LOCUS       sequence-457237-        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      complement(14..100)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             201..1703
                     /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      1813..1907
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     1920..1939
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(1978..3312,3313..3399))
                     /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(3569..3574,3575..3591,3592..3597)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             3786..4319
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     oriT            4530..4638
                     /note="incP origin of transfer"
     CDS             complement(4699..5847)
                     /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      6240..6771
                     /label=oriV
                     /note="incP origin of replication"
ORIGIN
        1 tctagggcgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac
       61 gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctttaa ttaatgtgac
      121 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc
      181 cctaggggag tcatgaacat atgagagccc tggtagtcat ccgcctgtcc cgcgtcaccg
      241 atgctacgac ttcaccggag cgtcagctgg agtcttgcca gcagctctgc gcccagcgcg
      301 gctgggacgt cgtcggggta gcggaggatc tggacgtctc cggggcggtc gatccgttcg
      361 accggaagcg cagaccgaac ctggcccggt ggctagcgtt cgaggagcaa ccgtttgacg
      421 tgatcgtggc gtaccgggta gatcggttga cccgatcgat ccggcatctt cagcagctgg
      481 tccactgggc cgaggaccac aagaagctgg tcgtctccgc gaccgaagcg cacttcgata
      541 cgacgacgcc gtttgcggcg gtcgtcatcg cgcttatggg aacggtggcg cagatggaat
      601 tagaagcgat caaagagcgg aaccgttcgg ctgcgcattt caatatccgc gccgggaaat
      661 accgaggatc cctgccgccg tggggatacc tgcctacgcg cgtggacggg gagtggcggc
      721 tggtgccgga ccctgtgcag cgagagcgca tcctcgaggt gtatcaccgc gtcgtcgaca
      781 accacgagcc gctgcatctg gtggcccacg acctgaaccg gcgtggtgtc ctgtcgccga
      841 aggactactt cgcgcagctg caaggccgcg agccgcaggg ccgggagtgg tcggctaccg
      901 cgctgaagcg atcgatgatc tccgaggcga tgctcgggta cgcgactctg aacggtaaga
      961 ccgtccgaga cgacgacgga gccccgctgg tgcgggctga gccgatcctg acccgtgagc
     1021 agctggaggc gctgcgcgcc gagctcgtga agacctcccg ggcgaagccc gcggtgtcta
     1081 ccccgtcgct gctgctgcgg gtgttgttct gcgcggtgtg cggggagccc gcgtacaagt
     1141 tcgccggggg aggacgtaag cacccgcgct accgctgccg ctcgatgggg ttcccgaagc
     1201 actgcgggaa cggcacggtg gcgatggccg agtgggacgc gttctgcgag gagcaggtac
     1261 tggatctgct cggggacgcg gagcgtctgg agaaagtctg ggtagcgggc tcggactccg
     1321 cggtcgaact cgcggaggtg aacgcggagc tggtggacct gacgtcgctg atcggctccc
     1381 cggcctaccg ggcgggctct ccgcagcgag aagcactgga tgcccgtatt gcggcgctgg
     1441 ccgcgcggca agaggagctg gagggcctgg aggctcgccc gtctggctgg gagtggcgcg
     1501 agaccgggca gcggttcggg gactggtggc gggagcagga caccgcggca aagaacacct
     1561 ggcttcggtc gatgaacgtt cggctgacgt tcgacgtccg cggcgggctg actcgcacga
     1621 tcgacttcgg ggatcttcag gagtacgagc agcatctcag gctcggcagc gtggtcgaac
     1681 ggctacacac cgggatgtcg taataacgct gatagtgcta gtgtagatcg ctacactagt
     1741 cctagagtcg acctgcaggc atgcaagctt gcggccgcgt cgtgactggg aaaaccctgg
