LOCUS       sequence-458899-        5663 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 (Am) 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 5663)
  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 5663)
  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-8
FEATURES             Location/Qualifiers
     source          1..5663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             168..176
                     /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    970..1017
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             1204..2004
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     protein_bind    2104..2141
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_feature    2147..2165
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     terminator      2176..2270
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2375..3802
                     /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        3859..3950
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3951..4019,4020..4811)
                     /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      4815..4854
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            5002..5110
                     /note="incP origin of transfer"
     rep_origin      5121..5509
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(5540..5626)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    5641..5659
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
ORIGIN
        1 gtctcaggcc gcctaggccg cggccgcgcg aattcgagct cggtacccgg ggatccgccc
       61 attgacaagg ctctcgcggc caggtataat tgcacgaggg cccaagttca cttaaaaagg
      121 agatcaacaa tgaaagcaat tttcgtactg aaacatctta atcatgctaa ggaggttttc
      181 taatgcgtaa aggtgaagaa ctgttcaccg gtgttgttcc gatcctggtt gaactggatg
      241 gtgatgttaa cggccacaaa ttctctgttc gtggtgaagg tgaaggtgat gcaaccaacg
      301 gtaaactgac cctgaaattc atctgcacta ccggtaaact gccggttcca tggccgactc
      361 tggtgactac cctgacctat ggtgttcagt gtttttctcg ttacccggat cacatgaagc
      421 agcatgattt cttcaaatct gcaatgccgg aaggttatgt acaggagcgc accatttctt
      481 tcaaagacga tggcacctac aaaacccgtg cagaggttaa atttgaaggt gatactctgg
      541 tgaaccgtat tgaactgaaa ggcattgatt tcaaagagga cggcaacatc ctgggccaca
      601 aactggaata taacttcaac tcccataacg tttacatcac cgcagacaaa cagaagaacg
      661 gtatcaaagc taacttcaaa attcgccata acgttgaaga cggtagcgta cagctggcgg
      721 accactacca gcagaacact ccgatcggtg atggtccggt tctgctgccg gataaccact
      781 acctgtccac ccagtctaaa ctgtccaaag acccgaacga aaagcgcgac cacatggtgc
      841 tgctggagtt cgttactgcc gcaggtatca cgcacggcat ggatgaactc tacaaataat
      901 ctagagtcga cctgcaggca tgcaagcttg cggccgccaa agcccgccga aaggcgggct
      961 tttctgtgtg gtttgtctgg tcaaccaccg cggtctcagt ggtgtacggt acaaacccaa
     1021 tttaaatctc acggtaactg atgccgtatt tgcagtacca gcgtacggcc cacagaatga
     1081 tgtcacgctg aaaatgccgg cctttgaatg ggttcatgtg cagctccatc agcaaaaggg
     1141 gatgataagt ttatcaccac cgactatttg caacagtgcc gttgatcgtg ctatgatcga
     1201 gtgatgtcat cagcggtgga gtgcaatgtc gtgcaatacg aatggcgaaa agccgagctg
     1261 atcggtcagc ttctcaacct tggggttacc cccggcggtg tgctgctggt ccacagctcc
     1321 ttccgtagcg tccggcccct cgaagatggg ccacttggac tgatcgaggc cctgcgtgct
     1381 gcgctgggtc cgggagggac gctcgtgatg ccctcgtggt caggtctgga cgacgagccg
     1441 ttcgatcctg ccacgtcgcc cgttacaccg gaccttggag ttgtctctga cacattctgg
     1501 cgcctgccaa atgtaaagcg cagcgcccat ccatttgcct ttgcggcagc ggggccacag
     1561 gcagagcaga tcatctctga tccattgccc ctgccacctc actcgcctgc aagcccggtc
     1621 gcccgtgtcc atgaactcga tgggcaggta cttctcctcg gcgtgggaca cgatgccaac
     1681 acgacgctgc atcttgccga gttgatggca aaggttccct atggggtgcc gagacactgc
     1741 accattcttc aggatggcaa gttggttcgc gtcgattatc tcgagaatga ccactgctgt
     1801 gagcgctttg ccttggcgga caggtggctc aaggagaaga gccttcagaa ggaaggtcca
     1861 gtcggtcatg cctttgctcg gttgatccgc tcccgcgaca ttgtggcgac agccctgggt
     1921 caactgggcc gagatccgtt gatcttcctg catccgccag aggcgggatg cgaagaatgc
     1981 gatgccgctc gccagtcgat tggctgagac cgttgtctaa ttaattgcgg accctagagg
     2041 tccccttttt tattttaaaa attttttcac aaaacggttt acaagcataa aatctctgag
     2101 gccggcttgt cgacgacggc ggtctccgtc gtcaggatca tccgggagat gtgtataaga
     2161 gacagactag tcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg
