LOCUS       sequence-442294-        7177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Flp and kanamycin to enable excision of antibiotic 
            resistance cassettes; flp-cl-sacB-pL1V-Lv1-neo-pBBR1-ELT3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7177)
  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 7177)
  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-Km
FEATURES             Location/Qualifiers
     source          1..7177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(56..1390,1391..1477))
                     /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(1478..1923)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             complement(2144..3415)
                     /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             3419..3427
                     /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             3516..4229
                     /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"
     terminator      4258..4352
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(4451..4470)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4462..5277
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     oriT            5428..5536
                     /note="incP origin of transfer"
     CDS             complement(5547..6209)
                     /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      6210..6981
                     /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(7086..7172)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tgccgaattc ggatccggag atgccaatag gatatcggca ttttcttttg cgtttttatt
       61 tgttaactgt taattgtcct tgttcaagga tgctgtcttt gacaacagat gttttcttgc
      121 ctttgatgtt cagcaggaag ctaggcgcaa acgttgattg tttgtctgcg tagaatcctc
      181 tgtttgtcat atagcttgta atcacgacat tgtttccttt cgcttgaggt acagcgaagt
      241 gtgagtaagt aaaggttaca tcgttaggat caagatccat ttttaacaca aggccagttt
      301 tgttcagcgg cttgtatggg ccagttaaag aattagaaac ataaccaagc atgtaaatat
      361 cgttagacgt aatgccgtca atcgtcattt ttgatccgcg ggagtcagtg aacagatacc
      421 atttgccgtt cattttaaag acgttcgcgc gttcaatttc atctgttact gtgttagatg
      481 caatcagcgg tttcatcact tttttcagtg tgtaatcatc gtttagctca atcataccga
      541 gagcgccgtt tgctaactca gccgtgcgtt ttttatcgct ttgcagaagt ttttgacttt
      601 cttgacggaa gaatgatgtg cttttgccat agtatgcttt gttaaataaa gattcttcgc
      661 cttggtagcc atcttcagtt ccagtgtttg cttcaaatac taagtatttg tggcctttat
      721 cttctacgta gtgaggatct ctcagcgtat ggttgtcgcc tgagctgtag ttgccttcat
      781 cgatgaactg ctgtacattt tgatacgttt ttccgtcacc gtcaaagatt gatttataat
      841 cctctacacc gttgatgttc aaagagctgt ctgatgctga tacgttaact tgtgcagttg
      901 tcagtgtttg tttgccgtaa tgtttaccgg agaaatcagt gtagaataaa cggatttttc
      961 cgtcagatgt aaatgtggct gaacctgacc attcttgtgt ttggtctttt aggatagaat
     1021 catttgcatc gaatttgtcg ctgtctttaa agacgcggcc agcgtttttc cagctgtcaa
     1081 tagaagtttc gccgactttt tgatagaaca tgtaaatcga tgtgtcatcc gcatttttag
     1141 gatctccggc taatgcaaag acgatgtggt agccgtgata gtttgcgaca gtgccgtcag
     1201 cgttttgtaa tggccagctg tcccaaacgt ccaggccttt tgcagaagag atatttttaa
     1261 ttgtggacga atcgaactca ggaacttgat atttttcatt tttttgctgt tcagggattt
     1321 gcagcatatc atggcgtgta atatgggaaa tgccgtatgt ttccttatat ggcttttggt
     1381 tcgtttcttt cgcaaacgct tgagttgcgc ctcctgccag cagtgcggta gtaaaggtta
     1441 atactgttgc ttgttttgca aactttttga tgttcatcgt tcatgtctcc ttttttatgt
     1501 actgtgttag cggtctgctt cttccagccc tcctgtttga agatggcaag ttagttacgc
