LOCUS       sequence-467796-        6862 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. CmR, 
            easily curable via sucrose counterselection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6862)
  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 6862)
  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-3
FEATURES             Location/Qualifiers
     source          1..6862
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        551..653
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             654..1313
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(720..739)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     oriT            1473..1581
                     /note="incP origin of transfer"
     CDS             complement(1642..2790)
                     /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      3183..3714
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(3841..3927)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             4028..5530
                     /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      5640..5734
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     5747..5766
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(5805..6862,1..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 aattggcgaa
      541 aatgagacgt tgatcggcac gtaagaggtt ccaactttca ccataatgaa ataagatcac
      601 taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct aaaatggaga
      661 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
      721 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
      781 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
      841 ttgcccgcct gatgaatgct catccggaat ttcgtatggc aatgaaagac ggtgagctgg
      901 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
      961 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     1021 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     1081 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     1141 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     1201 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     1261 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taatttgact
     1321 tcggtccgcg cgttgtcctt ttccgctgca taaccctgct tcggggtcat tatagcgatt
     1381 ttttcggtat atccatcctt tttcgcacga tatacaggat tttgccaaag ggttcgtgta
     1441 gactttcctt ggtgtatcca acggcgtcag ccgggcagga taggtgaagt aggcccaccc
     1501 gcgagcgggt gttccttctt cactgtccct tattcgcacc tggcggtgct caacgggaat
     1561 cctgctctgc gaggctggcc gtaggccggc cgcgatgcag gtggctgctg aacccccagc
     1621 cggaactgac cccacaaggc cctagcgttt gcaatgcacc aggtcaccat tgacccaggc
     1681 gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct tcgccgacct gctcgcgcca
     1741 cttcttcacg cgggtggaat ccgatccgca catgaggcgg aaggtttcca gcttgagcgg
     1801 gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt cgggccgtcg gcgacagctt
     1861 gcggtacttc tcccatatga atttcgtgta gtggtcgcca gcaaacagca cgacgatttc
     1921 ctcgtcgatc aggacctggc aacgggacgt tttcttgcca cggtccagga cgcggaagcg
     1981 gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg gacgtgaagc ccatcgccgt
     2041 cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa taccggccat tgatcgacca
     2101 gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc ggctcgccga taggggtgcg
     2161 cttcgcgtac tccaacacct gctgccacac cagttcgtca tcgtcggccc gcagctcgac
     2221 gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa atgaccttgt tttgcagcgc
     2281 ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca gagcgggccg tgtcgtttgg
     2341 catcgctcgc atcgtgtccg gccacggcgc aatatcgaac aaggaaagct gcatttcctt
     2401 gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg gcttcgctga cctgttttgc
     2461 caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata gttcctcgcg tgtcgatggt
     2521 catcgacttc gccaaacctg ccgcctcctg ttcgagacga cgcgaacgct ccacggcggc
     2581 cgatggcgcg ggcagggcag ggggagccag ttgcacgctg tcgcgctcga tcttggccgt
     2641 agcttgctgg actatcgagc cgacggactg gaaggtttcg cggggcgcac gcatgacggt
     2701 gcggcttgcg atggtttcgg catcctcggc ggaaaacccc gcgtcgatca gttcttgcct
     2761 gtatgccttc cggtcaaacg tccgattcat tcaccctcct tgcgggattg ccccggaatt
     2821 aattccccgg atcgatccgt cgatcttgat cccctgcgcc atcagatcct tggcggcaag
     2881 aaagccatcc agtttacttt gcagggcttc ccaaccttac cagagggcgc cccagctggc
     2941 aattccggtt cgcttgctgt ccataaaacc gcccagtcta gctatcgcca tgtaagccca
     3001 ctgcaagcta cctgctttct ctttgcgctt gcgttttccc ttgtccagat agcccagtag
     3061 ctgacattca tccggggtca gcaccgtttc tgcggactgg ctttctacgt ggctgccatt
     3121 tttggggtga ggccgttcgc ggccgagggg cgcagcccct ggggggatgg gaggcccgcg
