LOCUS       Exported                6698 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pOpt_mRuby2_Paro, complete sequence.
ACCESSION   KM061063
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6698)
  AUTHORS   Lauersen KJ, Mussgnug JH, Kruse O.
  TITLE     Efficient targeted expression of nuclear transgenes in Chlamydomonas
            reinhardtii with a novel, modular vector toolkit
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6698)
  AUTHORS   Lauersen KJ, Mussgnug JH, Kruse O.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-JUN-2014) Algae Biotechnology and Bioenergy, 
            University of Bielefeld Center for Biotechnology, Universitaetsstr 
            27, Bielefeld, NRW 33615, Germany
REFERENCE   3  (bases 1 to 6698)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6698
                     /organism="Cloning vector pOpt_mRuby2_Paro"
                     /mol_type="other DNA"
                     /db_xref="taxon:1549212"
     misc_feature    1..577
                     /label=pBluescript II KS(+)
                     /note="pBluescript II KS(+)"
     primer_bind     489..505
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        515..533
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      578..844
                     /regulatory_class="promoter"
                     /note="Chlamydomonas reinhardtii heat shock protein 70A 
                     (HSP70Ap) promoter"
     regulatory      851..1074
                     /regulatory_class="promoter"
                     /note="Chlamydomonas reinhardtii ribulose-1,5-bisphosphate 
                     carboxylase/oxygenase small subunit 2 promoter (RBCS2p)"
     CDS             join(1072..1079,1225..1562,1892..2346)
                     /codon_start=1
                     /product="mRuby2 red fluorescent protein"
                     /label=mRuby2 red fluorescent protein
                     /note="contains carboxy-terminal StrepII tag"
                     /protein_id="AIS68754.1"
                     /translation="MARMHMRSDVIEGRVSKGEELIKENMRMKVVMEGSVNGHQFKCTG
                     EGEGNPYMGTQTMRIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEG
                     FTWERVTRYEDGGVVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEM
                     MYPADGGLRGYTHMALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEES
                     DNEMFVVQREHAVAKFAGLGGGMDELYKIEGRDIEFWSHPQFEK"
     intron          1080..1224
                     /note="Chlamydomonas reinhardtii ribulose-1,5-bisphosphate 
                     carboxylase/oxygenase small subunit 2 intron 1 (RBCS2i1)"
     CDS             1247..1258
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     intron          1563..1891
                     /note="Chlamydomonas reinhardtii ribulose-1,5-bisphosphate 
                     carboxylase/oxygenase small subunit 2 intron 2 (RBCS2i2)"
     CDS             2296..2307
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    2319..4380
                     /label=pBluescript II KS(+)
                     /note="pBluescript II KS(+)"
     CDS             2320..2343
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     3UTR           2347..2580
                     /note="Chlamydomonas reinhardtii ribulose-1,5-bisphosphate 
                     carboxylase/oxygenase small subunit 2 3UTR"
     misc_feature    2581..2638
                     /label=multiple cloning site
                     /note="multiple cloning site"
     regulatory      2639..2905
                     /regulatory_class="promoter"
                     /note="HSP70Ap"
     regulatory      2912..3132
                     /regulatory_class="promoter"
                     /note="RBCS2p"
     intron          3141..3285
                     /note="RBCS2i1"
     CDS             3328..4131
                     /codon_start=1
                     /product="aminoglycoside 3-phosphotransferase VIII"
                     /label=aminoglycoside 3-phosphotransferase VIII
                     /note="derived from Streptomyces rimosus; aphVIII; 
                     paromomycin resistance"
                     /protein_id="AIS68755.1"
                     /translation="MDDALRALRGRYPGCEWVVVEDGASGAGVYRLRGGGRELFVKVAA
                     LGAGVGLLGEAERLVWLAEVGIPVPRVVEGGGDERVAWLVTEAVPGRPASARWPREQRL
                     DVAVALAGLARSLHALDWERCPFDRSLAVTVPQAARAVAEGSVDLEDLDEERKGWSGER
                     LLAELERTRPADEDLAVCHGHLCPDNVLLDPRTCEVTGLIDVGRVGRADRHSDLALVLR
                     ELAHEEDPWFGPECSAAFLREYGRGWDGAVSEEKLAFYRLLDEFF"
     3UTR           4141..4379
                     /label=RBCS2 3UTR
                     /note="RBCS2 3UTR"
     promoter        complement(4398..4416)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4437..4453)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4461..4477
                     /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(4485..4515)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4530..4551
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4839..5427)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5598..6458)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6459..6563)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6590..347)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"