LOCUS       sequence-115886-        5813 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Conjugally transferable Flp recombinase plasmid for unmarked 
            mutagenesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5813)
  AUTHORS   Hossain MJ, Thurlow CM, Sun D, Nasrin S, Liles MR
  TITLE     Genome modifications and cloning using a conjugally transferable 
            recombineering system.
  JOURNAL   Biotechnol Rep (Amst). 2015 Aug 28;8:24-35. doi: 
            10.1016/j.btre.2015.08.005. eCollection 2015 Dec.
  PUBMED    28352570
REFERENCE   2  (bases 1 to 5813)
  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: "Biotechnol 
            Rep (Amst)."; date: "2015-08-28"; volume: "8:24-35. doi"; pages: " 
            10.1016/j.btre.2015.08.005. eCollection 2015 Dec"
COMMENT     Sequence Label: pCMT-flp
FEATURES             Location/Qualifiers
     source          1..5813
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      48..79
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     rep_origin      641..863
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             911..1861
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     terminator      complement(2457..2486)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     terminator      complement(2584..2631)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2627..2645
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2660..3928)
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=FLP
                     /note="FLP is a site-specific recombinase from 
                     Saccharomyces cerevisiae. Recombination occurs at FRT 
                     sequences."
                     /translation="PQFGILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMIT
                     HNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTII
                     PYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNS
                     FEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETK
                     TSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRS
                     YNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVART
                     TYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRY
                     PAWNGIISQEVLDYLSSYINRRI"
     CDS             complement(3929..4216)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated and modified)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="modified to prevent cleavage by eukaryotic SUMO 
                     proteases"
                     /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
                     LMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(4229..4246)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        join(4340..4345,4346..4362,4363..4368)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             4416..5606
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(4768..4787)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tcgctcaata ctgaccattt aaatcatacc tgacctccat agcagaaagt caaaagcctc
       61 cgaccggagg cttttgactt gatcggcact aggtgaagta ggcccacccg cgagcgggtg
      121 ttccttcttc actgtccctt attcgttcca ctgagcgtca gaccccgttg atgataccgc
      181 tgccttactg ggtgcattag ccagtctgaa tgacctgtca cgggataatc cgaagtggtc
      241 agactggaaa atcagagggc aggaactgct gaacagcaaa aagtcagata gcaccacata
      301 gcagacccgc cataaaacgc cctgagaagc ccgtgacggg cttttcttgt attatgggta
      361 gtttccttgc atgaatccat aaaaggcgcc tgtagtgcca tttaccccca ttcactgcca
      421 gagccgtgag cgcagcgaac tgaatgtcac gaaaaagaca gcgactcagg tgcctgatgg
      481 tcggagacaa aaggaatatt cagcgatttg cccgattgcg gccgcaaccg agcttgcgag
      541 ggtgctactt aagcctttag ggttttaagg tctgttttgt agaggagcaa acagcgtttg
      601 cgacatcctt ttgtaatact gcggaactga ctaaagtagt gagttataca cagggctggg
      661 atctattctt tttatctttt tttattcttt ctttattcta taaattataa ccacttgaat
      721 ataaacaaaa aaaacacaca aaggtctagc ggaatttaca gagggtctag cagaatttac
      781 aagttttcca gcaaaggtct agcagaattt acagataccc acaactcaaa ggaaaaggac
      841 tagtaattat cattgactag cccatctcaa ttggtatagt gattaaaatc acctagacca
      901 attgagatgt atgtctgaat tagttgtttt caaagcaaat gaactagcga ttagtcgcta
      961 tgacttaacg gagcatgaaa ccaagctaat tttatgctgt gtggcactac tcaaccccac
     1021 gattgaaaac cctacaagga aagaacggac ggtatcgttc acttataacc aatacgttca
     1081 gatgatgaac atcagtaggg aaaatgctta tggtgtatta gctaaagcaa ccagagagct
     1141 gatgacgaga actgtggaaa tcaggaatcc tttggttaaa ggctttgaga ttttccagtg
     1201 gacaaactat gccaagttct caagcgaaaa attagaatta gtttttagtg aagagatatt
     1261 gccttatctt ttccagttaa aaaaattcat aaaatataat ctggaacatg ttaagtcttt
     1321 tgaaaacaaa tactctatga ggatttatga gtggttatta aaagaactaa cacaaaagaa
     1381 aactcacaag gcaaatatag agattagcct tgatgaattt aagttcatgt taatgcttga
     1441 aaataactac catgagttta aaaggcttaa ccaatgggtt ttgaaaccaa taagtaaaga
     1501 tttaaacact tacagcaata tgaaattggt ggttgataag cgaggccgcc cgactgatac
     1561 gttgattttc caagttgaac tagatagaca aatggatctc gtaaccgaac ttgagaacaa
     1621 ccagataaaa atgaatggtg acaaaatacc aacaaccatt acatcagatt cctacctaca
