LOCUS       Exported                6041 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pET24a_mCerulean3, complete sequence.
ACCESSION   KM504958
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6041)
  AUTHORS   Lauersen KJ, Kruse O, Mussgnug JH.
  TITLE     Targeted expression of nuclear transgenes in Chlamydomonas 
            reinhardtii with a versatile, modular vector toolkit
  JOURNAL   Appl. Microbiol. Biotechnol. (2015) In press
  PUBMED    25586579
REFERENCE   2  (bases 1 to 6041)
  AUTHORS   Lauersen KJ, Kruse O, Mussgnug JH.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-SEP-2014) Algae Biotechnology and Bioenergy, 
            University of Bielefeld Center for Biotechnology, Universitaetsstr 
            27, Bielefeld, NRW 33615, Germany
REFERENCE   3  (bases 1 to 6041)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6041
                     /organism="Expression vector pET24a_mCerulean3"
                     /mol_type="other DNA"
                     /label=for use in Escherichia coli
                     /note="for use in Escherichia coli"
                     /db_xref="taxon:1544372"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     rep_origin      complement(12..456)
                     /direction=LEFT
                     /label=M13 origin
                     /note="M13 origin"
     CDS             complement(560..1375)
                     /codon_start=1
                     /gene="aphIII"
                     /product="aminoglycoside 3-phosphotransferase III"
                     /function="kanamycin resistance"
                     /label=aphIII
                     /protein_id="AJG06995.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(560..1375)
                     /gene="aphIII"
                     /label=aphIII
     rep_origin      complement(1421..2103)
                     /direction=LEFT
                     /label=ColE1 rep origin
                     /note="ColE1 rep origin"
     rep_origin      1497..2085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2271..2413
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2515..2706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3515..4597)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(4598..4675)
                     /gene="lacI"
                     /label=lacI promoter
     regulatory      4984..5003
                     /regulatory_class="promoter"
                     /note="T7 promoter of expression vector multiple cloning 
                     site"
     promoter        4984..5002
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5003..5027
                     /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)."
     RBS             5042..5064
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5072..5878
                     /codon_start=1
                     /product="cyan fluorescent protein variant"
                     /label=cyan fluorescent protein variant
                     /note="includes C-terminal StrepII tag WSHPQFEK"
                     /protein_id="AJG06996.1"
                     /translation="MARMHMRSDVIEGRVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGN
                     VYITADKQKNGIKANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLS
                     KDPNEKRDHMVLLEFVTAAGITLGMDELYKIEGRDIEFWSHPQFEK"
     CDS             5102..5113
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             5828..5839
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             5852..5875
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5885..5902
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5969..6016
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"