     1561 acaataaaaa aagacctaaa atatgtaagg ggtgacgcca aagtatacac tttgcccttt
     1621 acacatttta ggtcttgcct gctttatcag taacaaaccc gcgcgattta cttttcgacc
     1681 tcattctatt agactctcgt ttggattgca actggtctat tttcctcttt tgtttgatag
     1741 aaaatcataa aaggatttgc agactacggg cctaaagaac taaaaaatct atctgtttct
     1801 tttcattctc tgtatttttt atagtttctg ttgcatgggc ataaagttgc ctttttaatc
     1861 acaattcaga aaatatcata atatctcatt tcactaaata atagtgaacg gcaggtatat
     1921 gtgatgggtt aaaaaggatc gatcctctag aggatccccc tagagaatag gaacttcgga
     1981 ataggaactt caaagcgttt ccgaaaacga gcgcttccga aaatgcaacg cgagctgcgc
     2041 acatacagct cactgttcac gtcgcaccta tatctgcgtg ttgcctgtat atatatatac
     2101 atgagaagaa cggcatagtg cgtgtttatg cttaaatgcg tacttatatg cgtctattta
     2161 tgtaggatga aaggtagtct agtacctcct gtgatattat cccattccat gcggggtatc
     2221 gtatgcttcc ttcagcacta ccctttagct gttctatatg ctgccactcc tcaattggat
     2281 tagtctcatc cttcaatgct atcatttcct ttgatattgg atcatatgca tagtaccgag
     2341 aaactagtgc gaagtagtga tcaggtattg ctgttatctg atgagtatac gttgtcctgg
     2401 ccacggcaga agcacgctta tcgctccaat ttcccacaac attagtcaac tccgttaggc
     2461 ccttcattga aagaaatgag gtcatcaaat gtcttccaat gtgagatttt gggccatttt
     2521 ttatagcaaa gattgaataa ggcgcatttt tcttcaaagc tttattgtac gatctgacta
     2581 agttatcttt taataattgg tattcctgtt tattgcttga agaattgccg gtcctattta
     2641 ctcgttttag gactggttca gaattcctca aaaattcatc caaatataca agtggatcga
     2701 tcctacccct tgcgctaaag aagtatatgt gcctactaac gcttgtcttt gtctctgtca
     2761 ctaaacactg gattattact cccagatact tattttggac taatttaaat gatttcggat
     2821 caacgttctt aatatcgctg aatcttccac aattgatgaa agtagctagg aagaggaatt
     2881 ggtataaagt ttttgttttt gtaaatctcg aagtatactc aaacgaattt agtattttct
     2941 cagtgatctc ccagatgctt tcaccctcac ttagaagtgc tttaagcatt tttttactgt
     3001 ggctatttcc cttatctgct tcttccgatg attcgaactg taattgcaaa ctacttacaa
     3061 tatcagtgat atcagattga tgtttttgtc catagtaagg aataattgta aattcccaag
     3121 caggaatcaa tttctttaat gaggcttcca gaattgttgc tttttgcgtc ttgtatttaa
     3181 actggagtga tttattgaca atatcgaaac tcagcgaatt gcttatgata gtattatagc
     3241 tcatgaatgt ggctctcttg attgctgttc cgttatgtgt aatcatccaa cataaatagg
     3301 ttagttcagc agcacataat gctattttct cacctgaagg tctttcaaac ctttccacaa
     3361 actgacgaac aagcacctta ggtggtgttt tacataatat atcaaattgt ggcatacaac
     3421 ctccttagta catgcaacca ttatcaccgc cagaggtaaa atagtcaaca cgcacggtgt
     3481 tagatattta tcccttgcgg tgatagattt aacgtatgag cacaaaaaag aaaccattaa
     3541 cacaagagca gcttgaggac gcacgtcgcc ttaaagcaat ttatgaaaaa aagaaaaatg
     3601 aacttggctt atcccaggaa tctgtcgcag acaagatggg gatggggcag tcaggcgttg
     3661 gtgctttatt taatggcatc aatgcattaa atgcttataa cgccgcattg cttacaaaaa
     3721 ttctcaaagt tagcgttgaa gaatttagcc cttcaatcgc cagagaaatc tacgagatgt
     3781 atgaagcggt tagtatgcag ccgtcactta gaagtgagta tgagtaccct gttttttctc
     3841 atgttcaggc agggatgttc tcacctaagc ttagaacctt taccaaaggt gatgcggaga
     3901 gatgggtaag cacaaccaaa aaagccagtg attctgcatt ctggcttgag gttgaaggta
     3961 attccatgac cgcaccaaca ggctccaagc caagctttcc tgacggaatg ttaattctcg
     4021 ttgaccctga gcaggctgtt gagccaggtg atttctgcat agccagactt gggggtgatg
     4081 agtttacctt caagaaactg atcagggata gcggtcaggt gtttttacaa ccactaaacc
     4141 cacagtaccc aatgatccca tgcaatgaga gttgttccgt tgtggggaaa gttatcgcta
     4201 gtcagtggcc tgaagagacg tttggctgat cggcaaggtg ttctggtcac gctcttggac
     4261 tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg
     4321 gttgccgccg ggcgtttttt attggtgaga atccaggcaa ttgtgtctca aaatctctga
