LOCUS       sequence-468478-        5950 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 5950)
  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 5950)
  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-5
FEATURES             Location/Qualifiers
     source          1..5950
                     /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             1104..2294
                     /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(1456..1475)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    2391..2428
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_feature    2434..2452
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     terminator      2463..2557
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2662..4089
                     /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        4146..4237
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4238..4306,4307..5098)
                     /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      5102..5141
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            5289..5397
                     /note="incP origin of transfer"
     rep_origin      5408..5796
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(5827..5913)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    5928..5946
                     /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 tttaaatttg acagcttatc atcgataaac tgtaatgcgg tagtttatca cagttaaatt
     1081 gctaacgcag tcaggcaccg tgtatgaaat ctaacaatgc gctcatcgtc atcctcggca
     1141 ccgtcaccct ggatgctgta ggcataggct tggttatgcc ggtactgccg ggcctcttgc
     1201 gggatatcgt ccattccgac agcatcgcca gtcactatgg cgtgctgcta gcgctatatg
     1261 cgttgatgca atttctatgc gcacccgttc tcggagcact gtccgaccgc tttggccgcc
     1321 gcccagtcct gctcgcttcg ctacttggag ccactatcga ctacgcgatc atggcgacca
     1381 cacccgtcct gtggattctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag
     1441 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
     1501 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
     1561 tgttgggcgc catctccctg cacgcaccat tccttgcggc ggcggtgctc aacggcctca
     1621 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgc cgtccgatgc
     1681 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg accattgtgg
     1741 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     1801 gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgagcggc ctgtcgcttg
     1861 cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac
     1921 gtttcggcga gaagcaggcc attatcgccg gcatggcggc cgacgcgctg ggctacgtct
     1981 tgctggcgtt cgcgacgcga ggctggatgg ccttccccat tatgattctt ctcgcttccg
     2041 gcggcatcgg gatgcccgcg ttgcaggcca tgctgtccag gcaggtagat gacgaccatc
     2101 agggacagct tcaaggatcg ctcgcggctc ttaccagcct aacttcgatc actggaccgc
     2161 tgatcgtcac ggcgatttat gccgcctcgg cgagcacatg gaacgggttg gcatggattg
     2221 taggcgccgc cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg
     2281 ccacctcgac ctgagacttc ggtctaatta attgcggacc ctagaggtcc ccttttttat
     2341 tttaaaaatt ttttcacaaa acggtttaca agcataaaat ctctgaggcc ggcttgtcga
     2401 cgacggcggt ctccgtcgtc aggatcatcc gggagatgtg tataagagac agactagtct
     2461 tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt tgttcagaac
     2521 gctcggttgc cgccgggcgt tttttattgg tgagaatcca ggggtcccct ggtttaaact
     2581 acacaagtag cgtcctgaac ggaacctttc ccgttttcca gaatctgatg ttccatgtga
     2641 cctcctaaca tggtaacgtt catgattacc agtgcactgc atcgtgcggc ggattgggcg
     2701 aaaagcgtgt tttctagtgc tgcgctgggt gatccgcgtc gtaccgcgcg tctggtgaat
     2761 gttgcggcgc aactggccaa atatagcggc aaaagcatta ccattagcag cgaaggcagc
     2821 aaagccatgc aggaaggcgc gtatcgtttt attcgtaatc cgaacgtgag cgcggaagcg
     2881 attcgtaaag cgggtgccat gcagaccgtg aaactggccc aggaatttcc ggaactgctg
