LOCUS       sequence-468477-        7346 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. TcR, 
            easily curable via sucrose counterselection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7346)
  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 7346)
  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-5
FEATURES             Location/Qualifiers
     source          1..7346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             610..1800
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMSGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(962..981)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     oriT            1957..2065
                     /note="incP origin of transfer"
     CDS             complement(2126..3274)
                     /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      3667..4198
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(4325..4411)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             4512..6014
                     /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      6124..6218
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     6231..6250
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(6289..7346,1..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 aatttgacag
      541 cttatcatcg ataaactgta atgcggtagt ttatcacagt taaattgcta acgcagtcag
      601 gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc tcggcaccgt caccctggat
      661 gctgtaggca taggcttggt tatgccggta ctgccgggcc tcttgcggga tatcgtccat
      721 tccgacagca tcgccagtca ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt
      781 ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg gccgccgccc agtcctgctc
      841 gcttcgctac ttggagccac tatcgactac gcgatcatgg cgaccacacc cgtcctgtgg
      901 attctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc
      961 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc
     1021 gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc
     1081 tccctgcacg caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc
     1141 tgcttcctaa tgcaggagtc gcataaggga gagcgccgtc cgatgccctt gagagccttc
     1201 aacccagtca gctccttccg gtgggcgcgg ggcatgacca ttgtggccgc acttatgact
     1261 gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc
     1321 gaggaccgct ttcgctggag cgcgacgatg agcggcctgt cgcttgcggt attcggaatc
     1381 ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag
     1441 caggccatta tcgccggcat ggcggccgac gcgctgggct acgtcttgct ggcgttcgcg
     1501 acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg catcgggatg
     1561 cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg acagcttcaa
     1621 ggatcgctcg cggctcttac cagcctaact tcgatcactg gaccgctgat cgtcacggcg
     1681 atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg cgccgcccta
     1741 taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga
     1801 gacttcggtc cgcgcgttgt ccttttccgc tgcataaccc tgcttcgggg tcattatagc
     1861 gattttttcg gtatatccat cctttttcgc acgatataca ggattttgcc aaagggttcg
     1921 tgtagacttt ccttggtgta tccaacggcg tcagccgggc aggataggtg aagtaggccc
     1981 acccgcgagc gggtgttcct tcttcactgt cccttattcg cacctggcgg tgctcaacgg
     2041 gaatcctgct ctgcgaggct ggccgtaggc cggccgcgat gcaggtggct gctgaacccc
