LOCUS       sequence-458897-        5672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of msfGFP (or your Module Of Interest) in a random 
            fashion via Tn5 insertion. The antibiotic cassette (Sm) used for 
            selecting for the integration can be removed in a later stage with 
            pFNC..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5672)
  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 5672)
  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: pCIFR-4
FEATURES             Location/Qualifiers
     source          1..5672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    10..28
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     RBS             200..208
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     protein_bind    1002..1049
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             1257..2048
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     protein_bind    2145..2182
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_feature    2188..2206
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     terminator      2217..2311
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2416..3843
                     /codon_start=1
                     /product="transposase from the bacterial Tn5 transposon 
                     (Reznikoff, 1993)"
                     /label=Tn5 transposase
                     /translation="MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKS
                     ITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYR
                     HQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESG
                     KWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGL
                     YLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNA
                     VLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAER
                     QRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTP
                     DECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQS
                     KLDGFLAAKDLMAQGIKI"
     promoter        3900..3991
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3992..4060,4061..4852)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     terminator      4856..4895
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            5043..5151
                     /note="incP origin of transfer"
     rep_origin      5162..5550
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(5581..5667)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tttaattaac tgtctcttat acacatcttt gtgtctcagg ccgcctaggc cgcggccgcg
       61 cgaattcgag ctcggtaccc ggggatccgc ccattgacaa ggctctcgcg gccaggtata
      121 attgcacgag ggcccaagtt cacttaaaaa ggagatcaac aatgaaagca attttcgtac
      181 tgaaacatct taatcatgct aaggaggttt tctaatgcgt aaaggtgaag aactgttcac
      241 cggtgttgtt ccgatcctgg ttgaactgga tggtgatgtt aacggccaca aattctctgt
      301 tcgtggtgaa ggtgaaggtg atgcaaccaa cggtaaactg accctgaaat tcatctgcac
      361 taccggtaaa ctgccggttc catggccgac tctggtgact accctgacct atggtgttca
      421 gtgtttttct cgttacccgg atcacatgaa gcagcatgat ttcttcaaat ctgcaatgcc
      481 ggaaggttat gtacaggagc gcaccatttc tttcaaagac gatggcacct acaaaacccg
      541 tgcagaggtt aaatttgaag gtgatactct ggtgaaccgt attgaactga aaggcattga
      601 tttcaaagag gacggcaaca tcctgggcca caaactggaa tataacttca actcccataa
      661 cgtttacatc accgcagaca aacagaagaa cggtatcaaa gctaacttca aaattcgcca
      721 taacgttgaa gacggtagcg tacagctggc ggaccactac cagcagaaca ctccgatcgg
      781 tgatggtccg gttctgctgc cggataacca ctacctgtcc acccagtcta aactgtccaa
      841 agacccgaac gaaaagcgcg accacatggt gctgctggag ttcgttactg ccgcaggtat
      901 cacgcacggc atggatgaac tctacaaata atctagagtc gacctgcagg catgcaagct
      961 tgcggccgcc aaagcccgcc gaaaggcggg cttttctgtg tggtttgtct ggtcaaccac
     1021 cgcggtctca gtggtgtacg gtacaaaccc aatttaaatg aaccttgacc gaacgcagcg
     1081 gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt
     1141 ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat
     1201 ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacatcatga
     1261 gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc
     1321 gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc
     1381 tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa
     1441 cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga
     1501 ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc
     1561 cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct
     1621 tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata
     1681 gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc
     1741 tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg
     1801 atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa
