LOCUS       sequence-442295-        8205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Flp and tetracycline to enable excision of antibiotic 
            resistance cassettes; flp-cl-sacB-pL1V-Lv1-TetAR-pBBR1-ELT3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8205)
  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 8205)
  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-Tc
FEATURES             Location/Qualifiers
     source          1..8205
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            136..244
                     /note="incP origin of transfer"
     CDS             complement(255..917)
                     /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      918..1689
                     /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(1794..1880)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(1941..3275,3276..3362))
                     /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(3363..3808)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             complement(4029..5300)
                     /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             5304..5312
                     /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             5401..6114
                     /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      6143..6237
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6246..6896)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline resistance regulatory protein"
                     /label=TetR
                     /translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
                     RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
                     PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
                     QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
     CDS             7002..8201
                     /codon_start=1
                     /gene="tetA"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
                     GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
                     VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
                     FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
                     QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
                     LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
                     AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
                     "
ORIGIN
        1 cttttccgct gcataaccct gcttcggggt cattatagcg attttttcgg tatatccatc
       61 ctttttcgca cgatatacag gattttgcca aagggttcgt gtagactttc cttggtgtat
      121 ccaacggcgt cagccgggca ggataggtga agtaggccca cccgcgagcg ggtgttcctt
      181 cttcactgtc ccttattcgc acctggcggt gctcaacggg aatcctgctc tgcgaggctg
      241 gccgtaggcc ggccctaccg gcgcggcagc gttacccgtg tcggcggctc caacggctcg
      301 ccatcgtcca gaaaacacgg ctcatcgggc atcggcaggc gctgctgccc gcgccgttcc
      361 cattcctccg tttcggtcaa ggctggcagg tctggttcca tgcccggaat gccgggctgg
      421 ctgggcggct cctcgccggg gccggtcggt agttgctgct cgcccggata cagggtcggg
      481 atgcggcgca ggtcgccatg ccccaacagc gattcgtcct ggtcgtcgtg atcaaccacc
      541 acggcggcac tgaacaccga caggcgcaac tggtcgcggg gctggcccca cgccacgcgg