     1801 cgactagtct tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt
     1861 tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca ggggtccccc
     1921 ggcatcagag cagattgtac tgagagtgca ccataatcgg cattttcttt tgcgttttta
     1981 tttgttaact gttaattgtc cttgttcaag gatgctgtct ttgacaacag atgttttctt
     2041 gcctttgatg ttcagcagga agctaggcgc aaacgttgat tgtttgtctg cgtagaatcc
     2101 tctgtttgtc atatagcttg taatcacgac attgtttcct ttcgcttgag gtacagcgaa
     2161 gtgtgagtaa gtaaaggtta catcgttagg atcaagatcc atttttaaca caaggccagt
     2221 tttgttcagc ggcttgtatg ggccagttaa agaattagaa acataaccaa gcatgtaaat
     2281 atcgttagac gtaatgccgt caatcgtcat ttttgatccg cgggagtcag tgaacagata
     2341 ccatttgccg ttcattttaa agacgttcgc gcgttcaatt tcatctgtta ctgtgttaga
     2401 tgcaatcagc ggtttcatca cttttttcag tgtgtaatca tcgtttagct caatcatacc
     2461 gagagcgccg tttgctaact cagccgtgcg ttttttatcg ctttgcagaa gtttttgact
     2521 ttcttgacgg aagaatgatg tgcttttgcc atagtatgct ttgttaaata aagattcttc
     2581 gccttggtag ccatcttcag ttccagtgtt tgcttcaaat actaagtatt tgtggccttt
     2641 atcttctacg tagtgaggat ctctcagcgt atggttgtcg cctgagctgt agttgccttc
     2701 atcgatgaac tgctgtacat tttgatacgt ttttccgtca ccgtcaaaga ttgatttata
     2761 atcctctaca ccgttgatgt tcaaagagct gtctgatgct gatacgttaa cttgtgcagt
     2821 tgtcagtgtt tgtttgccgt aatgtttacc ggagaaatca gtgtagaata aacggatttt
     2881 tccgtcagat gtaaatgtgg ctgaacctga ccattcttgt gtttggtctt ttaggataga
     2941 atcatttgca tcgaatttgt cgctgtcttt aaagacgcgg ccagcgtttt tccagctgtc
     3001 aatagaagtt tcgccgactt tttgatagaa catgtaaatc gatgtgtcat ccgcattttt
     3061 aggatctccg gctaatgcaa agacgatgtg gtagccgtga tagtttgcga cagtgccgtc
     3121 agcgttttgt aatggccagc tgtcccaaac gtccaggcct tttgcagaag agatattttt
     3181 aattgtggac gaatcgaact caggaacttg atatttttca tttttttgct gttcagggat
     3241 ttgcagcata tcatggcgtg taatatggga aatgccgtat gtttccttat atggcttttg
     3301 gttcgtttct ttcgcaaacg cttgagttgc gcctcctgcc agcagtgcgg tagtaaaggt
     3361 taatactgtt gcttgttttg caaacttttt gatgttcatc gttcatgtct ccttttttat
     3421 gtactgtgtt agcggtctgc ttcttccagc cctcctgttt gaagatggca agttagttac
     3481 gcacaataaa aaaagaccta aaatatgtaa ggggtgacgc caaagtatac actttgccct
     3541 ttacacattt taggtcttgc atttaaattt gacataagcc tgttcggttc gtaaactgta
     3601 atgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg
     3661 taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgtac agcctatgcc
     3721 tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag
     3781 caacgatgtt acgcagcagc aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt
     3841 taggtggctc aagtatgggc atcattcgca catgtaggct cggccctgac caagtcaaat
     3901 ccatgcgggc tgctcttgat cttttcggtc gtgagttcgg agacgtagcc acctactccc
     3961 aacatcagcc ggactccgat tacctcggga acttgctccg tagtaagaca ttcatcgcgc
     4021 ttgctgcctt cgaccaagaa gcggttgttg gcgctctcgc ggcttacgtt ctgcccaagt
     4081 ttgagcagcc gcgtagtgag atctatatct atgatctcgc agtctccgga gagcaccgga
     4141 ggcagggcat tgccaccgcg ctcatcaatc tcctcaagca tgaggccaac gcgcttggtg
     4201 cttatgtgat ctacgtgcaa gcagattacg gtgacgatcc cgcagtggct ctctatacaa
     4261 agttgggcat acgggaagaa gtgatgcact ttgatatcga cccaagtacc gccacctaac
     4321 aattcgttca agccgagatc ggcttcccgg ccgcggagtt gttcggtaaa ttggacaacg