     1861 tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc
     1921 ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc
     1981 gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg
     2041 gcaaataaga caattgtcta attaattgcg gaccctagag gtcccctttt ttattttaaa
     2101 aattttttca caaaacggtt tacaagcata aaatctctga ggccggcttg tcgacgacgg
     2161 cggtctccgt cgtcaggatc atccgggaga tgtgtataag agacagacta gtcttggact
     2221 cctgttgata gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg
     2281 ttgccgccgg gcgtttttta ttggtgagaa tccaggggtc ccctggttta aactacacaa
     2341 gtagcgtcct gaacggaacc tttcccgttt tccagaatct gatgttccat gtgacctcct
     2401 aacatggtaa cgttcatgat taccagtgca ctgcatcgtg cggcggattg ggcgaaaagc
     2461 gtgttttcta gtgctgcgct gggtgatccg cgtcgtaccg cgcgtctggt gaatgttgcg
     2521 gcgcaactgg ccaaatatag cggcaaaagc attaccatta gcagcgaagg cagcaaagcc
     2581 atgcaggaag gcgcgtatcg ttttattcgt aatccgaacg tgagcgcgga agcgattcgt
     2641 aaagcgggtg ccatgcagac cgtgaaactg gcccaggaat ttccggaact gctggcaatt
     2701 gaagatacca cctctctgag ctatcgtcat caggtggcgg aagaactggg caaactgggt
     2761 agcattcagg ataaaagccg tggttggtgg gtgcatagcg tgctgctgct ggaagcgacc
     2821 acctttcgta ccgtgggcct gctgcatcaa gaatggtgga tgcgtccgga tgatccggcg
     2881 gatgcggatg aaaaagaaag cggcaaatgg ctggccgctg ctgcaacttc gcgtctgaga
     2941 atgggcagca tgatgagcaa cgtgattgcg gtgtgcgatc gtgaagcgga tattcatgcg
     3001 tatctgcaag ataaactggc ccataacgaa cgttttgtgg tgcgtagcaa acatccgcgt
     3061 aaagatgtgg aaagcggcct gtatctgtat gatcacctga aaaaccagcc ggaactgggc
     3121 ggctatcaga ttagcattcc gcagaaaggc gtggtggata aacgtggcaa acgtaaaaac
     3181 cgtccggcgc gtaaagcgag cctgagcctg cgtagcggcc gtattaccct gaaacagggc
     3241 aacattaccc tgaacgcggt gctggccgaa gaaattaatc cgccgaaagg cgaaaccccg
     3301 ctgaaatggc tgctgctgac cagcgagccg gtggaaagtc tggcccaagc gctgcgtgtg
     3361 attgatattt atacccatcg ttggcgcatt gaagaatttc acaaagcgtg gaaaacgggt
     3421 gcgggtgcgg aacgtcagcg tatggaagaa ccggataacc tggaacgtat ggtgagcatt
     3481 ctgagctttg tggcggtgcg tctgctgcaa ctgcgtgaat cttttactcc gccgcaagca
     3541 ctgcgtgcgc agggcctgct gaaagaagcg gaacacgttg aaagccagag cgcggaaacc
     3601 gtgctgaccc cggatgaatg ccaactgctg ggctatctgg ataaaggcaa acgcaaacgc
     3661 aaagaaaaag cgggcagcct gcaatgggcg tatatggcga ttgcgcgtct gggcggcttt
     3721 atggatagca aacgtaccgg cattgcgagc tggggtgcgc tgtgggaagg ttgggaagcg
     3781 ctgcaaagca aactggatgg ctttctggcc gcgaaagacc tgatggcgca gggcattaaa
     3841 atctaatcgg cgatcgctag tagcccgcct aatgagcggg ctttttttta attcccctat
     3901 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3961 aatgcttcaa taatattgaa aaaggaagag tatgagcatt cagcattttc gtgtggcgct
     4021 gattccgttt tttgcggcgt tttgcctgcc ggtgtttgcg catccggaaa ccctggtgaa
     4081 agtgaaagat gcggaagatc aactgggtgc gcgcgtgggc tatattgaac tggatctgaa
     4141 cagcggcaaa attctggaat cttttcgtcc ggaagaacgt tttccgatga tgagcacctt
     4201 taaagtgctg ctgtgcggtg cggttctgag ccgtgtggat gcgggccagg aacaactggg
     4261 ccgtcgtatt cattatagcc agaacgatct ggtggaatat agcccggtga ccgaaaaaca
     4321 tctgaccgat ggcatgaccg tgcgtgaact gtgcagcgcg gcgattacca tgagcgataa
     4381 caccgcggcg aacctgctgc tgacgaccat tggcggtccg aaagaactga ccgcgtttct
     4441 gcataacatg ggcgatcatg tgacccgtct ggatcgttgg gaaccggaac tgaacgaagc
     4501 gattccgaac gatgaacgtg ataccaccat gccggcagca atggcgacca ccctgcgtaa
     4561 actgctgacg ggtgagctgc tgaccctggc aagccgccag caactgattg attggatgga
     4621 agcggataaa gtggcgggtc cgctgctgcg tagcgcgctg ccggctggct ggtttattgc
     4681 ggataaaagc ggtgcgggcg aacgtggcag ccgtggcatt attgcggcgc tgggcccgga
     4741 tggtaaaccg agccgtattg tggtgattta taccaccggc agccaggcga cgatggatga
     4801 acgtaaccgt cagattgcgg aaattggcgc gagcctgatt aaacattggt aaaccgatac
     4861 aattaaaggc tccttttgga gccttttttt ttggacacgc gtttgtcctt ttccgctgca
     4921 taaccctgct tcggggtcat tatagcgatt ttttcggtat atccatcctt tttcgcacga
     4981 tatacaggat tttgccaaag ggttcgtgta gactttcctt ggtgtatcca acggcgtcag
     5041 ccgggcagga taggtgaagt aggcccaccc gcgagcgggt gttccttctt cactgtccct
     5101 tattcgcacc tggcggtgct caacgggaat cctgctctgc gaggctggcc gtaggccggc
     5161 cgatctgaag atcagcagtt caacctgttg atagtacgta ctaagctctc atgtttcacg
     5221 tactaagctc tcatgtttaa cgtactaagc tctcatgttt aacgaactaa accctcatgg
     5281 ctaacgtact aagctctcat ggctaacgta ctaagctctc atgtttcacg tactaagctc
     5341 tcatgtttga acaataaaat taatataaat cagcaactta aatagcctct aaggttttaa
     5401 gttttataag aaaaaaaaga atatataagg cttttaaagc ctttaaggtt taacggttgt
     5461 ggacaacaag ccagggatgt aacgcactga gaagccctta gagcctctca aagcaatttt
     5521 gagtgacaca ggaacactta acggctgaca tggggcgcgc ccagctgtct agggcggcgg
     5581 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
     5641 tttcgactga gcctttcgtt ttatttgatg cc
//
