LOCUS       sequence-458626-        6884 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. GmR, 
            easily curable via sucrose counterselection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6884)
  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 6884)
  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-6
FEATURES             Location/Qualifiers
     source          1..6884
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      648..742
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     755..774
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(813..2147,2148..2234))
                     /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        join(2404..2409,2410..2426,2427..2432)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             2621..3154
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     oriT            3365..3473
                     /note="incP origin of transfer"
     CDS             complement(3534..4682)
                     /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      5075..5606
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(5733..5819)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             join(5920..6884,1..538)
                     /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 gcgctaccgc tgccgctcga tggggttccc gaagcactgc gggaacggca cggtggcgat
       61 ggccgagtgg gacgcgttct gcgaggagca ggtactggat ctgctcgggg acgcggagcg
      121 tctggagaaa gtctgggtag cgggctcgga ctccgcggtc gaactcgcgg aggtgaacgc
      181 ggagctggtg gacctgacgt cgctgatcgg ctccccggcc taccgggcgg gctctccgca
      241 gcgagaagca ctggatgccc gtattgcggc gctggccgcg cggcaagagg agctggaggg
      301 cctggaggct cgcccgtctg gctgggagtg gcgcgagacc gggcagcggt tcggggactg
      361 gtggcgggag caggacaccg cggcaaagaa cacctggctt cggtcgatga acgttcggct
      421 gacgttcgac gtccgcggcg ggctgactcg cacgatcgac ttcggggatc ttcaggagta
      481 cgagcagcat ctcaggctcg gcagcgtggt cgaacggcta cacaccggga tgtcgtaata
      541 acgctgatag tgctagtgta gatcgctaca ctagtcctag agtcgacctg caggcatgca
      601 agcttgcggc cgcgtcgtga ctgggaaaac cctggcgact agtcttggac tcctgttgat
      661 agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg
      721 ggcgtttttt attggtgaga atccaggggt cccccggcat cagagcagat tgtactgaga
      781 gtgcaccata atcggcattt tcttttgcgt ttttatttgt taactgttaa ttgtccttgt
      841 tcaaggatgc tgtctttgac aacagatgtt ttcttgcctt tgatgttcag caggaagcta
      901 ggcgcaaacg ttgattgttt gtctgcgtag aatcctctgt ttgtcatata gcttgtaatc
      961 acgacattgt ttcctttcgc ttgaggtaca gcgaagtgtg agtaagtaaa ggttacatcg
     1021 ttaggatcaa gatccatttt taacacaagg ccagttttgt tcagcggctt gtatgggcca
     1081 gttaaagaat tagaaacata accaagcatg taaatatcgt tagacgtaat gccgtcaatc
     1141 gtcatttttg atccgcggga gtcagtgaac agataccatt tgccgttcat tttaaagacg
     1201 ttcgcgcgtt caatttcatc tgttactgtg ttagatgcaa tcagcggttt catcactttt
     1261 ttcagtgtgt aatcatcgtt tagctcaatc ataccgagag cgccgtttgc taactcagcc
     1321 gtgcgttttt tatcgctttg cagaagtttt tgactttctt gacggaagaa tgatgtgctt
     1381 ttgccatagt atgctttgtt aaataaagat tcttcgcctt ggtagccatc ttcagttcca
     1441 gtgtttgctt caaatactaa gtatttgtgg cctttatctt ctacgtagtg aggatctctc
     1501 agcgtatggt tgtcgcctga gctgtagttg ccttcatcga tgaactgctg tacattttga
     1561 tacgtttttc cgtcaccgtc aaagattgat ttataatcct ctacaccgtt gatgttcaaa
