LOCUS       Exported                4531 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pwFRT-pheS-gat, complete sequence.
ACCESSION   GU074523
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4531)
  AUTHORS   Kang Y, Norris MH, Hoang TT.
  TITLE     Knock-out and pull-out recombineering protocols for natural 
            transformable Burkholderia thailandensis and Burkholderia 
            pseudomallei
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4531)
  AUTHORS   Kang Y, Norris MH, Hoang TT.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-OCT-2009) Molecular Biosciences and Biological 
            Engineering, University of Hawaii at Manoa, 2538 McCarthy Mall, 
            Snyder Hall 207, Honolulu, HI 96822, USA
REFERENCE   3  (bases 1 to 4531)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4531
                     /organism="Cloning vector pwFRT-pheS-gat"
                     /mol_type="other DNA"
                     /db_xref="taxon:908466"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             201..1061
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /protein_id="ADO63835.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            201..1061
                     /gene="bla"
                     /label=bla
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      1266..1884
                     /label=ColE1 origin of replication
                     /note="ColE1 origin of replication"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2144..2174
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2289..2356
                     /note="wild type FRT; Flp recognition target"
     protein_bind    complement(2306..2353)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     regulatory      2393..2434
                     /regulatory_class="promoter"
                     /note="PCS12; rpsL promoter from Burkholderia cenocepacia"
     RBS             2445..2467
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2478..3491
                     /codon_start=1
                     /gene="pheS"
                     /product="mutant phenylalanine tRNA synthetase alpha 
                     subunit"
                     /label=pheS
                     /note="incorporates phenylalanine and chlorinated 
                     phenylalanine on tRNA"
                     /protein_id="ADO63836.1"
                     /translation="MDLDQIVADAQQSFEGAADITTLENEKARFLGKSGALTELLKGLG
                     KLDPETRKTEGARINVAKQQVEAALNARRQALADALLNQRLAAEAIDVTLPGRGAGAGS
                     LHPVMRTWERVEQIFRSIGFDVADGPEIETDWYNFTALNSPENHPARSMQDTFYVDGKD
                     ADGRPLLLRTHTSPMQVRYARMNRPPIKVIAPGRTYRVDSDATHSPMFNQVEGLWIDEN
                     VSFADLKGAYTDFLKKFFERDDILVRFRPSYFPFTEPSAEIDMMFEHGKNAGKWLEISG
                     SGQVHPTVIRNMGLDPERYIGFGFGSGLERLTMLRYGVQDLRLFFENDLRFLRQFA"
     gene            2478..3491
                     /gene="pheS"
                     /label=pheS
     CDS             3527..3967
                     /codon_start=1
                     /gene="gat"
                     /product="glyphosate acetyl transferase"
                     /label=gat
                     /note="confers resistance to glyphosate"
                     /protein_id="ADO63837.1"
                     /translation="MIEVKPINAEDTYDLRHRVLRPNQPIEACMFESDLTRSAFHLGGF
                     YGGKLISVASFHQAEHSELQGKKQYQLRGVATLEGYREQKAGSSLVKHAEEILRKRGAD
                     MIWCNARTSASGYYRKLGFSEQGEVFDTPPVGPHILMYKRIT"
     gene            3527..3967
                     /gene="gat"
                     /label=gat
     misc_feature    4006..4079
                     /note="wild type FRT; Flp recognition target"
     protein_bind    complement(4023..4070)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     complement(4137..4153)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"