LOCUS       sequence-468581-        7006 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. KmR, 
            easily curable via sucrose counterselection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7006)
  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 7006)
  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-2
FEATURES             Location/Qualifiers
     source          1..7006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      169..700
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(827..913)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             1014..2516
                     /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      2626..2720
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     2733..2752
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(2791..4125,4126..4212))
                     /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"
     primer_bind     complement(4472..4491)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     oriT            5465..5573
                     /note="incP origin of transfer"
     CDS             complement(5634..6782)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNGDLVHCKR"
ORIGIN
        1 ctttctcttt gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg
       61 gggtcagcac cgtttctgcg gactggcttt ctacgtggct gccatttttg gggtgaggcc
      121 gttcgcggcc gaggggcgca gcccctgggg ggatgggagg cccgcgttag cgggccggga
      181 gggttcgaga agggggggca ccccccttcg gcgtgcgcgg tcacgcgcac agggcgcagc
      241 cctggttaaa aacaaggttt ataaatattg gtttaaaagc aggttaaaag acaggttagc
      301 ggtggccgaa aaacgggcgg aaacccttgc aaatgctgga ttttctgcct gtggacagcc
      361 cctcaaatgt caataggtgc gcccctcatc tgtcagcact ctgcccctca agtgtcaagg
      421 atcgcgcccc tcatctgtca gtagtcgcgc ccctcaagtg tcaataccgc agggcactta
      481 tccccaggct tgtccacatc atctgtggga aactcgcgta aaatcaggcg ttttcgccga
      541 tttgcgaggc tggccagctc cacgtcgccg gccgaaatcg agcctgcccc tcatctgtca
      601 acgccgcgcc gggtgagtcg gcccctcaag tgtcaacgtc cgcccctcat ctgtcagtga
      661 gggccaagtt ttccgcgagg tatccacaac gccggcggcc ctacatggct ctgctgtagt
      721 gagtgggttg cgcttcggca gcggtcctga tcccccgcag aaaaaaagga tctcaagaag
      781 atcctttgat cttttctacg gcgcgcccag ctgtctaggg cggcggattt gtcctactca
      841 ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct
      901 ttcgttttat ttgatgcctt taattaatgt gacaattaat catcggcata gtatatcggc
      961 atagtataat acgacaaggt gaggaactaa acccctaggg gagtcatgaa catatgagag
     1021 ccctggtagt catccgcctg tcccgcgtca ccgatgctac gacttcaccg gagcgtcagc
     1081 tggagtcttg ccagcagctc tgcgcccagc gcggctggga cgtcgtcggg gtagcggagg
     1141 atctggacgt ctccggggcg gtcgatccgt tcgaccggaa gcgcagaccg aacctggccc
     1201 ggtggctagc gttcgaggag caaccgtttg acgtgatcgt ggcgtaccgg gtagatcggt
     1261 tgacccgatc gatccggcat cttcagcagc tggtccactg ggccgaggac cacaagaagc
     1321 tggtcgtctc cgcgaccgaa gcgcacttcg atacgacgac gccgtttgcg gcggtcgtca
     1381 tcgcgcttat gggaacggtg gcgcagatgg aattagaagc gatcaaagag cggaaccgtt
     1441 cggctgcgca tttcaatatc cgcgccggga aataccgagg atccctgccg ccgtggggat
     1501 acctgcctac gcgcgtggac ggggagtggc ggctggtgcc ggaccctgtg cagcgagagc
     1561 gcatcctcga ggtgtatcac cgcgtcgtcg acaaccacga gccgctgcat ctggtggccc
     1621 acgacctgaa ccggcgtggt gtcctgtcgc cgaaggacta cttcgcgcag ctgcaaggcc