     2101 cagccggaac tgaccccaca aggccctagc gtttgcaatg caccaggtca ccattgaccc
     2161 aggcgtgttc caccaggccg ctgcctcgca actcttcgca ggcttcgccg acctgctcgc
     2221 gccacttctt cacgcgggtg gaatccgatc cgcacatgag gcggaaggtt tccagcttga
     2281 gcgggtacgg ctcccggtgc gagctgaaat agtcgaacat ccgtcgggcc gtcggcgaca
     2341 gcttgcggta cttctcccat atgaatttcg tgtagtggtc gccagcaaac agcacgacga
     2401 tttcctcgtc gatcaggacc tggcaacggg acgttttctt gccacggtcc aggacgcgga
     2461 agcggtgcag cagcgacacc gattccaggt gcccaacgcg gtcggacgtg aagcccatcg
     2521 ccgtcgcctg taggcgcgac aggcattcct cggccttcgt gtaataccgg ccattgatcg
     2581 accagcccag gtcctggcaa agctcgtaga acgtgaaggt gatcggctcg ccgatagggg
     2641 tgcgcttcgc gtactccaac acctgctgcc acaccagttc gtcatcgtcg gcccgcagct
     2701 cgacgccggt gtaggtgatc ttcacgtcct tgttgacgtg gaaaatgacc ttgttttgca
     2761 gcgcctcgcg cgggattttc ttgttgcgcg tggtgaacag ggcagagcgg gccgtgtcgt
     2821 ttggcatcgc tcgcatcgtg tccggccacg gcgcaatatc gaacaaggaa agctgcattt
     2881 ccttgatctg ctgcttcgtg tgtttcagca acgcggcctg cttggcttcg ctgacctgtt
     2941 ttgccaggtc ctcgccggcg gtttttcgct tcttggtcgt catagttcct cgcgtgtcga
     3001 tggtcatcga cttcgccaaa cctgccgcct cctgttcgag acgacgcgaa cgctccacgg
     3061 cggccgatgg cgcgggcagg gcagggggag ccagttgcac gctgtcgcgc tcgatcttgg
     3121 ccgtagcttg ctggactatc gagccgacgg actggaaggt ttcgcggggc gcacgcatga
     3181 cggtgcggct tgcgatggtt tcggcatcct cggcggaaaa ccccgcgtcg atcagttctt
     3241 gcctgtatgc cttccggtca aacgtccgat tcattcaccc tccttgcggg attgccccgg
     3301 aattaattcc ccggatcgat ccgtcgatct tgatcccctg cgccatcaga tccttggcgg
     3361 caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc
     3421 tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc gccatgtaag
     3481 cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc agatagccca
     3541 gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct acgtggctgc
     3601 catttttggg gtgaggccgt tcgcggccga ggggcgcagc ccctgggggg atgggaggcc
     3661 cgcgttagcg ggccgggagg gttcgagaag ggggggcacc ccccttcggc gtgcgcggtc
     3721 acgcgcacag ggcgcagccc tggttaaaaa caaggtttat aaatattggt ttaaaagcag
     3781 gttaaaagac aggttagcgg tggccgaaaa acgggcggaa acccttgcaa atgctggatt
     3841 ttctgcctgt ggacagcccc tcaaatgtca ataggtgcgc ccctcatctg tcagcactct
     3901 gcccctcaag tgtcaaggat cgcgcccctc atctgtcagt agtcgcgccc ctcaagtgtc
     3961 aataccgcag ggcacttatc cccaggcttg tccacatcat ctgtgggaaa ctcgcgtaaa
     4021 atcaggcgtt ttcgccgatt tgcgaggctg gccagctcca cgtcgccggc cgaaatcgag
     4081 cctgcccctc atctgtcaac gccgcgccgg gtgagtcggc ccctcaagtg tcaacgtccg
     4141 cccctcatct gtcagtgagg gccaagtttt ccgcgaggta tccacaacgc cggcggccct
     4201 acatggctct gctgtagtga gtgggttgcg cttcggcagc ggtcctgatc ccccgcagaa
     4261 aaaaaggatc tcaagaagat cctttgatct tttctacggc gcgcccagct gtctagggcg
     4321 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     4381 agtctttcga ctgagccttt cgttttattt gatgccttta attaatgtga caattaatca
     4441 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac ccctagggga
     4501 gtcatgaaca tatgagagcc ctggtagtca tccgcctgtc ccgcgtcacc gatgctacga
     4561 cttcaccgga gcgtcagctg gagtcttgcc agcagctctg cgcccagcgc ggctgggacg
     4621 tcgtcggggt agcggaggat ctggacgtct ccggggcggt cgatccgttc gaccggaagc
     4681 gcagaccgaa cctggcccgg tggctagcgt tcgaggagca accgtttgac gtgatcgtgg
