LOCUS       sequence-457238-        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"
     CDS             731..1522
                     /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"
     oriT            1679..1787
                     /note="incP origin of transfer"
     CDS             complement(1848..2996)
                     /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      3389..3920
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(4047..4133)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             4234..5736
                     /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      5846..5940
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     5953..5972
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(6011..7068,1..277,278..364))
                     /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"
ORIGIN
        1 aaatcgatgt gtcatccgca tttttaggat ctccggctaa tgcaaagacg atgtggtagc
       61 cgtgatagtt tgcgacagtg ccgtcagcgt tttgtaatgg ccagctgtcc caaacgtcca
      121 ggccttttgc agaagagata tttttaattg tggacgaatc gaactcagga acttgatatt
      181 tttcattttt ttgctgttca gggatttgca gcatatcatg gcgtgtaata tgggaaatgc
      241 cgtatgtttc cttatatggc ttttggttcg tttctttcgc aaacgcttga gttgcgcctc
      301 ctgccagcag tgcggtagta aaggttaata ctgttgcttg ttttgcaaac tttttgatgt
      361 tcatcgttca tgtctccttt tttatgtact gtgttagcgg tctgcttctt ccagccctcc
      421 tgtttgaaga tggcaagtta gttacgcaca ataaaaaaag acctaaaata tgtaaggggt
      481 gacgccaaag tatacacttt gccctttaca cattttaggt cttgcattta aatgaacctt
      541 gaccgaacgc agcggtggta acggcgcagt ggcggttttc atggcttgtt atgactgttt
      601 ttttggggta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg
      661 atgtttgatg ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa
      721 gttaaacatc atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt
      781 tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc
      841 agtggatggc ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag
      901 gcttgatgaa acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc
      961 tggagagagc gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat
     1021 tccgtggcgt tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat
     1081 tcttgcaggt atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa
     1141 agcaagagaa catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt
     1201 tcctgaacag gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc
     1261 cgactgggct ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc
     1321 agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc
     1381 ggcccagtat cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga
     1441 tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat
     1501 caccaaggta gtcggcaaat aagacaacgg tccgcgcgtt gtccttttcc gctgcataac
     1561 cctgcttcgg ggtcattata gcgatttttt cggtatatcc atcctttttc gcacgatata
     1621 caggattttg ccaaagggtt cgtgtagact ttccttggtg tatccaacgg cgtcagccgg
     1681 gcaggatagg tgaagtaggc ccacccgcga gcgggtgttc cttcttcact gtcccttatt
     1741 cgcacctggc ggtgctcaac gggaatcctg ctctgcgagg ctggccgtag gccggccgcg
     1801 atgcaggtgg ctgctgaacc cccagccgga actgacccca caaggcccta gcgtttgcaa
     1861 tgcaccaggt caccattgac ccaggcgtgt tccaccaggc cgctgcctcg caactcttcg
