LOCUS       sequence-458896-        5600 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Integration of msfGFP (or your Module Of Interest) in a random 
            fashion via Tn5 insertion. The antibiotic cassette (Km) 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 5600)
  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 5600)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-2
FEATURES             Location/Qualifiers
     source          1..5600
                     /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)"
     primer_bind     complement(1150..1169)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1161..1976
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     protein_bind    2073..2110
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_feature    2116..2134
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     terminator      2145..2239
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2344..3771
                     /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        3828..3919
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3920..3988,3989..4780)
                     /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      4784..4823
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            4971..5079
                     /note="incP origin of transfer"
     rep_origin      5090..5478
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(5509..5595)
                     /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 aatttaaatt tgtgtctcaa aatctctgat
     1081 gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa
     1141 acagtaatac aaggggtgtt atgagccata ttcagcgtga aacgagctgt agccgtccgc
     1201 gtctgaacag caacatggat gcggatctgt atggctataa atgggcgcgt gataacgtgg
     1261 gtcagagcgg cgcgaccatt tatcgtctgt atggcaaacc ggatgcgccg gaactgtttc
     1321 tgaaacatgg caaaggcagc gtggcgaacg atgtgaccga tgaaatggtg cgtctgaact
     1381 ggctgaccga atttatgccg ctgccgacca ttaaacattt tattcgcacc ccggatgatg
     1441 cgtggctgct gaccaccgcg attccgggca aaaccgcgtt tcaggtgctg gaagaatatc
     1501 cggatagcgg cgaaaacatt gtggatgcgc tggccgtgtt tctgcgtcgt ctgcatagca
     1561 ttccggtgtg caactgcccg tttaacagcg atcgtgtgtt tcgtctggcc caggcgcaga
     1621 gccgtatgaa caacggcctg gtggatgcga gcgattttga tgatgaacgt aacggctggc
     1681 cggtggaaca ggtgtggaaa gaaatgcata aactgctgcc gtttagcccg gatagcgtgg
     1741 tgacccacgg cgattttagc ctggataacc tgattttcga tgaaggcaaa ctgattggct
     1801 gcattgatgt gggccgtgtg ggcattgcgg atcgttatca ggatctggcc attctgtgga
     1861 actgcctggg cgaatttagc ccgagcctgc aaaaacgtct gtttcagaaa tatggcattg
     1921 ataatccgga tatgaacaaa ctgcaatttc atctgatgct ggatgaattt ttctaagacc
     1981 cttgtctaat taattgcgga ccctagaggt cccctttttt attttaaaaa ttttttcaca
     2041 aaacggttta caagcataaa atctctgagg ccggcttgtc gacgacggcg gtctccgtcg
     2101 tcaggatcat ccgggagatg tgtataagag acagactagt cttggactcc tgttgataga
     2161 tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt gccgccgggc
     2221 gttttttatt ggtgagaatc caggggtccc ctggtttaaa ctacacaagt agcgtcctga
     2281 acggaacctt tcccgttttc cagaatctga tgttccatgt gacctcctaa catggtaacg
     2341 ttcatgatta ccagtgcact gcatcgtgcg gcggattggg cgaaaagcgt gttttctagt
     2401 gctgcgctgg gtgatccgcg tcgtaccgcg cgtctggtga atgttgcggc gcaactggcc
     2461 aaatatagcg gcaaaagcat taccattagc agcgaaggca gcaaagccat gcaggaaggc
     2521 gcgtatcgtt ttattcgtaa tccgaacgtg agcgcggaag cgattcgtaa agcgggtgcc
     2581 atgcagaccg tgaaactggc ccaggaattt ccggaactgc tggcaattga agataccacc
     2641 tctctgagct atcgtcatca ggtggcggaa gaactgggca aactgggtag cattcaggat
