LOCUS       sequence-468475-        5466 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 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 5466)
  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 5466)
  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-3
FEATURES             Location/Qualifiers
     source          1..5466
                     /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)"
     promoter        1045..1147
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1148..1807
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1214..1233)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     protein_bind    1907..1944
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_feature    1950..1968
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     terminator      1979..2073
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2178..3605
                     /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        3662..3753
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3754..3822,3823..4614)
                     /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      4618..4657
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            4805..4913
                     /note="incP origin of transfer"
     rep_origin      4924..5312
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(5343..5429)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    5444..5462
                     /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 tttaaattgg cgaaaatgag acgttgatcg gcacgtaaga ggttccaact ttcaccataa
     1081 tgaaataaga tcactaccgg gcgtattttt tgagttatcg agattttcag gagctaagga
     1141 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg
     1201 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca
     1261 gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc
     1321 ctttattcac attcttgccc gcctgatgaa tgctcatccg gaatttcgta tggcaatgaa
     1381 agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca
     1441 aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca
     1501 catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt
     1561 tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt
     1621 aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac
     1681 gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg
     1741 cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg
     1801 ggcgtaattt gacttcggtc taattaattg cggaccctag aggtcccctt ttttatttta
     1861 aaaatttttt cacaaaacgg tttacaagca taaaatctct gaggccggct tgtcgacgac
     1921 ggcggtctcc gtcgtcagga tcatccggga gatgtgtata agagacagac tagtcttgga
     1981 ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc
     2041 ggttgccgcc gggcgttttt tattggtgag aatccagggg tcccctggtt taaactacac
     2101 aagtagcgtc ctgaacggaa cctttcccgt tttccagaat ctgatgttcc atgtgacctc
     2161 ctaacatggt aacgttcatg attaccagtg cactgcatcg tgcggcggat tgggcgaaaa
     2221 gcgtgttttc tagtgctgcg ctgggtgatc cgcgtcgtac cgcgcgtctg gtgaatgttg
     2281 cggcgcaact ggccaaatat agcggcaaaa gcattaccat tagcagcgaa ggcagcaaag
     2341 ccatgcagga aggcgcgtat cgttttattc gtaatccgaa cgtgagcgcg gaagcgattc
     2401 gtaaagcggg tgccatgcag accgtgaaac tggcccagga atttccggaa ctgctggcaa
     2461 ttgaagatac cacctctctg agctatcgtc atcaggtggc ggaagaactg ggcaaactgg
     2521 gtagcattca ggataaaagc cgtggttggt gggtgcatag cgtgctgctg ctggaagcga
     2581 ccacctttcg taccgtgggc ctgctgcatc aagaatggtg gatgcgtccg gatgatccgg
     2641 cggatgcgga tgaaaaagaa agcggcaaat ggctggccgc tgctgcaact tcgcgtctga