     1921 caggcttcgc cgacctgctc gcgccacttc ttcacgcggg tggaatccga tccgcacatg
     1981 aggcggaagg tttccagctt gagcgggtac ggctcccggt gcgagctgaa atagtcgaac
     2041 atccgtcggg ccgtcggcga cagcttgcgg tacttctccc atatgaattt cgtgtagtgg
     2101 tcgccagcaa acagcacgac gatttcctcg tcgatcagga cctggcaacg ggacgttttc
     2161 ttgccacggt ccaggacgcg gaagcggtgc agcagcgaca ccgattccag gtgcccaacg
     2221 cggtcggacg tgaagcccat cgccgtcgcc tgtaggcgcg acaggcattc ctcggccttc
     2281 gtgtaatacc ggccattgat cgaccagccc aggtcctggc aaagctcgta gaacgtgaag
     2341 gtgatcggct cgccgatagg ggtgcgcttc gcgtactcca acacctgctg ccacaccagt
     2401 tcgtcatcgt cggcccgcag ctcgacgccg gtgtaggtga tcttcacgtc cttgttgacg
     2461 tggaaaatga ccttgttttg cagcgcctcg cgcgggattt tcttgttgcg cgtggtgaac
     2521 agggcagagc gggccgtgtc gtttggcatc gctcgcatcg tgtccggcca cggcgcaata
     2581 tcgaacaagg aaagctgcat ttccttgatc tgctgcttcg tgtgtttcag caacgcggcc
     2641 tgcttggctt cgctgacctg ttttgccagg tcctcgccgg cggtttttcg cttcttggtc
     2701 gtcatagttc ctcgcgtgtc gatggtcatc gacttcgcca aacctgccgc ctcctgttcg
     2761 agacgacgcg aacgctccac ggcggccgat ggcgcgggca gggcaggggg agccagttgc
     2821 acgctgtcgc gctcgatctt ggccgtagct tgctggacta tcgagccgac ggactggaag
     2881 gtttcgcggg gcgcacgcat gacggtgcgg cttgcgatgg tttcggcatc ctcggcggaa
     2941 aaccccgcgt cgatcagttc ttgcctgtat gccttccggt caaacgtccg attcattcac
     3001 cctccttgcg ggattgcccc ggaattaatt ccccggatcg atccgtcgat cttgatcccc
     3061 tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag ggcttcccaa
     3121 ccttaccaga gggcgcccca gctggcaatt ccggttcgct tgctgtccat aaaaccgccc
     3181 agtctagcta tcgccatgta agcccactgc aagctacctg ctttctcttt gcgcttgcgt
     3241 tttcccttgt ccagatagcc cagtagctga cattcatccg gggtcagcac cgtttctgcg
     3301 gactggcttt ctacgtggct gccatttttg gggtgaggcc gttcgcggcc gaggggcgca
     3361 gcccctgggg ggatgggagg cccgcgttag cgggccggga gggttcgaga agggggggca
     3421 ccccccttcg gcgtgcgcgg tcacgcgcac agggcgcagc cctggttaaa aacaaggttt
     3481 ataaatattg gtttaaaagc aggttaaaag acaggttagc ggtggccgaa aaacgggcgg
     3541 aaacccttgc aaatgctgga ttttctgcct gtggacagcc cctcaaatgt caataggtgc
     3601 gcccctcatc tgtcagcact ctgcccctca agtgtcaagg atcgcgcccc tcatctgtca
     3661 gtagtcgcgc ccctcaagtg tcaataccgc agggcactta tccccaggct tgtccacatc
     3721 atctgtggga aactcgcgta aaatcaggcg ttttcgccga tttgcgaggc tggccagctc
     3781 cacgtcgccg gccgaaatcg agcctgcccc tcatctgtca acgccgcgcc gggtgagtcg
     3841 gcccctcaag tgtcaacgtc cgcccctcat ctgtcagtga gggccaagtt ttccgcgagg
     3901 tatccacaac gccggcggcc ctacatggct ctgctgtagt gagtgggttg cgcttcggca
     3961 gcggtcctga tcccccgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4021 gcgcgcccag ctgtctaggg cggcggattt gtcctactca ggagagcgtt caccgacaaa
     4081 caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcctt
     4141 taattaatgt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
     4201 gaggaactaa acccctaggg gagtcatgaa catatgagag ccctggtagt catccgcctg
     4261 tcccgcgtca ccgatgctac gacttcaccg gagcgtcagc tggagtcttg ccagcagctc
     4321 tgcgcccagc gcggctggga cgtcgtcggg gtagcggagg atctggacgt ctccggggcg
     4381 gtcgatccgt tcgaccggaa gcgcagaccg aacctggccc ggtggctagc gttcgaggag
     4441 caaccgtttg acgtgatcgt ggcgtaccgg gtagatcggt tgacccgatc gatccggcat
