LOCUS       Exported                4407 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pColdI DNA, complete sequence.
ACCESSION   AB186388
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4407)
  AUTHORS   Qing G, Ma LC, Khorchid A, Swapna GV, Mal TK, Takayama MM, Xia B, 
            Phadtare S, Ke H, Acton T, Montelione GT, Ikura M, Inouye M.
  TITLE     Cold-shock induced high-yield protein production in Escherichia coli
  JOURNAL   Nat. Biotechnol. 22 (7), 877-882 (2004)
  PUBMED    15195104
REFERENCE   2  (bases 1 to 4407)
  AUTHORS   Takayama MM, Inouye M.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-AUG-2004) Masanori Mitta Takayama, UMDNJ-Robert Wood 
            Johnson Medical School, Department of Biochemistry; 675 Hoes Lane, 
            Piscataway, NJ 08854-5635, USA (E-mail:mitsutam@takara-bio.co.jp, 
            Tel:81-77-543-7200, Fax:81-77-543-2494)
REFERENCE   3  (bases 1 to 4407)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4407
                     /organism="Expression vector pColdI"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /note="expresses an open reading frame as a factor Xa 
                     cleavable fusion to a His6 containing translation enhancing
                     element. This plasmid is derived from pUC118."
                     /db_xref="taxon:289229"
     gene            15..512
                     /gene="cspA"
                     /label=cspA
     regulatory      15..81
                     /regulatory_class="promoter"
                     /gene="cspA"
     promoter        15..81
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     misc_feature    82..121
                     /gene="cspA"
                     /label=synthetic lac operator sequence
                     /note="synthetic lac operator sequence"
     protein_bind    84..100
                     /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)."
     5UTR           122..255
                     /gene="cspA"
                     /label=modified cspA 5UTR
                     /note="modified cspA 5UTR"
     misc_feature    256..270
                     /gene="cspA"
                     /label=TEE (translation enhancing element)
                     /note="TEE (translation enhancing element)"
     CDS             256..270
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     misc_feature    271..288
                     /gene="cspA"
                     /label=His6 tag
                     /note="His6 tag"
     CDS             271..288
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    289..300
                     /gene="cspA"
                     /label=Factor Xa site
                     /note="Factor Xa site"
     CDS             289..300
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    301..360
                     /gene="cspA"
                     /note="MCS = Multiple cloning sites contain the follow 
                     restriction sites: NdeI, SacI, KpnI, XhoI, BamHI, EcoRI, 
                     HindIII, SalI, PstI, XbaI"
     3UTR           368..512
                     /gene="cspA"
                     /label=cspA 3UTR
                     /note="cspA 3UTR"
     misc_feature    complement(711..1184)
                     /label=M13 intergenic region
                     /note="M13 intergenic region"
     rep_origin      complement(711..1166)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        1266..1370
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1371..2231
                     /codon_start=1
                     /gene="Amp"
                     /product="beta-lactamase"
                     /label=Amp
                     /protein_id="BAD35134.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            1371..2231
                     /gene="Amp"
                     /label=Amp
     rep_origin      2391..3005
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      2402..2990
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3162..4244)
                     /codon_start=1
                     /gene="lacI"
                     /product="lactose operon repressor"
                     /label=lacI
                     /protein_id="BAD35135.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(3162..4244)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(4245..4322)
                     /gene="lacI"
                     /label=lacI promoter