     1681 gcgagccgca gggccgggag tggtcggcta ccgcgctgaa gcgatcgatg atctccgagg
     1741 cgatgctcgg gtacgcgact ctgaacggta agaccgtccg agacgacgac ggagccccgc
     1801 tggtgcgggc tgagccgatc ctgacccgtg agcagctgga ggcgctgcgc gccgagctcg
     1861 tgaagacctc ccgggcgaag cccgcggtgt ctaccccgtc gctgctgctg cgggtgttgt
     1921 tctgcgcggt gtgcggggag cccgcgtaca agttcgccgg gggaggacgt aagcacccgc
     1981 gctaccgctg ccgctcgatg gggttcccga agcactgcgg gaacggcacg gtggcgatgg
     2041 ccgagtggga cgcgttctgc gaggagcagg tactggatct gctcggggac gcggagcgtc
     2101 tggagaaagt ctgggtagcg ggctcggact ccgcggtcga actcgcggag gtgaacgcgg
     2161 agctggtgga cctgacgtcg ctgatcggct ccccggccta ccgggcgggc tctccgcagc
     2221 gagaagcact ggatgcccgt attgcggcgc tggccgcgcg gcaagaggag ctggagggcc
     2281 tggaggctcg cccgtctggc tgggagtggc gcgagaccgg gcagcggttc ggggactggt
     2341 ggcgggagca ggacaccgcg gcaaagaaca cctggcttcg gtcgatgaac gttcggctga
     2401 cgttcgacgt ccgcggcggg ctgactcgca cgatcgactt cggggatctt caggagtacg
     2461 agcagcatct caggctcggc agcgtggtcg aacggctaca caccgggatg tcgtaataac
     2521 gctgatagtg ctagtgtaga tcgctacact agtcctagag tcgacctgca ggcatgcaag
     2581 cttgcggccg cgtcgtgact gggaaaaccc tggcgactag tcttggactc ctgttgatag
     2641 atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg
     2701 cgttttttat tggtgagaat ccaggggtcc cccggcatca gagcagattg tactgagagt
     2761 gcaccataat cggcattttc ttttgcgttt ttatttgtta actgttaatt gtccttgttc
     2821 aaggatgctg tctttgacaa cagatgtttt cttgcctttg atgttcagca ggaagctagg
     2881 cgcaaacgtt gattgtttgt ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac
     2941 gacattgttt cctttcgctt gaggtacagc gaagtgtgag taagtaaagg ttacatcgtt
     3001 aggatcaaga tccattttta acacaaggcc agttttgttc agcggcttgt atgggccagt
     3061 taaagaatta gaaacataac caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt
     3121 catttttgat ccgcgggagt cagtgaacag ataccatttg ccgttcattt taaagacgtt
     3181 cgcgcgttca atttcatctg ttactgtgtt agatgcaatc agcggtttca tcactttttt
     3241 cagtgtgtaa tcatcgttta gctcaatcat accgagagcg ccgtttgcta actcagccgt
     3301 gcgtttttta tcgctttgca gaagtttttg actttcttga cggaagaatg atgtgctttt
     3361 gccatagtat gctttgttaa ataaagattc ttcgccttgg tagccatctt cagttccagt
     3421 gtttgcttca aatactaagt atttgtggcc tttatcttct acgtagtgag gatctctcag
     3481 cgtatggttg tcgcctgagc tgtagttgcc ttcatcgatg aactgctgta cattttgata
     3541 cgtttttccg tcaccgtcaa agattgattt ataatcctct acaccgttga tgttcaaaga
     3601 gctgtctgat gctgatacgt taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt
     3661 accggagaaa tcagtgtaga ataaacggat ttttccgtca gatgtaaatg tggctgaacc
     3721 tgaccattct tgtgtttggt cttttaggat agaatcattt gcatcgaatt tgtcgctgtc
     3781 tttaaagacg cggccagcgt ttttccagct gtcaatagaa gtttcgccga ctttttgata
     3841 gaacatgtaa atcgatgtgt catccgcatt tttaggatct ccggctaatg caaagacgat
     3901 gtggtagccg tgatagtttg cgacagtgcc gtcagcgttt tgtaatggcc agctgtccca
     3961 aacgtccagg ccttttgcag aagagatatt tttaattgtg gacgaatcga actcaggaac
     4021 ttgatatttt tcattttttt gctgttcagg gatttgcagc atatcatggc gtgtaatatg
     4081 ggaaatgccg tatgtttcct tatatggctt ttggttcgtt tctttcgcaa acgcttgagt
     4141 tgcgcctcct gccagcagtg cggtagtaaa ggttaatact gttgcttgtt ttgcaaactt
     4201 tttgatgttc atcgttcatg tctccttttt tatgtactgt gttagcggtc tgcttcttcc
     4261 agccctcctg tttgaagatg gcaagttagt tacgcacaat aaaaaaagac ctaaaatatg
     4321 taaggggtga cgccaaagta tacactttgc cctttacaca ttttaggtct tgcatttaaa