     4501 cttcagcagc tggtccactg ggccgaggac cacaagaagc tggtcgtctc cgcgaccgaa
     4561 gcgcacttcg atacgacgac gccgtttgcg gcggtcgtca tcgcgcttat gggaacggtg
     4621 gcgcagatgg aattagaagc gatcaaagag cggaaccgtt cggctgcgca tttcaatatc
     4681 cgcgccggga aataccgagg atccctgccg ccgtggggat acctgcctac gcgcgtggac
     4741 ggggagtggc ggctggtgcc ggaccctgtg cagcgagagc gcatcctcga ggtgtatcac
     4801 cgcgtcgtcg acaaccacga gccgctgcat ctggtggccc acgacctgaa ccggcgtggt
     4861 gtcctgtcgc cgaaggacta cttcgcgcag ctgcaaggcc gcgagccgca gggccgggag
     4921 tggtcggcta ccgcgctgaa gcgatcgatg atctccgagg cgatgctcgg gtacgcgact
     4981 ctgaacggta agaccgtccg agacgacgac ggagccccgc tggtgcgggc tgagccgatc
     5041 ctgacccgtg agcagctgga ggcgctgcgc gccgagctcg tgaagacctc ccgggcgaag
     5101 cccgcggtgt ctaccccgtc gctgctgctg cgggtgttgt tctgcgcggt gtgcggggag
     5161 cccgcgtaca agttcgccgg gggaggacgt aagcacccgc gctaccgctg ccgctcgatg
     5221 gggttcccga agcactgcgg gaacggcacg gtggcgatgg ccgagtggga cgcgttctgc
     5281 gaggagcagg tactggatct gctcggggac gcggagcgtc tggagaaagt ctgggtagcg
     5341 ggctcggact ccgcggtcga actcgcggag gtgaacgcgg agctggtgga cctgacgtcg
     5401 ctgatcggct ccccggccta ccgggcgggc tctccgcagc gagaagcact ggatgcccgt
     5461 attgcggcgc tggccgcgcg gcaagaggag ctggagggcc tggaggctcg cccgtctggc
     5521 tgggagtggc gcgagaccgg gcagcggttc ggggactggt ggcgggagca ggacaccgcg
     5581 gcaaagaaca cctggcttcg gtcgatgaac gttcggctga cgttcgacgt ccgcggcggg
     5641 ctgactcgca cgatcgactt cggggatctt caggagtacg agcagcatct caggctcggc
     5701 agcgtggtcg aacggctaca caccgggatg tcgtaataac gctgatagtg ctagtgtaga
     5761 tcgctacact agtcctagag tcgacctgca ggcatgcaag cttgcggccg cgtcgtgact
     5821 gggaaaaccc tggcgactag tcttggactc ctgttgatag atccagtaat gacctcagaa
     5881 ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat
     5941 ccaggggtcc cccggcatca gagcagattg tactgagagt gcaccataat cggcattttc
     6001 ttttgcgttt ttatttgtta actgttaatt gtccttgttc aaggatgctg tctttgacaa
     6061 cagatgtttt cttgcctttg atgttcagca ggaagctagg cgcaaacgtt gattgtttgt
     6121 ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac gacattgttt cctttcgctt
     6181 gaggtacagc gaagtgtgag taagtaaagg ttacatcgtt aggatcaaga tccattttta
     6241 acacaaggcc agttttgttc agcggcttgt atgggccagt taaagaatta gaaacataac
     6301 caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt catttttgat ccgcgggagt
     6361 cagtgaacag ataccatttg ccgttcattt taaagacgtt cgcgcgttca atttcatctg
     6421 ttactgtgtt agatgcaatc agcggtttca tcactttttt cagtgtgtaa tcatcgttta
     6481 gctcaatcat accgagagcg ccgtttgcta actcagccgt gcgtttttta tcgctttgca
     6541 gaagtttttg actttcttga cggaagaatg atgtgctttt gccatagtat gctttgttaa
     6601 ataaagattc ttcgccttgg tagccatctt cagttccagt gtttgcttca aatactaagt
     6661 atttgtggcc tttatcttct acgtagtgag gatctctcag cgtatggttg tcgcctgagc
     6721 tgtagttgcc ttcatcgatg aactgctgta cattttgata cgtttttccg tcaccgtcaa
     6781 agattgattt ataatcctct acaccgttga tgttcaaaga gctgtctgat gctgatacgt
     6841 taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt accggagaaa tcagtgtaga
     6901 ataaacggat ttttccgtca gatgtaaatg tggctgaacc tgaccattct tgtgtttggt
     6961 cttttaggat agaatcattt gcatcgaatt tgtcgctgtc tttaaagacg cggccagcgt
     7021 ttttccagct gtcaatagaa gtttcgccga ctttttgata gaacatgt
//
