LOCUS       Exported                7354 bp ds-DNA    circular SYN 01-1-2014
DEFINITION  Plasmid for transiently converting any E. coli strain to a RecA+ 
            phenotype for phage P1-mediated transduction.
ACCESSION   .
VERSION     .
KEYWORDS    pInvRecA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7354)
  AUTHORS   Sektas M, Gregorowicz M, Szybalski W.
  TITLE     Transient conversion to RecA+ phenotype to permit P1 transduction in
            any Escherichia coli recA- strains.
  JOURNAL   BioTechniques 1999;27:911-4.
  PUBMED    10572635
REFERENCE   2  (bases 1 to 7354)
  AUTHORS   Lucigen
  TITLE     Direct Submission
COMMENT     Addition of heated chlortetracycline induces synthesis of Flp 
            recombinase, thereby inverting the recA gene so that it is 
            expressed.
            
            Growth at 42(o)C inactivates the temperature-sensitive mutant Rep101
            protein, causing loss of the plasmid.
FEATURES             Location/Qualifiers
     source          1..7354
                     /organism="synthetic DNA construct"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
     CDS             complement(69..692)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /note="TetR"
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /protein_id="
                     "
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     promoter        711..766
                     /gene="tetR"
                     /note="tetR/tetA promoters"
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    717..735
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    747..765
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             783..2051
                     /codon_start=1
                     /product="site-specific recombinase"
                     /note="FLP"
                     /note="FLP is a site-specific recombinase from 
                     Saccharomyces cerevisiae. Recombination occurs at FRT 
                     sequences."
                     /protein_id="
                     "
                     /translation="PQFGILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMIT
                     HNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTII
                     PYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNS
                     FEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETK
                     TSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRS
                     YNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVART
                     TYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRY
                     PAWNGIISQEVLDYLSSYINRRI"
     protein_bind    complement(2251..2284)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FRT"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence (TTTCTAGA)."
     CDS             complement(2311..3372)
                     /codon_start=1
                     /gene="recA"
                     /product="DNA repair protein from E. coli"
                     /note="RecA"
                     /translation="MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGS
                     LSLDIALGAGGLPMGRIVEIYGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIY
                     ARKLGVDIDNLLCSQPDTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIGDS
                     HMGLAARMMSQAMRKLAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASV
                     RLDIRRIGAVKEGENVVGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVK
                     EKLIEKAGAWYSYKGEKIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSV
                     DDSEGVAETNEDF"
     protein_bind    3425..3458
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FRT"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence (TTTCTAGA)."
     terminator      3562..3648
                     /gene="Escherichia coli rrnB"
                     /note="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3743..3829
                     /gene="Escherichia coli rrnB"
                     /note="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3924..4010
                     /gene="Escherichia coli rrnB"
                     /note="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4105..4191
                     /gene="Escherichia coli rrnB"
                     /note="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4516..5466)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of a protein needed
                     for replication with the pSC101 origin"
                     /note="Rep101(Ts)"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      5514..5736
                     /note="pSC101 ori"
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(6358..7218)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7219..7323)
                     /gene="bla"
                     /note="AmpR promoter"