LOCUS       Exported               10526 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector LIC-pKLAC-LC2, complete sequence.
ACCESSION   JF327849
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10526)
  AUTHORS   Dortay H, Akula UM, Westphal C, Sittig M, Mueller-Roeber B.
  TITLE     High-Throughput Protein Expression Using a Combination of 
            Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein 
            (IFP) Detection
  JOURNAL   PLoS ONE 6 (4), E18900 (2011)
  PUBMED    21541323
REFERENCE   2  (bases 1 to 10526)
  AUTHORS   Dortay H, Mueller-Roeber B.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-FEB-2011) Molecular Biology, University of Potsdam, 
            Germany, Karl-Liebknecht-Str. 24-25, Haus 20, Potsdam, Brandenburg 
            14476, Germany
REFERENCE   3  (bases 1 to 10526)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10526
                     /organism="Expression vector LIC-pKLAC-LC2"
                     /mol_type="other DNA"
                     /label=Kluyveromyces lactis expression vector
                     /note="Kluyveromyces lactis expression vector"
                     /db_xref="taxon:1029640"
     misc_feature    14..16
                     /label=start codon
                     /note="start codon"
     misc_feature    26..33
                     /label=PmeI recognition site
                     /note="PmeI recognition site"
     misc_feature    34..700
                     /label=LIC stuffer fragment
                     /note="LIC stuffer fragment"
     misc_feature    701..708
                     /label=PmeI recognition site
                     /note="PmeI recognition site"
     misc_feature    714..734
                     /label=TEV recognition site
                     /note="TEV recognition site"
     CDS             714..734
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     misc_feature    744..1727
                     /label=infrared fluorescent protein
                     /note="infrared fluorescent protein"
     CDS             744..1727
                     /codon_start=1
                     /product="bacteriophytochrome-based monomeric infrared 
                     fluorescent protein (Shu et al., 2009)"
                     /label=IFP1.4
                     /translation="MARDPLPFFPPLYLGGPEITTENCEREPIHIPGSIQPHGALLTAD
                     GHSGEVLQVSLNAATFLGQEPTVLRGQTLAALLPEQWPALQAALPPGCPDALQYRATLD
                     WPAAGHLSLTVHRVAELLILEFEPTEAWDSIGPHALRNAMFALESAPNLRALAEVATQT
                     VRELTGFDRVMLYKFAPDATGEMIAEARREGMQAFLGHRFPASHTPAQARALYTRHLLR
                     LTADTRAAAVPLDPVLNPQTNAPTPLGGAVLRATSPMHMQYLRNMGVGSSLSVSVVVGG
                     QLWGLIVCHHQTPYVLPPDLRTTLEELGRKLSGQVQRKEAGMDELYK"
     misc_feature    1734..1754
                     /label=6xHis tag
                     /note="6xHis tag"
     CDS             1734..1751
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    1752..1757
                     /label=stop codon (TAA TAG)
                     /note="stop codon (TAA TAG)"
     terminator      1790..2408
                     /label=LAC4 terminator
                     /note="transcription terminator for Kluyveromyces lactis 
                     LAC4"
     regulatory      1790..2372
                     /regulatory_class="terminator"
                     /note="terminator"
     CDS             3132..4778
                     /codon_start=1
                     /product="acetamidase"
                     /label=acetamidase
                     /protein_id="AEE81074.1"
                     /translation="MPQSWEELAADKRARLAKTIPDEWKVQTLPAEDSVIDFPKKSGIL
                     SEAELKITEASAADLVSKLAAGELTSVEVTLAFCKRAAIAQQLTNCAHEFFPDAALAQA
                     RELDEYYAKHKRPVGPLHGLPISLKDQLRVKGYETSMGYISWLNKYDEGDSVLTTMLRK
                     AGAVFYVKTSVPQTLMVCETVNNIIGRTVNPRNKNWSCGGSSGGEGAIVGIRGGVIGVG
                     TDIGGSIRVPAAFNFLYGLRPSHGRLPYAKMANSMEGQETVHSVVGPITHSVEDLRLFT
                     KSVLGQEPWKYDSKVIPMPWRQSESDIIASKIKNGGLNIGYYNFDGNVLPHPPILRGVE
                     TTVAALAKAGHTVTPWTPYKHDFGHDLISHIYAADGSADVMRDISASGEPAIPNIKDLL
                     NPNIKAVNMNELWDTHLQKWNYQMEYLEKWREAEEKAGKELDAIIAPITPTAAVRHDQF
                     RYYGYASVINLLDFTSVVVPVTFADKNIDKKNESFKAVSELDALVQEEYDPEAYHGAPV
                     AVQVIGRRLSEERTLAIAEEVGKLLGNVVTP"
     promoter        5414..6070
                     /label=LAC4 promoter (5)
                     /note="5 portion of the Kluyveromyces lactis LAC4 
                     promoter"
     regulatory      5487..6067
                     /regulatory_class="promoter"
                     /note="5 promoter region"
     promoter        complement(6087..6105)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6119..6135)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6143..6159
                     /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(6167..6197)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6212..6233
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      6521..7109
                     /label=pMB1 origin
                     /note="pMB1 origin"
     CDS             complement(7280..8140)
                     /codon_start=1
                     /gene="ampR"
                     /product="beta-lactamase"
                     /label=ampR
                     /protein_id="AEE81075.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(7280..8140)
                     /gene="ampR"
                     /label=ampR
     promoter        complement(8141..8245)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      8272..8727
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     8868..8884
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      8894..10526
                     /regulatory_class="promoter"
                     /note="3 promoter region"
     promoter        8898..10426
                     /label=LAC4 promoter (3)
                     /note="3 portion of a modified Kluyveromyces lactis LAC4 
                     promoter"
     protein_bind    10432..10448
                     /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)."