pNYCOMPS-LIC-FH10T+ (N term) vector (V010575)

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LOCUS       pNYCOMPS-LIC-FH1        6858 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for high-throughput expression and purification of 
            membrane proteins with N-terminal FLAG(R) and 10xHis tags.
ACCESSION   .
VERSION     .
KEYWORDS    pNYCOMPS-LIC-FH10T+ (N term)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6858)
  AUTHORS   Mancia F, Love J.
  TITLE     High-throughput expression and purification of membrane proteins.
  JOURNAL   J. Struct. Biol. 2010;172:85-93.
  PUBMED    20394823
REFERENCE   2  (bases 1 to 6858)
  AUTHORS   New York Structural Biology Center
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6858)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Biol."; date: "2010"; volume: "172"; pages: "85-93"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SnaBI and 
            treat with T4 DNA polymerase plus dGTP.
FEATURES             Location/Qualifiers
     source          1..6858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             6..23
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      90..137
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      174..629
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(725..1537)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      1659..2247
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(2433..2575)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2680..2868)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(3643..3664)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3680..4759)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4760..4837)
                     /label=lacI promoter
     promoter        5146..5164
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5165..5189
                     /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)."
     RBS             5204..5226
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5233..5235
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             5236..5259
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             5260..5286
                     /label=9xHis
                     /note="9xHis affinity tag"
     CDS             5290..5310
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(5393..5695)
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
     CDS             complement(6040..6696)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(6750..6780)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"