     1681 taacggacta agaaaaacac tacacgatgc tttaactgca aaaattcagc tcaccagttt
     1741 tgaggcaaaa tttttgagtg acatgcaaag taagtatgat ctcaatggtt cgttctcatg
     1801 gctcacgcaa aaacaacgaa ccacactaga gaacatactg gctaaatacg gaaggatctg
     1861 aggttcttat ggctcttgta tctatcagtg aagcatcaag actaacaaac aaaagtagaa
     1921 caactgttca ccgttacata tcaaagggaa aactgtccat acccatgatc taatgaatcg
     1981 gccaacgcga accccttgcg gccgcccggg ccgtcgacca attctcatgt ttgacagctt
     2041 atcatcgaat ttctgccatt catccgctta ttatcactta ttcaggcgta gcaaccaggc
     2101 gtttaagggc accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag
     2161 tactgttgta attcattaag cattctgccg acatggaagc catctggtaa caggattagc
     2221 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2281 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctc ggaaaaagag
     2341 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     2401 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatt ttctaccgaa
     2461 gaaaggccca cccgtgaagg tgagccagtg agttgattgc agtccagtta cgctggagtc
     2521 tgaggctcgt cctgaatgat atcaagcttg aattcgttac ccggatatag ttcctccttt
     2581 cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta gttattgctc
     2641 agcggtggcg gccgctctat tatatgcgtc tatttatgta ggatgaaagg tagtctagta
     2701 cctcctgtga tattatccca ttccatgcgg ggtatcgtat gcttccttca gcactaccct
     2761 ttagctgttc tatatgctgc cactcctcaa ttggattagt ctcatccttc aatgctatca
     2821 tttcctttga tattggatca tatgcatagt accgagaaac tagtgcgaag tagtgatcag
     2881 gtattgctgt tatctgatga gtatacgttg tcctggccac ggcagaagca cgcttatcgc
     2941 tccaatttcc cacaacatta gtcaactccg ttaggccctt cattgaaaga aatgaggtca
     3001 tcaaatgtct tccaatgtga gattttgggc cattttttat agcaaagatt gaataaggcg
     3061 catttttctt caaagcttta ttgtacgatc tgactaagtt atcttttaat aattggtatt
     3121 cctgtttatt gcttgaagaa ttgccggtcc tatttactcg ttttaggact ggttcagaat
     3181 tcctcaaaaa ttcatccaaa tatacaagtg gatcgatcct accccttgcg ctaaagaagt
     3241 atatgtgcct actaacgctt gtctttgtct ctgtcactaa acactggatt attactccca
     3301 gatacttatt ttggactaat ttaaatgatt tcggatcaac gttcttaata tcgctgaatc
     3361 ttccacaatt gatgaaagta gctaggaaga ggaattggta taaagttttt gtttttgtaa
     3421 atctcgaagt atactcaaac gaatttagta ttttctcagt gatctcccag atgctttcac
     3481 cctcacttag aagtgcttta agcatttttt tactgtggct atttccctta tctgcttctt
     3541 ccgatgattc gaactgtaat tgcaaactac ttacaatatc agtgatatca gattgatgtt
     3601 tttgtccata gtaaggaata attgtaaatt cccaagcagg aatcaatttc tttaatgagg
     3661 cttccagaat tgttgctttt tgcgtcttgt atttaaactg gagtgattta ttgacaatat
     3721 cgaaactcag cgaattgctt atgatagtat tatagctcat gaatgtggct ctcttgattg
     3781 ctgttccgtt atgtgtaatc atccaacata aataggttag ttcagcagca cataatgcta
     3841 ttttctcacc tgaaggtctt tcaaaccttt ccacaaactg acgaacaagc accttaggtg
     3901 gtgttttaca taatatacca aattgtggac ctccaatctg ttcgcggtga gcctcaataa
     3961 tatcgttatc ctccatgtcc aaatcttcag gggtctgatc agcttgaatt tcaataccgt
     4021 cgtacaagaa cgttaaggag tccatttcct taccctgtct tttagcgaac gcttccatca
     4081 gccttcttaa aggagtggtc tttttgatct tgaagaagat ctctgaagat ccatcggaca
     4141 cctttaaatt gatgtgagtc tcaggcttga cttctggctt gacctctggc ttagcttctt
     4201 gattgacttc tgagtcctgc agggacccgt gatggtggtg atgatgcata tgtatatctc
     4261 cttcttatag ttaaacaaaa ttatttctag tgtagcacct gaagtcagcc ccatacgata
     4321 taagttgtaa ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat
     4381 cacagttaaa ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg
     4441 tcatcctcgg caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc
     4501 cgggcctctt gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc
     4561 tagcgctata tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc
     4621 gctttggccg ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga
     4681 tcatggcgac cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg gccggcatca
     4741 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     4801 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     4861 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     4921 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     4981 gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     5041 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     5101 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     5161 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     5221 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc
     5281 tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc
     5341 ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag
     5401 atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga
     5461 tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt
     5521 tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg
     5581 catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac
     5641 cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc aacccaaccc
     5701 ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg
     5761 cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgctag ata
//
