LOCUS       sequence-458895-        7059 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. AmR, 
            easily curable via sucrose counterselection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7059)
  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 7059)
  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-8
FEATURES             Location/Qualifiers
     source          1..7059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      1532..1626
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     1639..1658
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(1697..3031,3032..3118))
                     /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             3464..4264
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     oriT            4424..4532
                     /note="incP origin of transfer"
     CDS             complement(4593..5741)
                     /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      6134..6665
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(6792..6878)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             join(6979..7059,1..1422)
                     /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"
ORIGIN
        1 cagctctgcg cccagcgcgg ctgggacgtc gtcggggtag cggaggatct ggacgtctcc
       61 ggggcggtcg atccgttcga ccggaagcgc agaccgaacc tggcccggtg gctagcgttc
      121 gaggagcaac cgtttgacgt gatcgtggcg taccgggtag atcggttgac ccgatcgatc
      181 cggcatcttc agcagctggt ccactgggcc gaggaccaca agaagctggt cgtctccgcg
      241 accgaagcgc acttcgatac gacgacgccg tttgcggcgg tcgtcatcgc gcttatggga
      301 acggtggcgc agatggaatt agaagcgatc aaagagcgga accgttcggc tgcgcatttc
      361 aatatccgcg ccgggaaata ccgaggatcc ctgccgccgt ggggatacct gcctacgcgc
      421 gtggacgggg agtggcggct ggtgccggac cctgtgcagc gagagcgcat cctcgaggtg
      481 tatcaccgcg tcgtcgacaa ccacgagccg ctgcatctgg tggcccacga cctgaaccgg
      541 cgtggtgtcc tgtcgccgaa ggactacttc gcgcagctgc aaggccgcga gccgcagggc
      601 cgggagtggt cggctaccgc gctgaagcga tcgatgatct ccgaggcgat gctcgggtac
      661 gcgactctga acggtaagac cgtccgagac gacgacggag ccccgctggt gcgggctgag
      721 ccgatcctga cccgtgagca gctggaggcg ctgcgcgccg agctcgtgaa gacctcccgg
      781 gcgaagcccg cggtgtctac cccgtcgctg ctgctgcggg tgttgttctg cgcggtgtgc
      841 ggggagcccg cgtacaagtt cgccggggga ggacgtaagc acccgcgcta ccgctgccgc
      901 tcgatggggt tcccgaagca ctgcgggaac ggcacggtgg cgatggccga gtgggacgcg
      961 ttctgcgagg agcaggtact ggatctgctc ggggacgcgg agcgtctgga gaaagtctgg
     1021 gtagcgggct cggactccgc ggtcgaactc gcggaggtga acgcggagct ggtggacctg
     1081 acgtcgctga tcggctcccc ggcctaccgg gcgggctctc cgcagcgaga agcactggat
     1141 gcccgtattg cggcgctggc cgcgcggcaa gaggagctgg agggcctgga ggctcgcccg
     1201 tctggctggg agtggcgcga gaccgggcag cggttcgggg actggtggcg ggagcaggac
     1261 accgcggcaa agaacacctg gcttcggtcg atgaacgttc ggctgacgtt cgacgtccgc
     1321 ggcgggctga ctcgcacgat cgacttcggg gatcttcagg agtacgagca gcatctcagg
     1381 ctcggcagcg tggtcgaacg gctacacacc gggatgtcgt aataacgctg atagtgctag
     1441 tgtagatcgc tacactagtc ctagagtcga cctgcaggca tgcaagcttg cggccgcgtc
     1501 gtgactggga aaaccctggc gactagtctt ggactcctgt tgatagatcc agtaatgacc
     1561 tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt
     1621 gagaatccag gggtcccccg gcatcagagc agattgtact gagagtgcac cataatcggc
