LOCUS       Exported                8332 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pYOGM081, complete sequence.
ACCESSION   KJ094414
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8332)
  AUTHORS   Labunskyy VM, Suzuki Y, Hanly TJ, Murao A, Roth FP, Gladyshev VN.
  TITLE     The insertion Green Monster (iGM) method for expression of multiple 
            exogenous genes in yeast
  JOURNAL   G3 (Bethesda) 4 (7), 1183-1191 (2014)
  PUBMED    24776987
REFERENCE   2  (bases 1 to 8332)
  AUTHORS   Suzuki Y, Roth FP.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-JAN-2014) Synthetic Biology and Bioenergy, J. Craig 
            Venter Institute, 4120 Torrey Pines Road, La Jolla, CA 92037, USA
REFERENCE   3  (bases 1 to 8332)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8332
                     /organism="Cloning vector pYOGM081"
                     /mol_type="other DNA"
                     /db_xref="taxon:1445503"
     misc_feature    6..70
                     /label=upstream linearization site
                     /note="upstream linearization site"
     misc_feature    33..152
                     /label=similar to the KanMX4 cassette
                     /note="similar to the KanMX4 cassette"
     misc_feature    167..1247
                     /label=URA3 rescuing region
                     /note="URA3 rescuing region"
     promoter        167..383
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             384..1187
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLATGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     regulatory      1277..1488
                     /regulatory_class="terminator"
                     /note="ADH1 terminator"
     terminator      1295..1482
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     misc_feature    1494..1593
                     /label=contains two copies of tetO
                     /note="contains two copies of tetO"
     protein_bind    1499..1517
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1541..1559
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      1594..1746
                     /regulatory_class="TATA_box"
                     /note="CYC1 TATA region"
     CDS             1755..2471
                     /codon_start=1
                     /product="green fluorescent protein S65T"
                     /label=green fluorescent protein S65T
                     /protein_id="AIY31773.1"
                     /translation="MSKGEELFTGVVPVLVELDGDVNGQKFSVSGEGEGDATYGKLTLN
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFYKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNVYIMGDKPKNGIKV
                     NFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLE
                     FVTAARITHGMDELYK"
     regulatory      2483..2744
                     /regulatory_class="terminator"
                     /note="CYC1 terminator"
     terminator      2493..2740
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     regulatory      2755..3205
                     /regulatory_class="promoter"
                     /note="GAL1-10 promoter"
     promoter        2756..3197
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2756..2873
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3212..3228
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3236..3360
                     /label=Gateway cloning site
                     /note="Gateway cloning site"
     protein_bind    3236..3360
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3385..3415
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3469..4128
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             4473..4778
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB toxin"
                     /label=ccdB
                     /protein_id="AIY31772.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     gene            4473..4778
                     /gene="ccdB"
                     /label=ccdB
     protein_bind    complement(4819..4943)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    4819..4926
                     /label=Gateway cloning site
                     /note="Gateway cloning site"
     misc_feature    4984..5073
                     /label=HA tag
                     /note="HA tag"
     CDS             4984..5073
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     misc_feature    5102..5351
                     /label=similar to the KanMX4 cassette
                     /note="similar to the KanMX4 cassette"
     terminator      5107..5304
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     regulatory      5110..5304
                     /regulatory_class="terminator"
                     /note="TEF terminator"
     misc_feature    5346..5404
                     /label=downstream linearization site
                     /note="downstream linearization site"
     misc_feature    5405..8332
                     /label=pBluescript SK+ backbone
                     /note="pBluescript SK+ backbone"
     primer_bind     complement(5424..5440)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(5477..5495)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5516..5532)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5540..5556
                     /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(5564..5594)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5609..5630
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5918..6506)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6677..7537)
                     /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(7538..7642)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7668..8123)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     8265..8281
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8291..8309
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"