     1621 gagctgtctg atgctgatac gttaacttgt gcagttgtca gtgtttgttt gccgtaatgt
     1681 ttaccggaga aatcagtgta gaataaacgg atttttccgt cagatgtaaa tgtggctgaa
     1741 cctgaccatt cttgtgtttg gtcttttagg atagaatcat ttgcatcgaa tttgtcgctg
     1801 tctttaaaga cgcggccagc gtttttccag ctgtcaatag aagtttcgcc gactttttga
     1861 tagaacatgt aaatcgatgt gtcatccgca tttttaggat ctccggctaa tgcaaagacg
     1921 atgtggtagc cgtgatagtt tgcgacagtg ccgtcagcgt tttgtaatgg ccagctgtcc
     1981 caaacgtcca ggccttttgc agaagagata tttttaattg tggacgaatc gaactcagga
     2041 acttgatatt tttcattttt ttgctgttca gggatttgca gcatatcatg gcgtgtaata
     2101 tgggaaatgc cgtatgtttc cttatatggc ttttggttcg tttctttcgc aaacgcttga
     2161 gttgcgcctc ctgccagcag tgcggtagta aaggttaata ctgttgcttg ttttgcaaac
     2221 tttttgatgt tcatcgttca tgtctccttt tttatgtact gtgttagcgg tctgcttctt
     2281 ccagccctcc tgtttgaaga tggcaagtta gttacgcaca ataaaaaaag acctaaaata
     2341 tgtaaggggt gacgccaaag tatacacttt gccctttaca cattttaggt cttgcattta
     2401 aatttgacat aagcctgttc ggttcgtaaa ctgtaatgca agtagcgtat gcgctcacgc
     2461 aactggtcca gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg
     2521 gcttgttatg actgtttttt tgtacagcct atgcctcggg catccaagca gcaagcgcgt
     2581 tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg atgttacgca gcagcaacga
     2641 tgttacgcag cagggcagtc gccctaaaac aaagttaggt ggctcaagta tgggcatcat
     2701 tcgcacatgt aggctcggcc ctgaccaagt caaatccatg cgggctgctc ttgatctttt
     2761 cggtcgtgag ttcggagacg tagccaccta ctcccaacat cagccggact ccgattacct
     2821 cgggaacttg ctccgtagta agacattcat cgcgcttgct gccttcgacc aagaagcggt
     2881 tgttggcgct ctcgcggctt acgttctgcc caagtttgag cagccgcgta gtgagatcta
     2941 tatctatgat ctcgcagtct ccggagagca ccggaggcag ggcattgcca ccgcgctcat
     3001 caatctcctc aagcatgagg ccaacgcgct tggtgcttat gtgatctacg tgcaagcaga
     3061 ttacggtgac gatcccgcag tggctctcta tacaaagttg ggcatacggg aagaagtgat
     3121 gcactttgat atcgacccaa gtaccgccac ctaacaattc gttcaagccg agatcggctt
     3181 cccggccgcg gagttgttcg gtaaattgga caacggtccg cgcgttgtcc ttttccgctg
     3241 cataaccctg cttcggggtc attatagcga ttttttcggt atatccatcc tttttcgcac
     3301 gatatacagg attttgccaa agggttcgtg tagactttcc ttggtgtatc caacggcgtc
     3361 agccgggcag gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc ttcactgtcc
     3421 cttattcgca cctggcggtg ctcaacggga atcctgctct gcgaggctgg ccgtaggccg
     3481 gccgcgatgc aggtggctgc tgaaccccca gccggaactg accccacaag gccctagcgt
     3541 ttgcaatgca ccaggtcacc attgacccag gcgtgttcca ccaggccgct gcctcgcaac
     3601 tcttcgcagg cttcgccgac ctgctcgcgc cacttcttca cgcgggtgga atccgatccg
     3661 cacatgaggc ggaaggtttc cagcttgagc gggtacggct cccggtgcga gctgaaatag
     3721 tcgaacatcc gtcgggccgt cggcgacagc ttgcggtact tctcccatat gaatttcgtg
     3781 tagtggtcgc cagcaaacag cacgacgatt tcctcgtcga tcaggacctg gcaacgggac
     3841 gttttcttgc cacggtccag gacgcggaag cggtgcagca gcgacaccga ttccaggtgc
     3901 ccaacgcggt cggacgtgaa gcccatcgcc gtcgcctgta ggcgcgacag gcattcctcg
     3961 gccttcgtgt aataccggcc attgatcgac cagcccaggt cctggcaaag ctcgtagaac
     4021 gtgaaggtga tcggctcgcc gataggggtg cgcttcgcgt actccaacac ctgctgccac
     4081 accagttcgt catcgtcggc ccgcagctcg acgccggtgt aggtgatctt cacgtccttg
     4141 ttgacgtgga aaatgacctt gttttgcagc gcctcgcgcg ggattttctt gttgcgcgtg
     4201 gtgaacaggg cagagcgggc cgtgtcgttt ggcatcgctc gcatcgtgtc cggccacggc
