Basic Vector Information
LIC-pKLAC-LC2 vector Vector Map
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
LOCUS 40924_1774 10526 bp DNA circular SYN 17-DEC-2018 DEFINITION Expression vector LIC-pKLAC-LC2, complete sequence. ACCESSION . 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) TITLE Direct Submission REFERENCE 4 (bases 1 to 10526) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "PLoS ONE"; date: "2011"; volume: "6"; issue: "4"; pages: "E18900" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (14-FEB-2011) Molecular Biology, University of Potsdam, Germany, Karl-Liebknecht-Str. 24-25, Haus 20, Potsdam, Brandenburg 14476, Germany" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..10526 /mol_type="other DNA" /organism="synthetic DNA construct" 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" CDS 714..734 /codon_start=1 /label=TEV site /note="tobacco etch virus (TEV) protease recognition and cleavage site" /translation="ENLYFQG" CDS 744..1727 /codon_start=1 /label=IFP1.4 /note="bacteriophytochrome-based monomeric infrared fluorescent protein (Shu et al., 2009)" /translation="MARDPLPFFPPLYLGGPEITTENCEREPIHIPGSIQPHGALLTAD GHSGEVLQVSLNAATFLGQEPTVLRGQTLAALLPEQWPALQAALPPGCPDALQYRATLD WPAAGHLSLTVHRVAELLILEFEPTEAWDSIGPHALRNAMFALESAPNLRALAEVATQT VRELTGFDRVMLYKFAPDATGEMIAEARREGMQAFLGHRFPASHTPAQARALYTRHLLR LTADTRAAAVPLDPVLNPQTNAPTPLGGAVLRATSPMHMQYLRNMGVGSSLSVSVVVGG QLWGLIVCHHQTPYVLPPDLRTTLEELGRKLSGQVQRKEAGMDELYK" CDS 1734..1751 /codon_start=1 /label=6xHis /note="6xHis affinity tag" /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" promoter 2409..3111 /label=ADH1 promoter /note="promoter for alcohol dehydrogenase 1" CDS 3132..4775 /codon_start=1 /label=amdS /note="acetamidase from Aspergillus nidulans" /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" 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 complement(6212..6233) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(6521..7109) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(7283..8140) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(8141..8245) /label=AmpR promoter rep_origin 8272..8727 /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" promoter 8898..10426 /label=LAC4 promoter (3') /note="3' portion of a modified Kluyveromyces lactis LAC4 promoter" protein_bind 10432..10448 /label=lac operator /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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