LOCUS       Exported                5813 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Conjugally transferable flp vector pCMT-flp, complete sequence.
ACCESSION   KT072898
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     Conjugal Transfer of a Recombineering System to Generate and 
            Complement Markerless Mutants
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5813)
  AUTHORS   Hossain MJ, Thurlow CM, Sun D, Nasrin S, Liles MR.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-JUN-2015) Biological Sciences, Auburn University, 120 
            W Samford Avenue, Auburn, AL 36849, USA
REFERENCE   3  (bases 1 to 5813)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: CLC Genomics Workbench v. 8.1
            Sequencing Technology :: Illumina; Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5813
                     /organism="Conjugally transferable flp vector pCMT-flp"
                     /mol_type="other DNA"
                     /note="derived from Aeromonas hydrophila ML09-119 (pMJH97) 
                     and Escherichia coli SM10lambdapir (pCMT-flp); for unmarked
                     mutagenesis"
                     /db_xref="taxon:1752035"
     terminator      48..79
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     misc_feature    90..144
                     /note="oriT; origin of transfer sequence"
     rep_origin      complement(167..907)
                     /direction=LEFT
                     /label=oriR101
                     /note="oriR101"
     rep_origin      641..863
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             911..1861
                     /codon_start=1
                     /gene="repA101"
                     /product="repA101 protein"
                     /label=repA101
                     /protein_id="ALP68863.1"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     gene            911..1861
                     /gene="repA101"
                     /label=repA101
     terminator      2457..2486
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     terminator      2584..2631
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2660..4249)
                     /codon_start=1
                     /gene="flp"
                     /product="Site-specific recombinase Flp"
                     /label=flp
                     /note="Flp tagged with N-His6 SUMO"
                     /protein_id="ALP68864.1"
                     /translation="MHHHHHHGSLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIF
                     FKIKKTTPLRRLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQ
                     IGGPQFGILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMITHNGTAIKRATF
                     MSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYYGQKHQSDI
                     TDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTK
                     TLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFS
                     ARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPY
                     SIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQITAIPD
                     HYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEV
                     LDYLSSYINRRI"
     gene            complement(2660..4249)
                     /gene="flp"
                     /label=flp
     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)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
                     LMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(4229..4246)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        4340..4368
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             4416..5606
                     /codon_start=1
                     /gene="tetA; tetracycline resistance"
                     /product="tetracycline resistance protein"
                     /label=tetA
                     /note="tetA gene of pACYC184 origin"
                     /protein_id="ALP68865.1"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     gene            4416..5606
                     /gene="tetA; tetracycline resistance"
                     /label=tetA