     4741 cgtaccgggt agatcggttg acccgatcga tccggcatct tcagcagctg gtccactggg
     4801 ccgaggacca caagaagctg gtcgtctccg cgaccgaagc gcacttcgat acgacgacgc
     4861 cgtttgcggc ggtcgtcatc gcgcttatgg gaacggtggc gcagatggaa ttagaagcga
     4921 tcaaagagcg gaaccgttcg gctgcgcatt tcaatatccg cgccgggaaa taccgaggat
     4981 ccctgccgcc gtggggatac ctgcctacgc gcgtggacgg ggagtggcgg ctggtgccgg
     5041 accctgtgca gcgagagcgc atcctcgagg tgtatcaccg cgtcgtcgac aaccacgagc
     5101 cgctgcatct ggtggcccac gacctgaacc ggcgtggtgt cctgtcgccg aaggactact
     5161 tcgcgcagct gcaaggccgc gagccgcagg gccgggagtg gtcggctacc gcgctgaagc
     5221 gatcgatgat ctccgaggcg atgctcgggt acgcgactct gaacggtaag accgtccgag
     5281 acgacgacgg agccccgctg gtgcgggctg agccgatcct gacccgtgag cagctggagg
     5341 cgctgcgcgc cgagctcgtg aagacctccc gggcgaagcc cgcggtgtct accccgtcgc
     5401 tgctgctgcg ggtgttgttc tgcgcggtgt gcggggagcc cgcgtacaag ttcgccgggg
     5461 gaggacgtaa gcacccgcgc taccgctgcc gctcgatggg gttcccgaag cactgcggga
     5521 acggcacggt ggcgatggcc gagtgggacg cgttctgcga ggagcaggta ctggatctgc
     5581 tcggggacgc ggagcgtctg gagaaagtct gggtagcggg ctcggactcc gcggtcgaac
     5641 tcgcggaggt gaacgcggag ctggtggacc tgacgtcgct gatcggctcc ccggcctacc
     5701 gggcgggctc tccgcagcga gaagcactgg atgcccgtat tgcggcgctg gccgcgcggc
     5761 aagaggagct ggagggcctg gaggctcgcc cgtctggctg ggagtggcgc gagaccgggc
     5821 agcggttcgg ggactggtgg cgggagcagg acaccgcggc aaagaacacc tggcttcggt
     5881 cgatgaacgt tcggctgacg ttcgacgtcc gcggcgggct gactcgcacg atcgacttcg
     5941 gggatcttca ggagtacgag cagcatctca ggctcggcag cgtggtcgaa cggctacaca
     6001 ccgggatgtc gtaataacgc tgatagtgct agtgtagatc gctacactag tcctagagtc
     6061 gacctgcagg catgcaagct tgcggccgcg tcgtgactgg gaaaaccctg gcgactagtc
     6121 ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa
     6181 cgctcggttg ccgccgggcg ttttttattg gtgagaatcc aggggtcccc cggcatcaga
     6241 gcagattgta ctgagagtgc accataatcg gcattttctt ttgcgttttt atttgttaac
     6301 tgttaattgt ccttgttcaa ggatgctgtc tttgacaaca gatgttttct tgcctttgat
     6361 gttcagcagg aagctaggcg caaacgttga ttgtttgtct gcgtagaatc ctctgtttgt
     6421 catatagctt gtaatcacga cattgtttcc tttcgcttga ggtacagcga agtgtgagta
     6481 agtaaaggtt acatcgttag gatcaagatc catttttaac acaaggccag ttttgttcag
     6541 cggcttgtat gggccagtta aagaattaga aacataacca agcatgtaaa tatcgttaga
     6601 cgtaatgccg tcaatcgtca tttttgatcc gcgggagtca gtgaacagat accatttgcc
     6661 gttcatttta aagacgttcg cgcgttcaat ttcatctgtt actgtgttag atgcaatcag
     6721 cggtttcatc acttttttca gtgtgtaatc atcgtttagc tcaatcatac cgagagcgcc
     6781 gtttgctaac tcagccgtgc gttttttatc gctttgcaga agtttttgac tttcttgacg
     6841 gaagaatgat gtgcttttgc catagtatgc tttgttaaat aaagattctt cgccttggta
     6901 gccatcttca gttccagtgt ttgcttcaaa tactaagtat ttgtggcctt tatcttctac
     6961 gtagtgagga tctctcagcg tatggttgtc gcctgagctg tagttgcctt catcgatgaa
     7021 ctgctgtaca ttttgatacg tttttccgtc accgtcaaag attgatttat aatcctctac
     7081 accgttgatg ttcaaagagc tgtctgatgc tgatacgtta acttgtgcag ttgtcagtgt
     7141 ttgtttgccg taatgtttac cggagaaatc agtgtagaat aaacggattt ttccgtcaga
     7201 tgtaaatgtg gctgaacctg accattcttg tgtttggtct tttaggatag aatcatttgc
     7261 atcgaatttg tcgctgtctt taaagacgcg gccagcgttt ttccagctgt caatagaagt
     7321 ttcgccgact ttttgataga acatgt
//
