagagatgg gtaagcacaa
     6721 ccaaaaaagc cagtgattct gcattctggc ttgaggttga aggtaattcc atgaccgcac
     6781 caacaggctc caagccaagc tttcctgacg gaatgttaat tctcgttgac cctgagcagg
     6841 ctgttgagcc aggtgatttc tgcatagcca gacttggggg tgatgagttt accttcaaga
     6901 aactgatcag ggatagcggt caggtgtttt tacaaccact aaacccacag tacccaatga
     6961 tcccatgcaa tgagagttgt tccgttgtgg ggaaagttat cgctagtcag tggcctgaag
     7021 agacgtttgg ctgatcggca aggtgttctg gtca
//