     2221 atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaggggtcc
     2281 cctggtttaa actacacaag tagcgtcctg aacggaacct ttcccgtttt ccagaatctg
     2341 atgttccatg tgacctccta acatggtaac gttcatgatt accagtgcac tgcatcgtgc
     2401 ggcggattgg gcgaaaagcg tgttttctag tgctgcgctg ggtgatccgc gtcgtaccgc
     2461 gcgtctggtg aatgttgcgg cgcaactggc caaatatagc ggcaaaagca ttaccattag
     2521 cagcgaaggc agcaaagcca tgcaggaagg cgcgtatcgt tttattcgta atccgaacgt
     2581 gagcgcggaa gcgattcgta aagcgggtgc catgcagacc gtgaaactgg cccaggaatt
     2641 tccggaactg ctggcaattg aagataccac ctctctgagc tatcgtcatc aggtggcgga
     2701 agaactgggc aaactgggta gcattcagga taaaagccgt ggttggtggg tgcatagcgt
     2761 gctgctgctg gaagcgacca cctttcgtac cgtgggcctg ctgcatcaag aatggtggat
     2821 gcgtccggat gatccggcgg atgcggatga aaaagaaagc ggcaaatggc tggccgctgc
     2881 tgcaacttcg cgtctgagaa tgggcagcat gatgagcaac gtgattgcgg tgtgcgatcg
     2941 tgaagcggat attcatgcgt atctgcaaga taaactggcc cataacgaac gttttgtggt
     3001 gcgtagcaaa catccgcgta aagatgtgga aagcggcctg tatctgtatg atcacctgaa
     3061 aaaccagccg gaactgggcg gctatcagat tagcattccg cagaaaggcg tggtggataa
     3121 acgtggcaaa cgtaaaaacc gtccggcgcg taaagcgagc ctgagcctgc gtagcggccg
     3181 tattaccctg aaacagggca acattaccct gaacgcggtg ctggccgaag aaattaatcc
     3241 gccgaaaggc gaaaccccgc tgaaatggct gctgctgacc agcgagccgg tggaaagtct
     3301 ggcccaagcg ctgcgtgtga ttgatattta tacccatcgt tggcgcattg aagaatttca
     3361 caaagcgtgg aaaacgggtg cgggtgcgga acgtcagcgt atggaagaac cggataacct
     3421 ggaacgtatg gtgagcattc tgagctttgt ggcggtgcgt ctgctgcaac tgcgtgaatc
     3481 ttttactccg ccgcaagcac tgcgtgcgca gggcctgctg aaagaagcgg aacacgttga
     3541 aagccagagc gcggaaaccg tgctgacccc ggatgaatgc caactgctgg gctatctgga
     3601 taaaggcaaa cgcaaacgca aagaaaaagc gggcagcctg caatgggcgt atatggcgat
     3661 tgcgcgtctg ggcggcttta tggatagcaa acgtaccggc attgcgagct ggggtgcgct
     3721 gtgggaaggt tgggaagcgc tgcaaagcaa actggatggc tttctggccg cgaaagacct
     3781 gatggcgcag ggcattaaaa tctaatcggc gatcgctagt agcccgccta atgagcgggc
     3841 ttttttttaa ttcccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3901 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagcattc
     3961 agcattttcg tgtggcgctg attccgtttt ttgcggcgtt ttgcctgccg gtgtttgcgc
     4021 atccggaaac cctggtgaaa gtgaaagatg cggaagatca actgggtgcg cgcgtgggct
     4081 atattgaact ggatctgaac agcggcaaaa ttctggaatc ttttcgtccg gaagaacgtt
     4141 ttccgatgat gagcaccttt aaagtgctgc tgtgcggtgc ggttctgagc cgtgtggatg
     4201 cgggccagga acaactgggc cgtcgtattc attatagcca gaacgatctg gtggaatata
     4261 gcccggtgac cgaaaaacat ctgaccgatg gcatgaccgt gcgtgaactg tgcagcgcgg
     4321 cgattaccat gagcgataac accgcggcga acctgctgct gacgaccatt ggcggtccga
     4381 aagaactgac cgcgtttctg cataacatgg gcgatcatgt gacccgtctg gatcgttggg
     4441 aaccggaact gaacgaagcg attccgaacg atgaacgtga taccaccatg ccggcagcaa
     4501 tggcgaccac cctgcgtaaa ctgctgacgg gtgagctgct gaccctggca agccgccagc
     4561 aactgattga ttggatggaa gcggataaag tggcgggtcc gctgctgcgt agcgcgctgc
     4621 cggctggctg gtttattgcg gataaaagcg gtgcgggcga acgtggcagc cgtggcatta
     4681 ttgcggcgct gggcccggat ggtaaaccga gccgtattgt ggtgatttat accaccggca
     4741 gccaggcgac gatggatgaa cgtaaccgtc agattgcgga aattggcgcg agcctgatta
     4801 aacattggta aaccgataca attaaaggct ccttttggag cctttttttt tggacacgcg
     4861 tttgtccttt tccgctgcat aaccctgctt cggggtcatt atagcgattt tttcggtata
     4921 tccatccttt ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag actttccttg
     4981 gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta ggcccacccg cgagcgggtg
     5041 ttccttcttc actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg
     5101 aggctggccg taggccggcc gatctgaaga tcagcagttc aacctgttga tagtacgtac
     5161 taagctctca tgtttcacgt actaagctct catgtttaac gtactaagct ctcatgttta
     5221 acgaactaaa ccctcatggc taacgtacta agctctcatg gctaacgtac taagctctca
     5281 tgtttcacgt actaagctct catgtttgaa caataaaatt aatataaatc agcaacttaa
     5341 atagcctcta aggttttaag ttttataaga aaaaaaagaa tatataaggc ttttaaagcc
     5401 tttaaggttt aacggttgtg gacaacaagc cagggatgta acgcactgag aagcccttag
     5461 agcctctcaa agcaattttg agtgacacag gaacacttaa cggctgacat ggggcgcgcc
     5521 cagctgtcta gggcggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
     5581 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctttaattaa
     5641 ctgtctctta tacacatctt tgt
//