     4381 gtccgcgcgt tgtccttttc cgctgcataa ccctgcttcg gggtcattat agcgattttt
     4441 tcggtatatc catccttttt cgcacgatat acaggatttt gccaaagggt tcgtgtagac
     4501 tttccttggt gtatccaacg gcgtcagccg ggcaggatag gtgaagtagg cccacccgcg
     4561 agcgggtgtt ccttcttcac tgtcccttat tcgcacctgg cggtgctcaa cgggaatcct
     4621 gctctgcgag gctggccgta ggccggccgc gatgcaggtg gctgctgaac ccccagccgg
     4681 aactgacccc acaaggccct agcgtttgca atgcaccagg tcaccattga cccaggcgtg
     4741 ttccaccagg ccgctgcctc gcaactcttc gcaggcttcg ccgacctgct cgcgccactt
     4801 cttcacgcgg gtggaatccg atccgcacat gaggcggaag gtttccagct tgagcgggta
     4861 cggctcccgg tgcgagctga aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg
     4921 gtacttctcc catatgaatt tcgtgtagtg gtcgccagca aacagcacga cgatttcctc
     4981 gtcgatcagg acctggcaac gggacgtttt cttgccacgg tccaggacgc ggaagcggtg
     5041 cagcagcgac accgattcca ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc
     5101 ctgtaggcgc gacaggcatt cctcggcctt cgtgtaatac cggccattga tcgaccagcc
     5161 caggtcctgg caaagctcgt agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt
     5221 cgcgtactcc aacacctgct gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc
     5281 ggtgtaggtg atcttcacgt ccttgttgac gtggaaaatg accttgtttt gcagcgcctc
     5341 gcgcgggatt ttcttgttgc gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat
     5401 cgctcgcatc gtgtccggcc acggcgcaat atcgaacaag gaaagctgca tttccttgat
     5461 ctgctgcttc gtgtgtttca gcaacgcggc ctgcttggct tcgctgacct gttttgccag
     5521 gtcctcgccg gcggtttttc gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat
     5581 cgacttcgcc aaacctgccg cctcctgttc gagacgacgc gaacgctcca cggcggccga
     5641 tggcgcgggc agggcagggg gagccagttg cacgctgtcg cgctcgatct tggccgtagc
     5701 ttgctggact atcgagccga cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg
     5761 gcttgcgatg gtttcggcat cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta
     5821 tgccttccgg tcaaacgtcc gattcattca ccctccttgc gggattgccc cggaattaat
     5881 tccccggatc gatccgtcga tcttgatccc ctgcgccatc agatccttgg cggcaagaaa
     5941 gccatccagt ttactttgca gggcttccca accttaccag agggcgcccc agctggcaat
     6001 tccggttcgc ttgctgtcca taaaaccgcc cagtctagct atcgccatgt aagcccactg
     6061 caagctacct gctttctctt tgcgcttgcg ttttcccttg tccagatagc ccagtagctg
     6121 acattcatcc ggggtcagca ccgtttctgc ggactggctt tctacgtggc tgccattttt
     6181 ggggtgaggc cgttcgcggc cgaggggcgc agcccctggg gggatgggag gcccgcgtta
     6241 gcgggccggg agggttcgag aagggggggc accccccttc ggcgtgcgcg gtcacgcgca
     6301 cagggcgcag ccctggttaa aaacaaggtt tataaatatt ggtttaaaag caggttaaaa
     6361 gacaggttag cggtggccga aaaacgggcg gaaacccttg caaatgctgg attttctgcc
     6421 tgtggacagc ccctcaaatg tcaataggtg cgcccctcat ctgtcagcac tctgcccctc
     6481 aagtgtcaag gatcgcgccc ctcatctgtc agtagtcgcg cccctcaagt gtcaataccg
     6541 cagggcactt atccccaggc ttgtccacat catctgtggg aaactcgcgt aaaatcaggc
     6601 gttttcgccg atttgcgagg ctggccagct ccacgtcgcc ggccgaaatc gagcctgccc
     6661 ctcatctgtc aacgccgcgc cgggtgagtc ggcccctcaa gtgtcaacgt ccgcccctca
     6721 tctgtcagtg agggccaagt tttccgcgag gtatccacaa cgccggcggc cctacatggc
     6781 tctgctgtag tgagtgggtt gcgcttcggc agcggtcctg atcccccgca gaaaaaaagg
     6841 atctcaagaa gatcctttga tcttttctac ggcgcgccca gctg
//