     4261 gcaatatcga acaaggaaag ctgcatttcc ttgatctgct gcttcgtgtg tttcagcaac
     4321 gcggcctgct tggcttcgct gacctgtttt gccaggtcct cgccggcggt ttttcgcttc
     4381 ttggtcgtca tagttcctcg cgtgtcgatg gtcatcgact tcgccaaacc tgccgcctcc
     4441 tgttcgagac gacgcgaacg ctccacggcg gccgatggcg cgggcagggc agggggagcc
     4501 agttgcacgc tgtcgcgctc gatcttggcc gtagcttgct ggactatcga gccgacggac
     4561 tggaaggttt cgcggggcgc acgcatgacg gtgcggcttg cgatggtttc ggcatcctcg
     4621 gcggaaaacc ccgcgtcgat cagttcttgc ctgtatgcct tccggtcaaa cgtccgattc
     4681 attcaccctc cttgcgggat tgccccggaa ttaattcccc ggatcgatcc gtcgatcttg
     4741 atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact ttgcagggct
     4801 tcccaacctt accagagggc gccccagctg gcaattccgg ttcgcttgct gtccataaaa
     4861 ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt ctctttgcgc
     4921 ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt cagcaccgtt
     4981 tctgcggact ggctttctac gtggctgcca tttttggggt gaggccgttc gcggccgagg
     5041 ggcgcagccc ctggggggat gggaggcccg cgttagcggg ccgggagggt tcgagaaggg
     5101 ggggcacccc ccttcggcgt gcgcggtcac gcgcacaggg cgcagccctg gttaaaaaca
     5161 aggtttataa atattggttt aaaagcaggt taaaagacag gttagcggtg gccgaaaaac
     5221 gggcggaaac ccttgcaaat gctggatttt ctgcctgtgg acagcccctc aaatgtcaat
     5281 aggtgcgccc ctcatctgtc agcactctgc ccctcaagtg tcaaggatcg cgcccctcat
     5341 ctgtcagtag tcgcgcccct caagtgtcaa taccgcaggg cacttatccc caggcttgtc
     5401 cacatcatct gtgggaaact cgcgtaaaat caggcgtttt cgccgatttg cgaggctggc
     5461 cagctccacg tcgccggccg aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggt
     5521 gagtcggccc ctcaagtgtc aacgtccgcc cctcatctgt cagtgagggc caagttttcc
     5581 gcgaggtatc cacaacgccg gcggccctac atggctctgc tgtagtgagt gggttgcgct
     5641 tcggcagcgg tcctgatccc ccgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5701 tctacggcgc gcccagctgt ctagggcggc ggatttgtcc tactcaggag agcgttcacc
     5761 gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga
     5821 tgcctttaat taatgtgaca attaatcatc ggcatagtat atcggcatag tataatacga
     5881 caaggtgagg aactaaaccc ctaggggagt catgaacata tgagagccct ggtagtcatc
     5941 cgcctgtccc gcgtcaccga tgctacgact tcaccggagc gtcagctgga gtcttgccag
     6001 cagctctgcg cccagcgcgg ctgggacgtc gtcggggtag cggaggatct ggacgtctcc
     6061 ggggcggtcg atccgttcga ccggaagcgc agaccgaacc tggcccggtg gctagcgttc
     6121 gaggagcaac cgtttgacgt gatcgtggcg taccgggtag atcggttgac ccgatcgatc
     6181 cggcatcttc agcagctggt ccactgggcc gaggaccaca agaagctggt cgtctccgcg
     6241 accgaagcgc acttcgatac gacgacgccg tttgcggcgg tcgtcatcgc gcttatggga
     6301 acggtggcgc agatggaatt agaagcgatc aaagagcgga accgttcggc tgcgcatttc
     6361 aatatccgcg ccgggaaata ccgaggatcc ctgccgccgt ggggatacct gcctacgcgc
     6421 gtggacgggg agtggcggct ggtgccggac cctgtgcagc gagagcgcat cctcgaggtg
     6481 tatcaccgcg tcgtcgacaa ccacgagccg ctgcatctgg tggcccacga cctgaaccgg
     6541 cgtggtgtcc tgtcgccgaa ggactacttc gcgcagctgc aaggccgcga gccgcagggc
     6601 cgggagtggt cggctaccgc gctgaagcga tcgatgatct ccgaggcgat gctcgggtac
     6661 gcgactctga acggtaagac cgtccgagac gacgacggag ccccgctggt gcgggctgag
     6721 ccgatcctga cccgtgagca gctggaggcg ctgcgcgccg agctcgtgaa gacctcccgg
     6781 gcgaagcccg cggtgtctac cccgtcgctg ctgctgcggg tgttgttctg cgcggtgtgc
     6841 ggggagcccg cgtacaagtt cgccggggga ggacgtaagc accc
//