     2941 gcaattgaag ataccacctc tctgagctat cgtcatcagg tggcggaaga actgggcaaa
     3001 ctgggtagca ttcaggataa aagccgtggt tggtgggtgc atagcgtgct gctgctggaa
     3061 gcgaccacct ttcgtaccgt gggcctgctg catcaagaat ggtggatgcg tccggatgat
     3121 ccggcggatg cggatgaaaa agaaagcggc aaatggctgg ccgctgctgc aacttcgcgt
     3181 ctgagaatgg gcagcatgat gagcaacgtg attgcggtgt gcgatcgtga agcggatatt
     3241 catgcgtatc tgcaagataa actggcccat aacgaacgtt ttgtggtgcg tagcaaacat
     3301 ccgcgtaaag atgtggaaag cggcctgtat ctgtatgatc acctgaaaaa ccagccggaa
     3361 ctgggcggct atcagattag cattccgcag aaaggcgtgg tggataaacg tggcaaacgt
     3421 aaaaaccgtc cggcgcgtaa agcgagcctg agcctgcgta gcggccgtat taccctgaaa
     3481 cagggcaaca ttaccctgaa cgcggtgctg gccgaagaaa ttaatccgcc gaaaggcgaa
     3541 accccgctga aatggctgct gctgaccagc gagccggtgg aaagtctggc ccaagcgctg
     3601 cgtgtgattg atatttatac ccatcgttgg cgcattgaag aatttcacaa agcgtggaaa
     3661 acgggtgcgg gtgcggaacg tcagcgtatg gaagaaccgg ataacctgga acgtatggtg
     3721 agcattctga gctttgtggc ggtgcgtctg ctgcaactgc gtgaatcttt tactccgccg
     3781 caagcactgc gtgcgcaggg cctgctgaaa gaagcggaac acgttgaaag ccagagcgcg
     3841 gaaaccgtgc tgaccccgga tgaatgccaa ctgctgggct atctggataa aggcaaacgc
     3901 aaacgcaaag aaaaagcggg cagcctgcaa tgggcgtata tggcgattgc gcgtctgggc
     3961 ggctttatgg atagcaaacg taccggcatt gcgagctggg gtgcgctgtg ggaaggttgg
     4021 gaagcgctgc aaagcaaact ggatggcttt ctggccgcga aagacctgat ggcgcagggc
     4081 attaaaatct aatcggcgat cgctagtagc ccgcctaatg agcgggcttt tttttaattc
     4141 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4201 ctgataaatg cttcaataat attgaaaaag gaagagtatg agcattcagc attttcgtgt
     4261 ggcgctgatt ccgttttttg cggcgttttg cctgccggtg tttgcgcatc cggaaaccct
     4321 ggtgaaagtg aaagatgcgg aagatcaact gggtgcgcgc gtgggctata ttgaactgga
     4381 tctgaacagc ggcaaaattc tggaatcttt tcgtccggaa gaacgttttc cgatgatgag
     4441 cacctttaaa gtgctgctgt gcggtgcggt tctgagccgt gtggatgcgg gccaggaaca
     4501 actgggccgt cgtattcatt atagccagaa cgatctggtg gaatatagcc cggtgaccga
     4561 aaaacatctg accgatggca tgaccgtgcg tgaactgtgc agcgcggcga ttaccatgag
     4621 cgataacacc gcggcgaacc tgctgctgac gaccattggc ggtccgaaag aactgaccgc
     4681 gtttctgcat aacatgggcg atcatgtgac ccgtctggat cgttgggaac cggaactgaa
     4741 cgaagcgatt ccgaacgatg aacgtgatac caccatgccg gcagcaatgg cgaccaccct
     4801 gcgtaaactg ctgacgggtg agctgctgac cctggcaagc cgccagcaac tgattgattg
     4861 gatggaagcg gataaagtgg cgggtccgct gctgcgtagc gcgctgccgg ctggctggtt
     4921 tattgcggat aaaagcggtg cgggcgaacg tggcagccgt ggcattattg cggcgctggg
     4981 cccggatggt aaaccgagcc gtattgtggt gatttatacc accggcagcc aggcgacgat
     5041 ggatgaacgt aaccgtcaga ttgcggaaat tggcgcgagc ctgattaaac attggtaaac
     5101 cgatacaatt aaaggctcct tttggagcct ttttttttgg acacgcgttt gtccttttcc
     5161 gctgcataac cctgcttcgg ggtcattata gcgatttttt cggtatatcc atcctttttc
     5221 gcacgatata caggattttg ccaaagggtt cgtgtagact ttccttggtg tatccaacgg
     5281 cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga gcgggtgttc cttcttcact
     5341 gtcccttatt cgcacctggc ggtgctcaac gggaatcctg ctctgcgagg ctggccgtag
     5401 gccggccgat ctgaagatca gcagttcaac ctgttgatag tacgtactaa gctctcatgt
     5461 ttcacgtact aagctctcat gtttaacgta ctaagctctc atgtttaacg aactaaaccc
     5521 tcatggctaa cgtactaagc tctcatggct aacgtactaa gctctcatgt ttcacgtact
     5581 aagctctcat gtttgaacaa taaaattaat ataaatcagc aacttaaata gcctctaagg
     5641 ttttaagttt tataagaaaa aaaagaatat ataaggcttt taaagccttt aaggtttaac
     5701 ggttgtggac aacaagccag ggatgtaacg cactgagaag cccttagagc ctctcaaagc
     5761 aattttgagt gacacaggaa cacttaacgg ctgacatggg gcgcgcccag ctgtctaggg
     5821 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
     5881 ccagtctttc gactgagcct ttcgttttat ttgatgcctt taattaactg tctcttatac
     5941 acatctttgt
//
