LOCUS       Exported               10655 bp ds-DNA     circular SYN 30-NOV-2018
DEFINITION  Cloning vector pAW8-yEGFP, complete sequence.
ACCESSION   EU093099
VERSION     .
KEYWORDS    pAW8-yEGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10655)
  AUTHORS   Watson AT, Garcia V, Bone N, Carr AM, Armstrong J.
  TITLE     Gene tagging and gene replacement using recombinase-mediated 
            cassette exchange in Schizosaccharomyces pombe
  JOURNAL   Gene 407 (1-2), 63-74 (2008)
  PUBMED    18054176
REFERENCE   2  (bases 1 to 10655)
  AUTHORS   Watson AT.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-AUG-2007) Genome Damage and Stability Centre, 
            University of Sussex, Falmer, Brighton, East Sussex BN1 9RQ, UK
REFERENCE   3  (bases 1 to 10655)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10655
                     /organism="Cloning vector pAW8-yEGFP"
                     /mol_type="other DNA"
                     /note="CRE expression plasmid"
                     /db_xref="taxon:461531"
     rep_origin      231..1424
                     /note="fission yeast ars1"
     rep_origin      575..1356
                     /note="ars1"
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     misc_feature    1425..3860
                     /note="pUC19 vector backbone"
     promoter        1716..1820
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             1821..2681
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2852..3440
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    3728..3749
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3764..3794
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    3802..3818
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="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)."
     primer_bind     3826..3842
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     gene            complement(3976..5332)
                     /note="kanMX"
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      3976..4173
                     /note="TEF terminator"
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4179..4988)
                     /codon_start=1
                     /transl_table=11
                     /gene="kanR"
                     /product="kanamycin and geneticin G418 resistance protein"
                     /note="kanR"
                     /protein_id="ABU48525.1"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(4179..4988)
                     /gene="kanR"
                     /note="kanR"
     promoter        complement(4989..5332)
                     /note="TEF promoter"
                     /note="Ashbya gossypii TEF promoter"
     regulatory      complement(5378..5606)
                     /regulatory_class="terminator"
                     /note="Saccharomyces cerevisiae ADH1"
     terminator      5407..5594
                     /gene="S. cerevisiae ADH1"
                     /note="ADH1 terminator"
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     CDS             complement(5615..6331)
                     /codon_start=1
                     /transl_table=11
                     /gene="GFP"
                     /product="green fluorescent protein"
                     /note="GFP"
                     /note="yEGFP; codon optimized variant of GFP"
                     /protein_id="ABU48522.1"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     gene            complement(5615..6331)
                     /gene="GFP"
                     /note="GFP"
     misc_recomb     complement(6340..6373)
                     /note="loxP"
     protein_bind    complement(6340..6373)
                     /bound_moiety="Cre recombinase"
                     /note="loxP"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(6389..6405)
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      6612..7168
                     /regulatory_class="terminator"
                     /note="nmt1"
     CDS             complement(join(7174..8166,8216..8254))
                     /codon_start=1
                     /transl_table=11
                     /gene="cre"
                     /product="Cre"
                     /note="cre"
                     /note="bacteriophage P1 recombinase"
                     /protein_id="ABU48523.1"
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     gene            complement(7174..8254)
                     /gene="cre"
                     /note="cre"
     intron          complement(8167..8215)
                     /gene="cre"
                     /note="Schizosaccharomyces pombe rad50 intron 1"
     regulatory      complement(8255..8865)
                     /regulatory_class="promoter"
                     /note="nmt41"
     gene            8884..10655
                     /gene="LEU2"
                     /note="LEU2"
     regulatory      8884..9303
                     /regulatory_class="promoter"
                     /gene="LEU2"
     CDS             9304..10398
                     /codon_start=1
                     /transl_table=11
                     /gene="LEU2"
                     /product="beta-isopropyl-malate dehydrogenase"
                     /note="LEU2"
                     /note="involved in leucine biosynthesis"
                     /protein_id="ABU48524.1"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     regulatory      10399..10655
                     /regulatory_class="terminator"
                     /gene="LEU2"