LOCUS       Exported                9988 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pKLMF-FX, complete sequence.
ACCESSION   FJ010197
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9988)
  AUTHORS   Ganatra MB, Read JD, Colussi PA, Taron CH.
  TITLE     Vectors for intracellular expression of maltose binding protein 
            fusions in the yeast Kluyveromyces lactis
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9988)
  AUTHORS   Ganatra MB, Read JD, Colussi PA, Taron CH.
  TITLE     Direct Submission
  JOURNAL   Submitted (11-AUG-2008) Gene Expression Division, New England 
            Biolabs, 240 County Road, Ipswich, MA 01938, USA
REFERENCE   3  (bases 1 to 9988)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9988
                     /organism="Expression vector pKLMF-FX"
                     /mol_type="other DNA"
                     /db_xref="taxon:568094"
     CDS             13..1137
                     /codon_start=1
                     /gene="malE"
                     /product="maltose binding protein"
                     /label=malE
                     /note="confers binding of expressed fusion proteins to 
                     amylose chromatography resin"
                     /protein_id="ACI46135.1"
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQTNSSSIEGR"
     gene            13..1137
                     /gene="malE"
                     /label=malE
     CDS             13..1113
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     misc_feature    1114..1137
                     /gene="malE"
                     /note="spacer peptide containing a factor X (IEGR) in frame
                     with the maltose binding protein"
     CDS             1126..1137
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    1138..1234
                     /label=multiple cloning site
                     /note="multiple cloning site"
     regulatory      1251..1834
                     /regulatory_class="terminator"
                     /note="Kluyveromyces lactis LAC4 transcription terminator"
     terminator      1252..1870
                     /label=LAC4 terminator
                     /note="transcription terminator for Kluyveromyces lactis 
                     LAC4"
     regulatory      1890..2593
                     /regulatory_class="promoter"
                     /note="Saccharomyces cerevisiae ADH1 promoter"
     CDS             2594..4240
                     /codon_start=1
                     /gene="amdS"
                     /product="acetamidase"
                     /label=amdS
                     /note="confers ability to process acetamide for selection 
                     in Kluyveromyces lactis"
                     /protein_id="ACI46136.1"
                     /translation="MPQSWEELAADKRARLAKTIPDEWKVQTLPAEDSVIDFPKKSGIL
                     SEAELKITEASAADLVSKLAAGELTSVEVTLAFCKRAAIAQQLTNCAHEFFPDAALAQA
                     RELDEYYAKHKRPVGPLHGLPISLKDQLRVKGYETSMGYISWLNKYDEGDSVLTTMLRK
                     AGAVFYVKTSVPQTLMVCETVNNIIGRTVNPRNKNWSCGGSSGGEGAIVGIRGGVIGVG
                     TDIGGSIRVPAAFNFLYGLRPSHGRLPYAKMANSMEGQETVHSVVGPITHSVEDLRLFT
                     KSVLGQEPWKYDSKVIPMPWRQSESDIIASKIKNGGLNIGYYNFDGNVLPHPPILRGVE
                     TTVAALAKAGHTVTPWTPYKHDFGHDLISHIYAADGSADVMRDISASGEPAIPNIKDLL
                     NPNIKAVNMNELWDTHLQKWNYQMEYLEKWREAEEKAGKELDAIIAPITPTAAVRHDQF
                     RYYGYASVINLLDFTSVVVPVTFADKNIDKKNESFKAVSELDALVQEEYDPEAYHGAPV
                     AVQVIGRRLSEERTLAIAEEVGKLLGNVVTP"
     gene            2594..4240
                     /gene="amdS"
                     /label=amdS
     promoter        4876..5532
                     /label=LAC4 promoter (5)
                     /note="5 portion of the Kluyveromyces lactis LAC4 
                     promoter"
     regulatory      4948..5528
                     /regulatory_class="promoter"
                     /note="modified Kluyveromyces lactis LAC4 promoter 5 end"
     promoter        complement(5549..5567)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5581..5597)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5605..5621
                     /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)."
     promoter        complement(5629..5659)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5674..5695
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5983..6571)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6742..7602)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /note="confers ampicillin resistance in Escherichia coli"
                     /protein_id="ACI46137.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(6742..7602)
                     /gene="bla"
                     /label=bla
     promoter        complement(7603..7707)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7734..8189
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     8330..8346
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      8356..9988
                     /regulatory_class="promoter"
                     /note="modified Kluyveromyces lactis LAC4 promoter 3 end"
     promoter        8360..9888
                     /label=LAC4 promoter (3)
                     /note="3 portion of a modified Kluyveromyces lactis LAC4 
                     promoter"
     protein_bind    9894..9910
                     /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)."