     2701 gaatgggcag catgatgagc aacgtgattg cggtgtgcga tcgtgaagcg gatattcatg
     2761 cgtatctgca agataaactg gcccataacg aacgttttgt ggtgcgtagc aaacatccgc
     2821 gtaaagatgt ggaaagcggc ctgtatctgt atgatcacct gaaaaaccag ccggaactgg
     2881 gcggctatca gattagcatt ccgcagaaag gcgtggtgga taaacgtggc aaacgtaaaa
     2941 accgtccggc gcgtaaagcg agcctgagcc tgcgtagcgg ccgtattacc ctgaaacagg
     3001 gcaacattac cctgaacgcg gtgctggccg aagaaattaa tccgccgaaa ggcgaaaccc
     3061 cgctgaaatg gctgctgctg accagcgagc cggtggaaag tctggcccaa gcgctgcgtg
     3121 tgattgatat ttatacccat cgttggcgca ttgaagaatt tcacaaagcg tggaaaacgg
     3181 gtgcgggtgc ggaacgtcag cgtatggaag aaccggataa cctggaacgt atggtgagca
     3241 ttctgagctt tgtggcggtg cgtctgctgc aactgcgtga atcttttact ccgccgcaag
     3301 cactgcgtgc gcagggcctg ctgaaagaag cggaacacgt tgaaagccag agcgcggaaa
     3361 ccgtgctgac cccggatgaa tgccaactgc tgggctatct ggataaaggc aaacgcaaac
     3421 gcaaagaaaa agcgggcagc ctgcaatggg cgtatatggc gattgcgcgt ctgggcggct
     3481 ttatggatag caaacgtacc ggcattgcga gctggggtgc gctgtgggaa ggttgggaag
     3541 cgctgcaaag caaactggat ggctttctgg ccgcgaaaga cctgatggcg cagggcatta
     3601 aaatctaatc ggcgatcgct agtagcccgc ctaatgagcg ggcttttttt taattcccct
     3661 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3721 taaatgcttc aataatattg aaaaaggaag agtatgagca ttcagcattt tcgtgtggcg
     3781 ctgattccgt tttttgcggc gttttgcctg ccggtgtttg cgcatccgga aaccctggtg
     3841 aaagtgaaag atgcggaaga tcaactgggt gcgcgcgtgg gctatattga actggatctg
     3901 aacagcggca aaattctgga atcttttcgt ccggaagaac gttttccgat gatgagcacc
     3961 tttaaagtgc tgctgtgcgg tgcggttctg agccgtgtgg atgcgggcca ggaacaactg
     4021 ggccgtcgta ttcattatag ccagaacgat ctggtggaat atagcccggt gaccgaaaaa
     4081 catctgaccg atggcatgac cgtgcgtgaa ctgtgcagcg cggcgattac catgagcgat
     4141 aacaccgcgg cgaacctgct gctgacgacc attggcggtc cgaaagaact gaccgcgttt
     4201 ctgcataaca tgggcgatca tgtgacccgt ctggatcgtt gggaaccgga actgaacgaa
     4261 gcgattccga acgatgaacg tgataccacc atgccggcag caatggcgac caccctgcgt
     4321 aaactgctga cgggtgagct gctgaccctg gcaagccgcc agcaactgat tgattggatg
     4381 gaagcggata aagtggcggg tccgctgctg cgtagcgcgc tgccggctgg ctggtttatt
     4441 gcggataaaa gcggtgcggg cgaacgtggc agccgtggca ttattgcggc gctgggcccg
     4501 gatggtaaac cgagccgtat tgtggtgatt tataccaccg gcagccaggc gacgatggat
     4561 gaacgtaacc gtcagattgc ggaaattggc gcgagcctga ttaaacattg gtaaaccgat
     4621 acaattaaag gctccttttg gagccttttt ttttggacac gcgtttgtcc ttttccgctg
     4681 cataaccctg cttcggggtc attatagcga ttttttcggt atatccatcc tttttcgcac
     4741 gatatacagg attttgccaa agggttcgtg tagactttcc ttggtgtatc caacggcgtc
     4801 agccgggcag gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc ttcactgtcc
     4861 cttattcgca cctggcggtg ctcaacggga atcctgctct gcgaggctgg ccgtaggccg
     4921 gccgatctga agatcagcag ttcaacctgt tgatagtacg tactaagctc tcatgtttca
     4981 cgtactaagc tctcatgttt aacgtactaa gctctcatgt ttaacgaact aaaccctcat
     5041 ggctaacgta ctaagctctc atggctaacg tactaagctc tcatgtttca cgtactaagc
     5101 tctcatgttt gaacaataaa attaatataa atcagcaact taaatagcct ctaaggtttt
     5161 aagttttata agaaaaaaaa gaatatataa ggcttttaaa gcctttaagg tttaacggtt
     5221 gtggacaaca agccagggat gtaacgcact gagaagccct tagagcctct caaagcaatt
     5281 ttgagtgaca caggaacact taacggctga catggggcgc gcccagctgt ctagggcggc
     5341 ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag
     5401 tctttcgact gagcctttcg ttttatttga tgcctttaat taactgtctc ttatacacat
     5461 ctttgt
//
