LOCUS       Exported                9107 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pKLAC2, complete sequence.
ACCESSION   EU196354
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9107)
  AUTHORS   Colussi PA, Read JD, Taron CH.
  TITLE     An integrative expression vector for the yeast Kluyveromyces lactis
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9107)
  AUTHORS   Colussi PA, Read JD, Taron CH.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-OCT-2007) Gene Expression Division, New England 
            Biolabs, 240 County Road, Ipswich, MA 01938, USA
REFERENCE   3  (bases 1 to 9107)
  AUTHORS   Colussi PA, Read JD, Taron CH.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-AUG-2008) Gene Expression Division, New England 
            Biolabs, 240 County Road, Ipswich, MA 01938, USA
REFERENCE   4  (bases 1 to 9107)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     On Aug 14, 2008 this sequence version replaced EU196354.1.
FEATURES             Location/Qualifiers
     source          1..9107
                     /organism="Expression vector pKLAC2"
                     /mol_type="other DNA"
                     /db_xref="taxon:478548"
     misc_feature    14..349
                     /note="Kluyveromyces lactis alpha mating factor pre-pro 
                     secretion leader"
     misc_feature    257..354
                     /label=multiple cloning site
                     /note="multiple cloning site"
     terminator      371..989
                     /label=LAC4 terminator
                     /note="transcription terminator for Kluyveromyces lactis 
                     LAC4"
     regulatory      371..953
                     /regulatory_class="terminator"
                     /note="Kluyveromyces lactis LAC4 transcription terminator"
     regulatory      1010..1712
                     /regulatory_class="promoter"
                     /note="Saccharomyces cerevisiae ADH1 promoter"
     CDS             1713..3359
                     /codon_start=1
                     /gene="amdS"
                     /product="acetamidase"
                     /label=amdS
                     /note="confers ability to process acetamide for selection 
                     in Kluyveromyces lactis"
                     /protein_id="ABW72883.1"
                     /translation="MPQSWEELAADKRARLAKTIPDEWKVQTLPAEDSVIDFPKKSGIL
                     SEAELKITEASAADLVSKLAAGELTSVEVTLAFCKRAAIAQQLTNCAHEFFPDAALAQA
                     RELDEYYAKHKRPVGPLHGLPISLKDQLRVKGYETSMGYISWLNKYDEGDSVLTTMLRK
                     AGAVFYVKTSVPQTLMVCETVNNIIGRTVNPRNKNWSCGGSSGGEGAIVGIRGGVIGVG
                     TDIGGSIRVPAAFNFLYGLRPSHGRLPYAKMANSMEGQETVHSVVGPITHSVEDLRLFT
                     KSVLGQEPWKYDSKVIPMPWRQSESDIIASKIKNGGLNIGYYNFDGNVLPHPPILRGVE
                     TTVAALAKAGHTVTPWTPYKHDFGHDLISHIYAADGSADVMRDISASGEPAIPNIKDLL
                     NPNIKAVNMNELWDTHLQKWNYQMEYLEKWREAEEKAGKELDAIIAPITPTAAVRHDQF
                     RYYGYASVINLLDFTSVVVPVTFADKNIDKKNESFKAVSELDALVQEEYDPEAYHGAPV
                     AVQVIGRRLSEERTLAIAEEVGKLLGNVVTP"
     gene            1713..3359
                     /gene="amdS"
                     /label=amdS
     promoter        3995..4651
                     /label=LAC4 promoter (5)
                     /note="5 portion of the Kluyveromyces lactis LAC4 
                     promoter"
     regulatory      4068..4648
                     /regulatory_class="promoter"
                     /note="modified Kluyveromyces lactis LAC4 promoter 5 end"
     promoter        complement(4668..4686)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4700..4716)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4724..4740
                     /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(4748..4778)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4793..4814
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      5102..5690
                     /label=Escherichia coli pMB1 origin of replication
                     /note="Escherichia coli pMB1 origin of replication"
     CDS             complement(5861..6721)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /note="confers ampicillin resistance in Escherichia coli"
                     /protein_id="ABW72884.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(5861..6721)
                     /gene="bla"
                     /label=bla
     promoter        complement(6722..6826)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6853..7308
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     7449..7465
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      7475..9107
                     /regulatory_class="promoter"
                     /note="modified Kluyveromyces lactis LAC4 promoter 3 end"
     promoter        7479..9007
                     /label=LAC4 promoter (3)
                     /note="3 portion of a modified Kluyveromyces lactis LAC4 
                     promoter"
     protein_bind    9013..9029
                     /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)."