LIC-pKLAC-LC2 vector (V009866)

Basic Vector Information

      • Vector Name:
      • LIC-pKLAC-LC2
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 10526 bp
      • Type:
      • Expression vector
      • Source/Author:
      • Dortay H, Akula UM, Westphal C, Sittig M, Mueller-Roeber B.

LIC-pKLAC-LC2 vector Vector Map

LIC-pKLAC-LC210526 bp5001000150020002500300035004000450050005500600065007000750080008500900095001000010500start codonPmeI recognition siteLIC stuffer fragmentPmeI recognition siteTEV recognition siteinfrared fluorescent protein6xHis tagLAC4 terminatoracetamidaseLAC4 promoter (5')T7 promoterM13 revlac operatorlac promoterCAP binding sitepMB1 originampRAmpR promoterf1 oriM13 fwd3' promoter region

Plasmid Resuspension Protocol:

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5.Store the plasmid at -20 ℃.

LIC-pKLAC-LC2 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

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)."

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