     1681 attttctttt gcgtttttat ttgttaactg ttaattgtcc ttgttcaagg atgctgtctt
     1741 tgacaacaga tgttttcttg cctttgatgt tcagcaggaa gctaggcgca aacgttgatt
     1801 gtttgtctgc gtagaatcct ctgtttgtca tatagcttgt aatcacgaca ttgtttcctt
     1861 tcgcttgagg tacagcgaag tgtgagtaag taaaggttac atcgttagga tcaagatcca
     1921 tttttaacac aaggccagtt ttgttcagcg gcttgtatgg gccagttaaa gaattagaaa
     1981 cataaccaag catgtaaata tcgttagacg taatgccgtc aatcgtcatt tttgatccgc
     2041 gggagtcagt gaacagatac catttgccgt tcattttaaa gacgttcgcg cgttcaattt
     2101 catctgttac tgtgttagat gcaatcagcg gtttcatcac ttttttcagt gtgtaatcat
     2161 cgtttagctc aatcataccg agagcgccgt ttgctaactc agccgtgcgt tttttatcgc
     2221 tttgcagaag tttttgactt tcttgacgga agaatgatgt gcttttgcca tagtatgctt
     2281 tgttaaataa agattcttcg ccttggtagc catcttcagt tccagtgttt gcttcaaata
     2341 ctaagtattt gtggccttta tcttctacgt agtgaggatc tctcagcgta tggttgtcgc
     2401 ctgagctgta gttgccttca tcgatgaact gctgtacatt ttgatacgtt tttccgtcac
     2461 cgtcaaagat tgatttataa tcctctacac cgttgatgtt caaagagctg tctgatgctg
     2521 atacgttaac ttgtgcagtt gtcagtgttt gtttgccgta atgtttaccg gagaaatcag
     2581 tgtagaataa acggattttt ccgtcagatg taaatgtggc tgaacctgac cattcttgtg
     2641 tttggtcttt taggatagaa tcatttgcat cgaatttgtc gctgtcttta aagacgcggc
     2701 cagcgttttt ccagctgtca atagaagttt cgccgacttt ttgatagaac atgtaaatcg
     2761 atgtgtcatc cgcattttta ggatctccgg ctaatgcaaa gacgatgtgg tagccgtgat
     2821 agtttgcgac agtgccgtca gcgttttgta atggccagct gtcccaaacg tccaggcctt
     2881 ttgcagaaga gatattttta attgtggacg aatcgaactc aggaacttga tatttttcat
     2941 ttttttgctg ttcagggatt tgcagcatat catggcgtgt aatatgggaa atgccgtatg
     3001 tttccttata tggcttttgg ttcgtttctt tcgcaaacgc ttgagttgcg cctcctgcca
     3061 gcagtgcggt agtaaaggtt aatactgttg cttgttttgc aaactttttg atgttcatcg
     3121 ttcatgtctc cttttttatg tactgtgtta gcggtctgct tcttccagcc ctcctgtttg
     3181 aagatggcaa gttagttacg cacaataaaa aaagacctaa aatatgtaag gggtgacgcc
     3241 aaagtataca ctttgccctt tacacatttt aggtcttgca tttaaatctc acggtaactg
     3301 atgccgtatt tgcagtacca gcgtacggcc cacagaatga tgtcacgctg aaaatgccgg
     3361 cctttgaatg ggttcatgtg cagctccatc agcaaaaggg gatgataagt ttatcaccac
     3421 cgactatttg caacagtgcc gttgatcgtg ctatgatcga gtgatgtcat cagcggtgga
     3481 gtgcaatgtc gtgcaatacg aatggcgaaa agccgagctg atcggtcagc ttctcaacct
     3541 tggggttacc cccggcggtg tgctgctggt ccacagctcc ttccgtagcg tccggcccct
     3601 cgaagatggg ccacttggac tgatcgaggc cctgcgtgct gcgctgggtc cgggagggac
     3661 gctcgtgatg ccctcgtggt caggtctgga cgacgagccg ttcgatcctg ccacgtcgcc
     3721 cgttacaccg gaccttggag ttgtctctga cacattctgg cgcctgccaa atgtaaagcg
     3781 cagcgcccat ccatttgcct ttgcggcagc ggggccacag gcagagcaga tcatctctga
     3841 tccattgccc ctgccacctc actcgcctgc aagcccggtc gcccgtgtcc atgaactcga
     3901 tgggcaggta cttctcctcg gcgtgggaca cgatgccaac acgacgctgc atcttgccga
     3961 gttgatggca aaggttccct atggggtgcc gagacactgc accattcttc aggatggcaa
     4021 gttggttcgc gtcgattatc tcgagaatga ccactgctgt gagcgctttg ccttggcgga
     4081 caggtggctc aaggagaaga gccttcagaa ggaaggtcca gtcggtcatg cctttgctcg
     4141 gttgatccgc tcccgcgaca ttgtggcgac agccctgggt caactgggcc gagatccgtt
     4201 gatcttcctg catccgccag aggcgggatg cgaagaatgc gatgccgctc gccagtcgat
     4261 tggctgagac cgcggtccgc gcgttgtcct tttccgctgc ataaccctgc ttcggggtca
     4321 ttatagcgat tttttcggta tatccatcct ttttcgcacg atatacagga ttttgccaaa
