LOCUS       sequence-458909-        7068 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the BxbI phage integrase. It can be used to remove 
            the antibiotic resistance cassette integrated with pCIFR. SmR, 
            easily curable via sucrose counterselection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7068)
  AUTHORS   Federici F, Luppino F, Aguilar-Vilar C, Mazaraki ME, Petersen LB, 
            Ahonen L, Nikel PI
  TITLE     CIFR (Clone-Integrate-Flip-out-Repeat): A toolset for iterative 
            genome and pathway engineering of Gram-negative bacteria.
  JOURNAL   Metab Eng. 2025 Jan 6;88:180-195. doi: 10.1016/j.ymben.2025.01.001.
  PUBMED    39778677
REFERENCE   2  (bases 1 to 7068)
  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: "Metab 
            Eng."; date: "2025-01-6"; volume: "88:180-195. doi"; pages: " 
            10.1016/j.ymben.2025.01.001"
COMMENT     Sequence Label: pFNC-4
FEATURES             Location/Qualifiers
     source          1..7068
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            595..703
                     /note="incP origin of transfer"
     CDS             complement(764..1912)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNGDLVHCKR"
     rep_origin      2305..2836
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(2963..3049)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             3150..4652
                     /codon_start=1
                     /transl_table=11
                     /gene="Bxb1"
                     /product="Bxb1 integrase"
                     /label=Bxb1 integrase
                     /note="Bxb1 large serine integrase from Mycobacterium 
                     bacteriophage Bxb1 (Ghosh et al. 2003)."
                     /protein_id="AFF61396.1"
                     /translation="MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLD
                     VSGAVDPFDRKRRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLV
                     VSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGY
                     LPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQG
                     REPQGREWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAE
                     LVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTV
                     AMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNAELVDLTSLIGSPAYRAG
                     SPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSM
                     NVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
     terminator      4762..4856
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     4869..4888
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(4927..6261,6262..6348))
                     /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"
     CDS             join(6715..7068,1..438)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca
       61 agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct
      121 gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac
      181 tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta
      241 accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc
      301 cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc
      361 ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc
      421 aaggtagtcg gcaaataaga caacggtccg cgcgttgtcc ttttccgctg cataaccctg
      481 cttcggggtc attatagcga ttttttcggt atatccatcc tttttcgcac gatatacagg
      541 attttgccaa agggttcgtg tagactttcc ttggtgtatc caacggcgtc agccgggcag
      601 gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc ttcactgtcc cttattcgca
      661 cctggcggtg ctcaacggga atcctgctct gcgaggctgg ccgtaggccg gccgcgatgc
      721 aggtggctgc tgaaccccca gccggaactg accccacaag gccctagcgt ttgcaatgca
      781 ccaggtcacc attgacccag gcgtgttcca ccaggccgct gcctcgcaac tcttcgcagg
      841 cttcgccgac ctgctcgcgc cacttcttca cgcgggtgga atccgatccg cacatgaggc