     4381 tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata
     4441 aacagtaata caaggggtgt tatgagccat attcagcgtg aaacgagctg tagccgtccg
     4501 cgtctgaaca gcaacatgga tgcggatctg tatggctata aatgggcgcg tgataacgtg
     4561 ggtcagagcg gcgcgaccat ttatcgtctg tatggcaaac cggatgcgcc ggaactgttt
     4621 ctgaaacatg gcaaaggcag cgtggcgaac gatgtgaccg atgaaatggt gcgtctgaac
     4681 tggctgaccg aatttatgcc gctgccgacc attaaacatt ttattcgcac cccggatgat
     4741 gcgtggctgc tgaccaccgc gattccgggc aaaaccgcgt ttcaggtgct ggaagaatat
     4801 ccggatagcg gcgaaaacat tgtggatgcg ctggccgtgt ttctgcgtcg tctgcatagc
     4861 attccggtgt gcaactgccc gtttaacagc gatcgtgtgt ttcgtctggc ccaggcgcag
     4921 agccgtatga acaacggcct ggtggatgcg agcgattttg atgatgaacg taacggctgg
     4981 ccggtggaac aggtgtggaa agaaatgcat aaactgctgc cgtttagccc ggatagcgtg
     5041 gtgacccacg gcgattttag cctggataac ctgattttcg atgaaggcaa actgattggc
     5101 tgcattgatg tgggccgtgt gggcattgcg gatcgttatc aggatctggc cattctgtgg
     5161 aactgcctgg gcgaatttag cccgagcctg caaaaacgtc tgtttcagaa atatggcatt
     5221 gataatccgg atatgaacaa actgcaattt catctgatgc tggatgaatt tttctaataa
     5281 ttaattgaac tacttttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc
     5341 ggtatatcca tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt
     5401 tccttggtgt atccaacggc gtcagccggg caggataggt gaagtaggcc cacccgcgag
     5461 cgggtgttcc ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc
     5521 tctgcgaggc tggccgtagg ccggccctac cggcgcggca gcgttacccg tgtcggcggc
     5581 tccaacggct cgccatcgtc cagaaaacac ggctcatcgg gcatcggcag gcgctgctgc
     5641 ccgcgccgtt cccattcctc cgtttcggtc aaggctggca ggtctggttc catgcccgga
     5701 atgccgggct ggctgggcgg ctcctcgccg gggccggtcg gtagttgctg ctcgcccgga
     5761 tacagggtcg ggatgcggcg caggtcgcca tgccccaaca gcgattcgtc ctggtcgtcg
     5821 tgatcaacca ccacggcggc actgaacacc gacaggcgca actggtcgcg gggctggccc
     5881 cacgccacgc ggtcattgac cacgtaggcc gacacggtgc cggggccgtt gagcttcacg
     5941 acggagatcc agcgctcggc caccaagtcc ttgactgcgt attggaccgt ccgcaaagaa
     6001 cgtccgatga gcttggaaag agtcttttgg ctgaccacca cggcgttctg gtggcccatc
     6061 tgcgcgacga ggtgatgcag cagcattgcc gccgtgggtt tcctcgcaat aagcccggcc
     6121 cacgcctcat gcgctttgcg ttccgtttgg acccagtgac cgggcttgtt cttggcttga
     6181 atgccgattt ctctggactg cgtggccatg cttatctcca tgcggtaggg gtgccgcacg
     6241 gttgcggcac catgcgcaat cagctgcaac ttttcggcag cgcgacaaca attatgcgtt
     6301 gcgtaaaagt ggcagtcaat tacagatttt ctttaaccta cgcaatgagc tattgcgggg
     6361 ggtgccgcaa tgagctgttg cgtacccccc ttttttaagt tgttgatttt taagtctttc
     6421 gcatttcgcc ctatatctag ttctttggtg cccaaagaag ggcacccctg cggggttccc
     6481 ccacgccttc ggcgcggctc cccctccggc aaaaagtggc ccctccgggg cttgttgatc
     6541 gactgcgcgg ccttcggcct tgcccaaggt ggcgctgccc ccttggaacc cccgcactcg
     6601 ccgccgtgag gctcgggggg caggcgggcg ggcttcgccc ttcgactgcc cccactcgca
     6661 taggcttggg tcgttccagg cgcgtcaagg ccaagccgct gcgcggtcgc tgcgcgagcc
     6721 ttgacccgcc ttcctcttgg tgtccaaccg gcaagcgaag cgcgcaggcc gcaggccgga
     6781 ggcttttccc cagagaaaat taaaaaaatt gatggggcaa ggccgcaggc cgcgcagttg
     6841 gagccggtgg gtatgtggtc gaaggctggg tagccggtgg gcaatccctg tggtcaagct
     6901 cgtgggcagg cgcagcctgt ccatcagctt gtccagcagg gttgtccacg ggccgagcga
     6961 agcgagccag ccggtggccg ctcgcggcca tcgtccacat atccacgggc tggcaaggga
     7021 gcgcagcgac cgcgcagggc gaagcccgga gagcaagccc gtagggggtt actctagggc
     7081 ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc
     7141 cagtctttcg actgagcctt tcgttttatt tgatgcc
//