     3181 ttagcgggcc gggagggttc gagaaggggg ggcacccccc ttcggcgtgc gcggtcacgc
     3241 gcacagggcg cagccctggt taaaaacaag gtttataaat attggtttaa aagcaggtta
     3301 aaagacaggt tagcggtggc cgaaaaacgg gcggaaaccc ttgcaaatgc tggattttct
     3361 gcctgtggac agcccctcaa atgtcaatag gtgcgcccct catctgtcag cactctgccc
     3421 ctcaagtgtc aaggatcgcg cccctcatct gtcagtagtc gcgcccctca agtgtcaata
     3481 ccgcagggca cttatcccca ggcttgtcca catcatctgt gggaaactcg cgtaaaatca
     3541 ggcgttttcg ccgatttgcg aggctggcca gctccacgtc gccggccgaa atcgagcctg
     3601 cccctcatct gtcaacgccg cgccgggtga gtcggcccct caagtgtcaa cgtccgcccc
     3661 tcatctgtca gtgagggcca agttttccgc gaggtatcca caacgccggc ggccctacat
     3721 ggctctgctg tagtgagtgg gttgcgcttc ggcagcggtc ctgatccccc gcagaaaaaa
     3781 aggatctcaa gaagatcctt tgatcttttc tacggcgcgc ccagctgtct agggcggcgg
     3841 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
     3901 tttcgactga gcctttcgtt ttatttgatg cctttaatta atgtgacaat taatcatcgg
     3961 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaacccct aggggagtca
     4021 tgaacatatg agagccctgg tagtcatccg cctgtcccgc gtcaccgatg ctacgacttc
     4081 accggagcgt cagctggagt cttgccagca gctctgcgcc cagcgcggct gggacgtcgt
     4141 cggggtagcg gaggatctgg acgtctccgg ggcggtcgat ccgttcgacc ggaagcgcag
     4201 accgaacctg gcccggtggc tagcgttcga ggagcaaccg tttgacgtga tcgtggcgta
     4261 ccgggtagat cggttgaccc gatcgatccg gcatcttcag cagctggtcc actgggccga
     4321 ggaccacaag aagctggtcg tctccgcgac cgaagcgcac ttcgatacga cgacgccgtt
     4381 tgcggcggtc gtcatcgcgc ttatgggaac ggtggcgcag atggaattag aagcgatcaa
     4441 agagcggaac cgttcggctg cgcatttcaa tatccgcgcc gggaaatacc gaggatccct
     4501 gccgccgtgg ggatacctgc ctacgcgcgt ggacggggag tggcggctgg tgccggaccc
     4561 tgtgcagcga gagcgcatcc tcgaggtgta tcaccgcgtc gtcgacaacc acgagccgct
     4621 gcatctggtg gcccacgacc tgaaccggcg tggtgtcctg tcgccgaagg actacttcgc
     4681 gcagctgcaa ggccgcgagc cgcagggccg ggagtggtcg gctaccgcgc tgaagcgatc
     4741 gatgatctcc gaggcgatgc tcgggtacgc gactctgaac ggtaagaccg tccgagacga
     4801 cgacggagcc ccgctggtgc gggctgagcc gatcctgacc cgtgagcagc tggaggcgct
     4861 gcgcgccgag ctcgtgaaga cctcccgggc gaagcccgcg gtgtctaccc cgtcgctgct
     4921 gctgcgggtg ttgttctgcg cggtgtgcgg ggagcccgcg tacaagttcg ccgggggagg
     4981 acgtaagcac ccgcgctacc gctgccgctc gatggggttc ccgaagcact gcgggaacgg
     5041 cacggtggcg atggccgagt gggacgcgtt ctgcgaggag caggtactgg atctgctcgg
     5101 ggacgcggag cgtctggaga aagtctgggt agcgggctcg gactccgcgg tcgaactcgc
     5161 ggaggtgaac gcggagctgg tggacctgac gtcgctgatc ggctccccgg cctaccgggc
     5221 gggctctccg cagcgagaag cactggatgc ccgtattgcg gcgctggccg cgcggcaaga
     5281 ggagctggag ggcctggagg ctcgcccgtc tggctgggag tggcgcgaga ccgggcagcg
     5341 gttcggggac tggtggcggg agcaggacac cgcggcaaag aacacctggc ttcggtcgat
     5401 gaacgttcgg ctgacgttcg acgtccgcgg cgggctgact cgcacgatcg acttcgggga
     5461 tcttcaggag tacgagcagc atctcaggct cggcagcgtg gtcgaacggc tacacaccgg
     5521 gatgtcgtaa taacgctgat agtgctagtg tagatcgcta cactagtcct agagtcgacc
     5581 tgcaggcatg caagcttgcg gccgcgtcgt gactgggaaa accctggcga ctagtcttgg
     5641 actcctgttg atagatccag taatgacctc agaactccat ctggatttgt tcagaacgct
     5701 cggttgccgc cgggcgtttt ttattggtga gaatccaggg gtcccccggc atcagagcag
     5761 attgtactga gagtgcacca taatcggcat tttcttttgc gtttttattt gttaactgtt
     5821 aattgtcctt gttcaaggat gctgtctttg acaacagatg ttttcttgcc tttgatgttc
     5881 agcaggaagc taggcgcaaa cgttgattgt ttgtctgcgt agaatcctct gtttgtcata
     5941 tagcttgtaa tcacgacatt gtttcctttc gcttgaggta cagcgaagtg tgagtaagta
     6001 aaggttacat cgttaggatc aagatccatt tttaacacaa ggccagtttt gttcagcggc
     6061 ttgtatgggc cagttaaaga attagaaaca taaccaagca tgtaaatatc gttagacgta
     6121 atgccgtcaa tcgtcatttt tgatccgcgg gagtcagtga acagatacca tttgccgttc
     6181 attttaaaga cgttcgcgcg ttcaatttca tctgttactg tgttagatgc aatcagcggt
     6241 ttcatcactt ttttcagtgt gtaatcatcg tttagctcaa tcataccgag agcgccgttt
     6301 gctaactcag ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc ttgacggaag
     6361 aatgatgtgc ttttgccata gtatgctttg ttaaataaag attcttcgcc ttggtagcca
     6421 tcttcagttc cagtgtttgc ttcaaatact aagtatttgt ggcctttatc ttctacgtag
     6481 tgaggatctc tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc gatgaactgc
     6541 tgtacatttt gatacgtttt tccgtcaccg tcaaagattg atttataatc ctctacaccg
     6601 ttgatgttca aagagctgtc tgatgctgat acgttaactt gtgcagttgt cagtgtttgt
     6661 ttgccgtaat gtttaccgga gaaatcagtg tagaataaac ggatttttcc gtcagatgta
     6721 aatgtggctg aacctgacca ttcttgtgtt tggtctttta ggatagaatc atttgcatcg
     6781 aatttgtcgc tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat agaagtttcg
     6841 ccgacttttt gatagaacat gt
//