      601 tcattgacca cgtaggccga cacggtgccg gggccgttga gcttcacgac ggagatccag
      661 cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc gcaaagaacg tccgatgagc
      721 ttggaaagag tcttttggct gaccaccacg gcgttctggt ggcccatctg cgcgacgagg
      781 tgatgcagca gcattgccgc cgtgggtttc ctcgcaataa gcccggccca cgcctcatgc
      841 gctttgcgtt ccgtttggac ccagtgaccg ggcttgttct tggcttgaat gccgatttct
      901 ctggactgcg tggccatgct tatctccatg cggtaggggt gccgcacggt tgcggcacca
      961 tgcgcaatca gctgcaactt ttcggcagcg cgacaacaat tatgcgttgc gtaaaagtgg
     1021 cagtcaatta cagattttct ttaacctacg caatgagcta ttgcgggggg tgccgcaatg
     1081 agctgttgcg tacccccctt ttttaagttg ttgattttta agtctttcgc atttcgccct
     1141 atatctagtt ctttggtgcc caaagaaggg cacccctgcg gggttccccc acgccttcgg
     1201 cgcggctccc cctccggcaa aaagtggccc ctccggggct tgttgatcga ctgcgcggcc
     1261 ttcggccttg cccaaggtgg cgctgccccc ttggaacccc cgcactcgcc gccgtgaggc
     1321 tcggggggca ggcgggcggg cttcgccctt cgactgcccc cactcgcata ggcttgggtc
     1381 gttccaggcg cgtcaaggcc aagccgctgc gcggtcgctg cgcgagcctt gacccgcctt
     1441 cctcttggtg tccaaccggc aagcgaagcg cgcaggccgc aggccggagg cttttcccca
     1501 gagaaaatta aaaaaattga tggggcaagg ccgcaggccg cgcagttgga gccggtgggt
     1561 atgtggtcga aggctgggta gccggtgggc aatccctgtg gtcaagctcg tgggcaggcg
     1621 cagcctgtcc atcagcttgt ccagcagggt tgtccacggg ccgagcgaag cgagccagcc
     1681 ggtggccgct cgcggccatc gtccacatat ccacgggctg gcaagggagc gcagcgaccg
     1741 cgcagggcga agcccggaga gcaagcccgt agggggttac tctagggcgg cggatttgtc
     1801 ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
     1861 tgagcctttc gttttatttg atgcctgccg aattcggatc cggagatgcc aataggatat
     1921 cggcattttc ttttgcgttt ttatttgtta actgttaatt gtccttgttc aaggatgctg
     1981 tctttgacaa cagatgtttt cttgcctttg atgttcagca ggaagctagg cgcaaacgtt
     2041 gattgtttgt ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac gacattgttt
     2101 cctttcgctt gaggtacagc gaagtgtgag taagtaaagg ttacatcgtt aggatcaaga
     2161 tccattttta acacaaggcc agttttgttc agcggcttgt atgggccagt taaagaatta
     2221 gaaacataac caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt catttttgat
     2281 ccgcgggagt cagtgaacag ataccatttg ccgttcattt taaagacgtt cgcgcgttca
     2341 atttcatctg ttactgtgtt agatgcaatc agcggtttca tcactttttt cagtgtgtaa
     2401 tcatcgttta gctcaatcat accgagagcg ccgtttgcta actcagccgt gcgtttttta
     2461 tcgctttgca gaagtttttg actttcttga cggaagaatg atgtgctttt gccatagtat
     2521 gctttgttaa ataaagattc ttcgccttgg tagccatctt cagttccagt gtttgcttca
     2581 aatactaagt atttgtggcc tttatcttct acgtagtgag gatctctcag cgtatggttg
     2641 tcgcctgagc tgtagttgcc ttcatcgatg aactgctgta cattttgata cgtttttccg
     2701 tcaccgtcaa agattgattt ataatcctct acaccgttga tgttcaaaga gctgtctgat
     2761 gctgatacgt taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt accggagaaa
     2821 tcagtgtaga ataaacggat ttttccgtca gatgtaaatg tggctgaacc tgaccattct
     2881 tgtgtttggt cttttaggat agaatcattt gcatcgaatt tgtcgctgtc tttaaagacg
     2941 cggccagcgt ttttccagct gtcaatagaa gtttcgccga ctttttgata gaacatgtaa
     3001 atcgatgtgt catccgcatt tttaggatct ccggctaatg caaagacgat gtggtagccg