      901 ggaaggtttc cagcttgagc gggtacggct cccggtgcga gctgaaatag tcgaacatcc
      961 gtcgggccgt cggcgacagc ttgcggtact tctcccatat gaatttcgtg tagtggtcgc
     1021 cagcaaacag cacgacgatt tcctcgtcga tcaggacctg gcaacgggac gttttcttgc
     1081 cacggtccag gacgcggaag cggtgcagca gcgacaccga ttccaggtgc ccaacgcggt
     1141 cggacgtgaa gcccatcgcc gtcgcctgta ggcgcgacag gcattcctcg gccttcgtgt
     1201 aataccggcc attgatcgac cagcccaggt cctggcaaag ctcgtagaac gtgaaggtga
     1261 tcggctcgcc gataggggtg cgcttcgcgt actccaacac ctgctgccac accagttcgt
     1321 catcgtcggc ccgcagctcg acgccggtgt aggtgatctt cacgtccttg ttgacgtgga
     1381 aaatgacctt gttttgcagc gcctcgcgcg ggattttctt gttgcgcgtg gtgaacaggg
     1441 cagagcgggc cgtgtcgttt ggcatcgctc gcatcgtgtc cggccacggc gcaatatcga
     1501 acaaggaaag ctgcatttcc ttgatctgct gcttcgtgtg tttcagcaac gcggcctgct
     1561 tggcttcgct gacctgtttt gccaggtcct cgccggcggt ttttcgcttc ttggtcgtca
     1621 tagttcctcg cgtgtcgatg gtcatcgact tcgccaaacc tgccgcctcc tgttcgagac
     1681 gacgcgaacg ctccacggcg gccgatggcg cgggcagggc agggggagcc agttgcacgc
     1741 tgtcgcgctc gatcttggcc gtagcttgct ggactatcga gccgacggac tggaaggttt
     1801 cgcggggcgc acgcatgacg gtgcggcttg cgatggtttc ggcatcctcg gcggaaaacc
     1861 ccgcgtcgat cagttcttgc ctgtatgcct tccggtcaaa cgtccgattc attcaccctc
     1921 cttgcgggat tgccccggaa ttaattcccc ggatcgatcc gtcgatcttg atcccctgcg
     1981 ccatcagatc cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt
     2041 accagagggc gccccagctg gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc
     2101 tagctatcgc catgtaagcc cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc
     2161 ccttgtccag atagcccagt agctgacatt catccggggt cagcaccgtt tctgcggact
     2221 ggctttctac gtggctgcca tttttggggt gaggccgttc gcggccgagg ggcgcagccc
     2281 ctggggggat gggaggcccg cgttagcggg ccgggagggt tcgagaaggg ggggcacccc
     2341 ccttcggcgt gcgcggtcac gcgcacaggg cgcagccctg gttaaaaaca aggtttataa
     2401 atattggttt aaaagcaggt taaaagacag gttagcggtg gccgaaaaac gggcggaaac
     2461 ccttgcaaat gctggatttt ctgcctgtgg acagcccctc aaatgtcaat aggtgcgccc
     2521 ctcatctgtc agcactctgc ccctcaagtg tcaaggatcg cgcccctcat ctgtcagtag
     2581 tcgcgcccct caagtgtcaa taccgcaggg cacttatccc caggcttgtc cacatcatct
     2641 gtgggaaact cgcgtaaaat caggcgtttt cgccgatttg cgaggctggc cagctccacg
     2701 tcgccggccg aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggt gagtcggccc
     2761 ctcaagtgtc aacgtccgcc cctcatctgt cagtgagggc caagttttcc gcgaggtatc
     2821 cacaacgccg gcggccctac atggctctgc tgtagtgagt gggttgcgct tcggcagcgg
     2881 tcctgatccc ccgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggcgc
     2941 gcccagctgt ctagggcggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac
     3001 agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctttaat
     3061 taatgtgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
     3121 aactaaaccc ctaggggagt catgaacata tgagagccct ggtagtcatc cgcctgtccc
     3181 gcgtcaccga tgctacgact tcaccggagc gtcagctgga gtcttgccag cagctctgcg
     3241 cccagcgcgg ctgggacgtc gtcggggtag cggaggatct ggacgtctcc ggggcggtcg
     3301 atccgttcga ccggaagcgc agaccgaacc tggcccggtg gctagcgttc gaggagcaac
     3361 cgtttgacgt gatcgtggcg taccgggtag atcggttgac ccgatcgatc cggcatcttc
     3421 agcagctggt ccactgggcc gaggaccaca agaagctggt cgtctccgcg accgaagcgc
     3481 acttcgatac gacgacgccg tttgcggcgg tcgtcatcgc gcttatggga acggtggcgc
     3541 agatggaatt agaagcgatc aaagagcgga accgttcggc tgcgcatttc aatatccgcg
     3601 ccgggaaata ccgaggatcc ctgccgccgt ggggatacct gcctacgcgc gtggacgggg
     3661 agtggcggct ggtgccggac cctgtgcagc gagagcgcat cctcgaggtg tatcaccgcg
     3721 tcgtcgacaa ccacgagccg ctgcatctgg tggcccacga cctgaaccgg cgtggtgtcc
     3781 tgtcgccgaa ggactacttc gcgcagctgc aaggccgcga gccgcagggc cgggagtggt
     3841 cggctaccgc gctgaagcga tcgatgatct ccgaggcgat gctcgggtac gcgactctga
     3901 acggtaagac cgtccgagac gacgacggag ccccgctggt gcgggctgag ccgatcctga
