LOCUS       Exported                5357 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pbleCrCre DNA, complete sequence.
ACCESSION   LC150885
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5357)
  AUTHORS   Kasai Y, Harayama S.
  TITLE     Construction of marker-free transgenic strains of Chlamydomonas 
            reinhardtii using a Cre/loxP-mediated recombinase system
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5357)
  AUTHORS   Kasai Y, Harayama S.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-MAY-2016) Contact:Yuki Kasai Chuo University, 
            Department of Biological sciences; Kasuga 1-13-27, Bunkyo-ku, Tokyo 
            112-8551, Japan
REFERENCE   3  (bases 1 to 5357)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5357
                     /organism="Expression vector pbleCrCre"
                     /mol_type="other DNA"
                     /db_xref="taxon:1849579"
     primer_bind     18..34
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      43..317
                     /regulatory_class="promoter"
                     /note="includes promoter region of Chlamydomonas 
                     reinhardtii nuclear gene hsp70A for cytosolic HSP70"
     regulatory      325..540
                     /regulatory_class="promoter"
                     /note="includes promoter region and start codon of 
                     Chlamydomonas reinhardtii nuclear gene rbcS2 for ribulose 
                     bisphosphate carboxylase/oxygenase small subunit"
     CDS             join(533..540,686..854,1000..2235)
                     /codon_start=1
                     /gene="ble::CrCRE"
                     /product="fusion Ble/CrCRE protein"
                     /label=ble::CrCRE
                     /protein_id="BAV82215.1"
                     /translation="MARMAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVV
                     RDDVTLFISAVQDQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGE
                     QPWGREFALRDPAGNCVHFVAEEQDNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQA
                     FSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQL
                     NMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDR
                     CQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKAL
                     SLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLI
                     YGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSE
                     TGAMVRLLEDGD"
     gene            533..2235
                     /gene="ble::CrCRE"
                     /label=ble::CrCRE
     intron          541..685
                     /gene="ble::CrCRE"
                     /note="includes intron 1 from Chlamydomonas reinhardtii 
                     rbcS2 gene"
     exon            686..854
                     /gene="ble::CrCRE"
                     /note="includes a fragment of Streptoalloteichus 
                     hindustanus Ble gene"
     intron          855..999
                     /gene="ble::CrCRE"
                     /note="includes intron 1 from Chlamydomonas reinhardtii 
                     rbcS2 gene"
     exon            1000..2235
                     /gene="ble::CrCRE"
                     /note="includes a fragment of Streptoalloteichus 
                     hindustanus Ble gene fused with codon optimized cre 
                     recombinase gene CrCRE"
     misc_feature    1210..2235
                     /gene="ble::CrCRE"
                     /label=synthetic CrCRE gene coding for cre recombinase
                     /note="synthetic CrCRE gene coding for cre recombinase"
     regulatory      2236..2476
                     /regulatory_class="terminator"
                     /note="includes terminator region from Chlamydomonas 
                     reinhardtii rbcS2 gene"
     misc_feature    2477..5357
                     /label=derived from plasmid pBluescript SK(+)
                     /note="derived from plasmid pBluescript SK(+)"
     promoter        complement(2513..2531)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2541..2557)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(2698..3153)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        3180..3284
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3285..4145
                     /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"
     rep_origin      4316..4904
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    5192..5213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5228..5258
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5266..5282
                     /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)."
     primer_bind     5290..5306
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5327..5345
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"