     4381 tttgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgaaca
     4441 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcagcgt
     4501 gaaacgagct gtagccgtcc gcgtctgaac agcaacatgg atgcgaatct gtatggctat
     4561 aaatgggcgc gtgataacgt gggtcagagc ggcgcgacca tttatcgtct gtatggcaaa
     4621 ccggatgcgc cggaactgtt tctgaaacat ggcaaaggca gcgtggcgaa cgatgtgacc
     4681 gatgaaatgg tgcgtctgaa ctggctgacc gaatttatgc cgctgccgac cattaaacat
     4741 tttattcgca ccccggatga tgcgtggctg ctgaccaccg cgattccggg caaaaccgcg
     4801 tttcaggtgc tggaagaata tccggatagc ggcgaaaaca ttgtggatgc gctggccgtg
     4861 tttctgcgtc gtctgcatag cattccggtg tgcaactgcc cgtttaacag cgatcgtgtg
     4921 tttcgtctgg cccaggcgca gagccgtatg aacaacggcc tggtggatgc gagcgatttt
     4981 gatgatgaac gtaacggctg gccggtggaa caggtgtgga aagaaatgca taaactgctg
     5041 ccgtttagcc cggatagcgt ggtgacccac ggcgatttta gcctggataa cctgattttc
     5101 gatgaaggca aactgattgg ctgcattgat gtgggccgtg tgggcattgc ggatcgttat
     5161 caggatctgg ccattctgtg gaactgcctg ggcgaattta gcccgagcct gcaaaaacgt
     5221 ctgtttcaga aatatggcat tgataatccg gatatgaaca aactgcaatt tcatctgatg
     5281 ctggatgaat ttttctaata attaattgga ccgcggtccg cgcgttgtcc ttttccgctg
     5341 cataaccctg cttcggggtc attatagcga ttttttcggt atatccatcc tttttcgcac
     5401 gatatacagg attttgccaa agggttcgtg tagactttcc ttggtgtatc caacggcgtc
     5461 agccgggcag gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc ttcactgtcc
     5521 cttattcgca cctggcggtg ctcaacggga atcctgctct gcgaggctgg ccgtaggccg
     5581 gccgcgatgc aggtggctgc tgaaccccca gccggaactg accccacaag gccctagcgt
     5641 ttgcaatgca ccaggtcacc attgacccag gcgtgttcca ccaggccgct gcctcgcaac
     5701 tcttcgcagg cttcgccgac ctgctcgcgc cacttcttca cgcgggtgga atccgatccg
     5761 cacatgaggc ggaaggtttc cagcttgagc gggtacggct cccggtgcga gctgaaatag
     5821 tcgaacatcc gtcgggccgt cggcgacagc ttgcggtact tctcccatat gaatttcgtg
     5881 tagtggtcgc cagcaaacag cacgacgatt tcctcgtcga tcaggacctg gcaacgggac
     5941 gttttcttgc cacggtccag gacgcggaag cggtgcagca gcgacaccga ttccaggtgc
     6001 ccaacgcggt cggacgtgaa gcccatcgcc gtcgcctgta ggcgcgacag gcattcctcg
     6061 gccttcgtgt aataccggcc attgatcgac cagcccaggt cctggcaaag ctcgtagaac
     6121 gtgaaggtga tcggctcgcc gataggggtg cgcttcgcgt actccaacac ctgctgccac
     6181 accagttcgt catcgtcggc ccgcagctcg acgccggtgt aggtgatctt cacgtccttg
     6241 ttgacgtgga aaatgacctt gttttgcagc gcctcgcgcg ggattttctt gttgcgcgtg
     6301 gtgaacaggg cagagcgggc cgtgtcgttt ggcatcgctc gcatcgtgtc cggccacggc
     6361 gcaatatcga acaaggaaag ctgcatttcc ttgatctgct gcttcgtgtg tttcagcaac
     6421 gcggcctgct tggcttcgct gacctgtttt gccaggtcct cgccggcggt ttttcgcttc
     6481 ttggtcgtca tagttcctcg cgtgtcgatg gtcatcgact tcgccaaacc tgccgcctcc
     6541 tgttcgagac gacgcgaacg ctccacggcg gccgatggcg cgggcagggc agggggagcc
     6601 agttgcacgc tgtcgcgctc gatcttggcc gtagcttgct ggactatcga gccgacggac
     6661 tggaaggttt cgcggggcgc acgcatgacg gtgcggcttg cgatggtttc ggcatcctcg
     6721 gcggaaaacc ccgcgtcgat cagttcttgc ctgtatgcct tccggtcaaa cgtccgattc
     6781 attcaccctc cttgcgggat tgccccggaa ttaattcccc ggatcgatcc gtcgatcttg
     6841 atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact ttgcagggct
     6901 tcccaacctt accagagggc gccccagctg gcaattccgg ttcgcttgct gtccataaaa
     6961 ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctg
//