     3961 cccgtgagca gctggaggcg ctgcgcgccg agctcgtgaa gacctcccgg gcgaagcccg
     4021 cggtgtctac cccgtcgctg ctgctgcggg tgttgttctg cgcggtgtgc ggggagcccg
     4081 cgtacaagtt cgccggggga ggacgtaagc acccgcgcta ccgctgccgc tcgatggggt
     4141 tcccgaagca ctgcgggaac ggcacggtgg cgatggccga gtgggacgcg ttctgcgagg
     4201 agcaggtact ggatctgctc ggggacgcgg agcgtctgga gaaagtctgg gtagcgggct
     4261 cggactccgc ggtcgaactc gcggaggtga acgcggagct ggtggacctg acgtcgctga
     4321 tcggctcccc ggcctaccgg gcgggctctc cgcagcgaga agcactggat gcccgtattg
     4381 cggcgctggc cgcgcggcaa gaggagctgg agggcctgga ggctcgcccg tctggctggg
     4441 agtggcgcga gaccgggcag cggttcgggg actggtggcg ggagcaggac accgcggcaa
     4501 agaacacctg gcttcggtcg atgaacgttc ggctgacgtt cgacgtccgc ggcgggctga
     4561 ctcgcacgat cgacttcggg gatcttcagg agtacgagca gcatctcagg ctcggcagcg
     4621 tggtcgaacg gctacacacc gggatgtcgt aataacgctg atagtgctag tgtagatcgc
     4681 tacactagtc ctagagtcga cctgcaggca tgcaagcttg cggccgcgtc gtgactggga
     4741 aaaccctggc gactagtctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc
     4801 atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccag
     4861 gggtcccccg gcatcagagc agattgtact gagagtgcac cataatcggc attttctttt
     4921 gcgtttttat ttgttaactg ttaattgtcc ttgttcaagg atgctgtctt tgacaacaga
     4981 tgttttcttg cctttgatgt tcagcaggaa gctaggcgca aacgttgatt gtttgtctgc
     5041 gtagaatcct ctgtttgtca tatagcttgt aatcacgaca ttgtttcctt tcgcttgagg
     5101 tacagcgaag tgtgagtaag taaaggttac atcgttagga tcaagatcca tttttaacac
     5161 aaggccagtt ttgttcagcg gcttgtatgg gccagttaaa gaattagaaa cataaccaag
     5221 catgtaaata tcgttagacg taatgccgtc aatcgtcatt tttgatccgc gggagtcagt
     5281 gaacagatac catttgccgt tcattttaaa gacgttcgcg cgttcaattt catctgttac
     5341 tgtgttagat gcaatcagcg gtttcatcac ttttttcagt gtgtaatcat cgtttagctc
     5401 aatcataccg agagcgccgt ttgctaactc agccgtgcgt tttttatcgc tttgcagaag
     5461 tttttgactt tcttgacgga agaatgatgt gcttttgcca tagtatgctt tgttaaataa
     5521 agattcttcg ccttggtagc catcttcagt tccagtgttt gcttcaaata ctaagtattt
     5581 gtggccttta tcttctacgt agtgaggatc tctcagcgta tggttgtcgc ctgagctgta
     5641 gttgccttca tcgatgaact gctgtacatt ttgatacgtt tttccgtcac cgtcaaagat
     5701 tgatttataa tcctctacac cgttgatgtt caaagagctg tctgatgctg atacgttaac
     5761 ttgtgcagtt gtcagtgttt gtttgccgta atgtttaccg gagaaatcag tgtagaataa
     5821 acggattttt ccgtcagatg taaatgtggc tgaacctgac cattcttgtg tttggtcttt
     5881 taggatagaa tcatttgcat cgaatttgtc gctgtcttta aagacgcggc cagcgttttt
     5941 ccagctgtca atagaagttt cgccgacttt ttgatagaac atgtaaatcg atgtgtcatc
     6001 cgcattttta ggatctccgg ctaatgcaaa gacgatgtgg tagccgtgat agtttgcgac
     6061 agtgccgtca gcgttttgta atggccagct gtcccaaacg tccaggcctt ttgcagaaga
     6121 gatattttta attgtggacg aatcgaactc aggaacttga tatttttcat ttttttgctg
     6181 ttcagggatt tgcagcatat catggcgtgt aatatgggaa atgccgtatg tttccttata
     6241 tggcttttgg ttcgtttctt tcgcaaacgc ttgagttgcg cctcctgcca gcagtgcggt
     6301 agtaaaggtt aatactgttg cttgttttgc aaactttttg atgttcatcg ttcatgtctc
     6361 cttttttatg tactgtgtta gcggtctgct tcttccagcc ctcctgtttg aagatggcaa
     6421 gttagttacg cacaataaaa aaagacctaa aatatgtaag gggtgacgcc aaagtataca
     6481 ctttgccctt tacacatttt aggtcttgca tttaaatgaa ccttgaccga acgcagcggt
     6541 ggtaacggcg cagtggcggt tttcatggct tgttatgact gtttttttgg ggtacagtct
     6601 atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg
     6661 agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttaaa catcatgagg
     6721 gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc
     6781 catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg
     6841 aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg
     6901 cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt
     6961 ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca
     7021 gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattctt
//