     3061 tgatagtttg cgacagtgcc gtcagcgttt tgtaatggcc agctgtccca aacgtccagg
     3121 ccttttgcag aagagatatt tttaattgtg gacgaatcga actcaggaac ttgatatttt
     3181 tcattttttt gctgttcagg gatttgcagc atatcatggc gtgtaatatg ggaaatgccg
     3241 tatgtttcct tatatggctt ttggttcgtt tctttcgcaa acgcttgagt tgcgcctcct
     3301 gccagcagtg cggtagtaaa ggttaatact gttgcttgtt ttgcaaactt tttgatgttc
     3361 atcgttcatg tctccttttt tatgtactgt gttagcggtc tgcttcttcc agccctcctg
     3421 tttgaagatg gcaagttagt tacgcacaat aaaaaaagac ctaaaatatg taaggggtga
     3481 cgccaaagta tacactttgc cctttacaca ttttaggtct tgcctgcttt atcagtaaca
     3541 aacccgcgcg atttactttt cgacctcatt ctattagact ctcgtttgga ttgcaactgg
     3601 tctattttcc tcttttgttt gatagaaaat cataaaagga tttgcagact acgggcctaa
     3661 agaactaaaa aatctatctg tttcttttca ttctctgtat tttttatagt ttctgttgca
     3721 tgggcataaa gttgcctttt taatcacaat tcagaaaata tcataatatc tcatttcact
     3781 aaataatagt gaacggcagg tatatgtgat gggttaaaaa ggatcgatcc tctagaggat
     3841 ccccctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct
     3901 tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct
     3961 gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa
     4021 atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat
     4081 attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct
     4141 atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat
     4201 attggatcat atgcatagta ccgagaaact agtgcgaagt agtgatcagg tattgctgtt
     4261 atctgatgag tatacgttgt cctggccacg gcagaagcac gcttatcgct ccaatttccc
     4321 acaacattag tcaactccgt taggcccttc attgaaagaa atgaggtcat caaatgtctt
     4381 ccaatgtgag attttgggcc attttttata gcaaagattg aataaggcgc atttttcttc
     4441 aaagctttat tgtacgatct gactaagtta tcttttaata attggtattc ctgtttattg
     4501 cttgaagaat tgccggtcct atttactcgt tttaggactg gttcagaatt cctcaaaaat
     4561 tcatccaaat atacaagtgg atcgatccta ccccttgcgc taaagaagta tatgtgccta
     4621 ctaacgcttg tctttgtctc tgtcactaaa cactggatta ttactcccag atacttattt
     4681 tggactaatt taaatgattt cggatcaacg ttcttaatat cgctgaatct tccacaattg
     4741 atgaaagtag ctaggaagag gaattggtat aaagtttttg tttttgtaaa tctcgaagta
     4801 tactcaaacg aatttagtat tttctcagtg atctcccaga tgctttcacc ctcacttaga
     4861 agtgctttaa gcattttttt actgtggcta tttcccttat ctgcttcttc cgatgattcg
     4921 aactgtaatt gcaaactact tacaatatca gtgatatcag attgatgttt ttgtccatag
     4981 taaggaataa ttgtaaattc ccaagcagga atcaatttct ttaatgaggc ttccagaatt
     5041 gttgcttttt gcgtcttgta tttaaactgg agtgatttat tgacaatatc gaaactcagc
     5101 gaattgctta tgatagtatt atagctcatg aatgtggctc tcttgattgc tgttccgtta
     5161 tgtgtaatca tccaacataa ataggttagt tcagcagcac ataatgctat tttctcacct
     5221 gaaggtcttt caaacctttc cacaaactga cgaacaagca ccttaggtgg tgttttacat
     5281 aatatatcaa attgtggcat acaacctcct tagtacatgc aaccattatc accgccagag
     5341 gtaaaatagt caacacgcac ggtgttagat atttatccct tgcggtgata gatttaacgt
     5401 atgagcacaa aaaagaaacc attaacacaa gagcagcttg aggacgcacg tcgccttaaa
     5461 gcaatttatg aaaaaaagaa aaatgaactt ggcttatccc aggaatctgt cgcagacaag
     5521 atggggatgg ggcagtcagg cgttggtgct ttatttaatg gcatcaatgc attaaatgct
     5581 tataacgccg cattgcttac aaaaattctc aaagttagcg ttgaagaatt tagcccttca