     4381 gggttcgtgt agactttcct tggtgtatcc aacggcgtca gccgggcagg ataggtgaag
     4441 taggcccacc cgcgagcggg tgttccttct tcactgtccc ttattcgcac ctggcggtgc
     4501 tcaacgggaa tcctgctctg cgaggctggc cgtaggccgg ccgcgatgca ggtggctgct
     4561 gaacccccag ccggaactga ccccacaagg ccctagcgtt tgcaatgcac caggtcacca
     4621 ttgacccagg cgtgttccac caggccgctg cctcgcaact cttcgcaggc ttcgccgacc
     4681 tgctcgcgcc acttcttcac gcgggtggaa tccgatccgc acatgaggcg gaaggtttcc
     4741 agcttgagcg ggtacggctc ccggtgcgag ctgaaatagt cgaacatccg tcgggccgtc
     4801 ggcgacagct tgcggtactt ctcccatatg aatttcgtgt agtggtcgcc agcaaacagc
     4861 acgacgattt cctcgtcgat caggacctgg caacgggacg ttttcttgcc acggtccagg
     4921 acgcggaagc ggtgcagcag cgacaccgat tccaggtgcc caacgcggtc ggacgtgaag
     4981 cccatcgccg tcgcctgtag gcgcgacagg cattcctcgg ccttcgtgta ataccggcca
     5041 ttgatcgacc agcccaggtc ctggcaaagc tcgtagaacg tgaaggtgat cggctcgccg
     5101 ataggggtgc gcttcgcgta ctccaacacc tgctgccaca ccagttcgtc atcgtcggcc
     5161 cgcagctcga cgccggtgta ggtgatcttc acgtccttgt tgacgtggaa aatgaccttg
     5221 ttttgcagcg cctcgcgcgg gattttcttg ttgcgcgtgg tgaacagggc agagcgggcc
     5281 gtgtcgtttg gcatcgctcg catcgtgtcc ggccacggcg caatatcgaa caaggaaagc
     5341 tgcatttcct tgatctgctg cttcgtgtgt ttcagcaacg cggcctgctt ggcttcgctg
     5401 acctgttttg ccaggtcctc gccggcggtt tttcgcttct tggtcgtcat agttcctcgc
     5461 gtgtcgatgg tcatcgactt cgccaaacct gccgcctcct gttcgagacg acgcgaacgc
     5521 tccacggcgg ccgatggcgc gggcagggca gggggagcca gttgcacgct gtcgcgctcg
     5581 atcttggccg tagcttgctg gactatcgag ccgacggact ggaaggtttc gcggggcgca
     5641 cgcatgacgg tgcggcttgc gatggtttcg gcatcctcgg cggaaaaccc cgcgtcgatc
     5701 agttcttgcc tgtatgcctt ccggtcaaac gtccgattca ttcaccctcc ttgcgggatt
     5761 gccccggaat taattccccg gatcgatccg tcgatcttga tcccctgcgc catcagatcc
     5821 ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg
     5881 ccccagctgg caattccggt tcgcttgctg tccataaaac cgcccagtct agctatcgcc
     5941 atgtaagccc actgcaagct acctgctttc tctttgcgct tgcgttttcc cttgtccaga
     6001 tagcccagta gctgacattc atccggggtc agcaccgttt ctgcggactg gctttctacg
     6061 tggctgccat ttttggggtg aggccgttcg cggccgaggg gcgcagcccc tggggggatg
     6121 ggaggcccgc gttagcgggc cgggagggtt cgagaagggg gggcaccccc cttcggcgtg
     6181 cgcggtcacg cgcacagggc gcagccctgg ttaaaaacaa ggtttataaa tattggttta
     6241 aaagcaggtt aaaagacagg ttagcggtgg ccgaaaaacg ggcggaaacc cttgcaaatg
     6301 ctggattttc tgcctgtgga cagcccctca aatgtcaata ggtgcgcccc tcatctgtca
     6361 gcactctgcc cctcaagtgt caaggatcgc gcccctcatc tgtcagtagt cgcgcccctc
     6421 aagtgtcaat accgcagggc acttatcccc aggcttgtcc acatcatctg tgggaaactc
     6481 gcgtaaaatc aggcgttttc gccgatttgc gaggctggcc agctccacgt cgccggccga
     6541 aatcgagcct gcccctcatc tgtcaacgcc gcgccgggtg agtcggcccc tcaagtgtca
     6601 acgtccgccc ctcatctgtc agtgagggcc aagttttccg cgaggtatcc acaacgccgg
     6661 cggccctaca tggctctgct gtagtgagtg ggttgcgctt cggcagcggt cctgatcccc
     6721 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggcgcg cccagctgtc
     6781 tagggcggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga
     6841 aaggcccagt ctttcgactg agcctttcgt tttatttgat gcctttaatt aatgtgacaa
     6901 ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaacccc
     6961 taggggagtc atgaacatat gagagccctg gtagtcatcc gcctgtcccg cgtcaccgat
     7021 gctacgactt caccggagcg tcagctggag tcttgccag
//
