LOCUS       Exported                4928 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pEMY128AD_pY128, complete sequence.
ACCESSION   MH374490
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4928)
  AUTHORS   Young EM, Zhao Z, Gielesen BE, Wu L, Benjamin Gordon D, Roubos JA, 
            Voigt CA.
  TITLE     Iterative algorithm-guided design of massive strain libraries, 
            applied to itaconic acid production in yeast
  JOURNAL   Metab. Eng. 48, 33-43 (2018)
  PUBMED    29753070
REFERENCE   2  (bases 1 to 4928)
  AUTHORS   Young E, Zhao Z, Gielesen B, Wu L, Gordon DB, Roubos J, Voigt C.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-MAY-2018) Genetics, DSM Biotechnology Center, 
            Alexander Fleminglaan 1, Delft, South Holland 2613AX, Netherlands
REFERENCE   3  (bases 1 to 4928)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4928
                     /organism="Cloning vector pEMY128AD_pY128"
                     /mol_type="other DNA"
                     /db_xref="taxon:2218584"
     primer_bind     229..245
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    246..302
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(315..331)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    339..355
                     /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(363..393)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    408..429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(659..1247)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    1496..1543
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     rep_origin      1524..2505
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     gene            complement(2547..3903)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      2547..2744
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2750..3559)
                     /codon_start=1
                     /gene="aph(3)-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(3561..3903)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             complement(4072..4731)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             complement(4876..319)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSILEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCRSLHRSS
                     NQLTGSPSRVGLSSARANC"
     terminator      4894..4923
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"