     5641 atcgccagag aaatctacga gatgtatgaa gcggttagta tgcagccgtc acttagaagt
     5701 gagtatgagt accctgtttt ttctcatgtt caggcaggga tgttctcacc taagcttaga
     5761 acctttacca aaggtgatgc ggagagatgg gtaagcacaa ccaaaaaagc cagtgattct
     5821 gcattctggc ttgaggttga aggtaattcc atgaccgcac caacaggctc caagccaagc
     5881 tttcctgacg gaatgttaat tctcgttgac cctgagcagg ctgttgagcc aggtgatttc
     5941 tgcatagcca gacttggggg tgatgagttt accttcaaga aactgatcag ggatagcggt
     6001 caggtgtttt tacaaccact aaacccacag tacccaatga tcccatgcaa tgagagttgt
     6061 tccgttgtgg ggaaagttat cgctagtcag tggcctgaag agacgtttgg ctgatcggca
     6121 aggtgttctg gtcacgctct tggactcctg ttgatagatc cagtaatgac ctcagaactc
     6181 catctggatt tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca
     6241 ggcaatcaat cgtcaccctt tctcggtcct tcaacgttcc tgacaacgag cctccttttc
     6301 gccaatccat cgacaatcac cgcgagtccc tgctcgaacg ctgcgtccgg accggcttcg
     6361 tcgaaggcgt ctatcgcggc ccgcaacagc ggcgagagcg gagcctgttc aacggtgccg
     6421 ccgcgctcgc cggcatcgct gtcgccggcc tgctcctcaa gcacggcccc aacagtgaag
     6481 tagctgattg tcatcagcgc attgacggcg tccccggccg aaaaacccgc ctcgcagagg
     6541 aagcgaagct gcgcgtcggc cgtttccatc tgcggtgcgc ccggtcgcgt gccggcatgg
     6601 atgcgcgcgc catcgcggta ggcgagcagc gcctgcctga agctgcgggc attcccgatc
     6661 agaaatgagc gccagtcgtc gtcggctctc ggcaccgaat gcgtatgatt ctccgccagc
     6721 atggcttcgg ccagtgcgtc gagcagcgcc cgcttgttcc tgaagtgcca gtaaagcgcc
     6781 ggctgctgaa cccccaaccg ttccgccagt ttgcgtgtcg tcagaccgtc tacgccgacc
     6841 tcgttcaaca ggtccagggc ggcacggatc actgtattcg gctgcaactt tgtcatgctt
     6901 gacactttat cactgataaa cataatatgt ccaccaactt atcagtgata aagaatccgc
     6961 gcgttcaatc ggaccagcgg aggctggtcc ggaggccaga cgtgaaaccc aacatacccc
     7021 tgatcgtaat tctgagcact gtcgcgctcg acgctgtcgg catcggcctg attatgccgg
     7081 tgctgccggg cctcctgcgc gatctggttc actcgaacga cgtcaccgcc cactatggca
     7141 ttctgctggc gctgtatgcg ttggtgcaat ttgcctgcgc acctgtgctg ggcgcgctgt
     7201 cggatcgttt cgggcggcgg ccaatcttgc tcgtgtcgct ggccggcgcc actgtcgact
     7261 acgccatcat ggcgacagcg cctttccttt gggttctcta tatcgggcgg atcgtggccg
     7321 gcatcaccgg ggcgactggg gcggtagccg gcgcttatat tgccgatatc actgatggcg
     7381 atgagcgcgc gcggcacttc ggcttcatga gcgcctgttt cgggttcggg atggtcgcgg
     7441 gacctgtgct cggtgggctg atgggcggtt tctcccccca cgctccgttc ttcgccgcgg
     7501 cagccttgaa cggcctcaat ttcctgacgg gctgtttcct tttgccggag tcgcacaaag
     7561 gcgaacgccg gccgttacgc cgggaggctc tcaacccgct cgcttcgttc cggtgggccc
     7621 ggggcatgac cgtcgtcgcc gccctgatgg cggtgttctt catcatgcaa cttgtcggac
     7681 aggtgccggc cgcgctttgg gtcattttcg gcgaggatcg ctttcactgg gacgcgacca
     7741 cgatcggcat ttcgcttgcc gcatttggca ttctgcattc actcgcccag gcaatgatca
     7801 ccggccctgt agccgcccgg ctcggcgaaa ggcgggcact catgctcgga atgattgccg
     7861 acggcacagg ctacatcctg cttgccttcg cgacacgggg atggatggcg ttcccgatca
     7921 tggtcctgct tgcttcgggt ggcatcggaa tgccggcgct gcaagcaatg ttgtccaggc
     7981 aggtggatga ggaacgtcag gggcagctgc aaggctcact ggcggcgctc accagcctga
     8041 cctcgatcgt cggacccctc ctcttcacgg cgatctatgc ggcttctata acaacgtgga
     8101 acgggtgggc atggattgca ggcgctgccc tctacttgct ctgcctgccg gcgctgcgtc
     8161 gcgggctttg gagcggcgca gggcaacgag ccgatcgctg aacta
//