     2701 aaaagccgtg gttggtgggt gcatagcgtg ctgctgctgg aagcgaccac ctttcgtacc
     2761 gtgggcctgc tgcatcaaga atggtggatg cgtccggatg atccggcgga tgcggatgaa
     2821 aaagaaagcg gcaaatggct ggccgctgct gcaacttcgc gtctgagaat gggcagcatg
     2881 atgagcaacg tgattgcggt gtgcgatcgt gaagcggata ttcatgcgta tctgcaagat
     2941 aaactggccc ataacgaacg ttttgtggtg cgtagcaaac atccgcgtaa agatgtggaa
     3001 agcggcctgt atctgtatga tcacctgaaa aaccagccgg aactgggcgg ctatcagatt
     3061 agcattccgc agaaaggcgt ggtggataaa cgtggcaaac gtaaaaaccg tccggcgcgt
     3121 aaagcgagcc tgagcctgcg tagcggccgt attaccctga aacagggcaa cattaccctg
     3181 aacgcggtgc tggccgaaga aattaatccg ccgaaaggcg aaaccccgct gaaatggctg
     3241 ctgctgacca gcgagccggt ggaaagtctg gcccaagcgc tgcgtgtgat tgatatttat
     3301 acccatcgtt ggcgcattga agaatttcac aaagcgtgga aaacgggtgc gggtgcggaa
     3361 cgtcagcgta tggaagaacc ggataacctg gaacgtatgg tgagcattct gagctttgtg
     3421 gcggtgcgtc tgctgcaact gcgtgaatct tttactccgc cgcaagcact gcgtgcgcag
     3481 ggcctgctga aagaagcgga acacgttgaa agccagagcg cggaaaccgt gctgaccccg
     3541 gatgaatgcc aactgctggg ctatctggat aaaggcaaac gcaaacgcaa agaaaaagcg
     3601 ggcagcctgc aatgggcgta tatggcgatt gcgcgtctgg gcggctttat ggatagcaaa
     3661 cgtaccggca ttgcgagctg gggtgcgctg tgggaaggtt gggaagcgct gcaaagcaaa
     3721 ctggatggct ttctggccgc gaaagacctg atggcgcagg gcattaaaat ctaatcggcg
     3781 atcgctagta gcccgcctaa tgagcgggct tttttttaat tcccctattt gtttattttt
     3841 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3901 atattgaaaa aggaagagta tgagcattca gcattttcgt gtggcgctga ttccgttttt
     3961 tgcggcgttt tgcctgccgg tgtttgcgca tccggaaacc ctggtgaaag tgaaagatgc
     4021 ggaagatcaa ctgggtgcgc gcgtgggcta tattgaactg gatctgaaca gcggcaaaat
     4081 tctggaatct tttcgtccgg aagaacgttt tccgatgatg agcaccttta aagtgctgct
     4141 gtgcggtgcg gttctgagcc gtgtggatgc gggccaggaa caactgggcc gtcgtattca
     4201 ttatagccag aacgatctgg tggaatatag cccggtgacc gaaaaacatc tgaccgatgg
     4261 catgaccgtg cgtgaactgt gcagcgcggc gattaccatg agcgataaca ccgcggcgaa
     4321 cctgctgctg acgaccattg gcggtccgaa agaactgacc gcgtttctgc ataacatggg
     4381 cgatcatgtg acccgtctgg atcgttggga accggaactg aacgaagcga ttccgaacga
     4441 tgaacgtgat accaccatgc cggcagcaat ggcgaccacc ctgcgtaaac tgctgacggg
     4501 tgagctgctg accctggcaa gccgccagca actgattgat tggatggaag cggataaagt
     4561 ggcgggtccg ctgctgcgta gcgcgctgcc ggctggctgg tttattgcgg ataaaagcgg
     4621 tgcgggcgaa cgtggcagcc gtggcattat tgcggcgctg ggcccggatg gtaaaccgag
     4681 ccgtattgtg gtgatttata ccaccggcag ccaggcgacg atggatgaac gtaaccgtca
     4741 gattgcggaa attggcgcga gcctgattaa acattggtaa accgatacaa ttaaaggctc
     4801 cttttggagc cttttttttt ggacacgcgt ttgtcctttt ccgctgcata accctgcttc
     4861 ggggtcatta tagcgatttt ttcggtatat ccatcctttt tcgcacgata tacaggattt
     4921 tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc gggcaggata
     4981 ggtgaagtag gcccacccgc gagcgggtgt tccttcttca ctgtccctta ttcgcacctg
     5041 gcggtgctca acgggaatcc tgctctgcga ggctggccgt aggccggccg atctgaagat
     5101 cagcagttca acctgttgat agtacgtact aagctctcat gtttcacgta ctaagctctc
     5161 atgtttaacg tactaagctc tcatgtttaa cgaactaaac cctcatggct aacgtactaa
     5221 gctctcatgg ctaacgtact aagctctcat gtttcacgta ctaagctctc atgtttgaac
     5281 aataaaatta atataaatca gcaacttaaa tagcctctaa ggttttaagt tttataagaa
     5341 aaaaaagaat atataaggct tttaaagcct ttaaggttta acggttgtgg acaacaagcc
     5401 agggatgtaa cgcactgaga agcccttaga gcctctcaaa gcaattttga gtgacacagg
     5461 aacacttaac ggctgacatg gggcgcgccc agctgtctag ggcggcggat ttgtcctact
     5521 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc
     5581 ctttcgtttt atttgatgcc
//
