LOCUS       sequence-402857-        8999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast expression vector; To express proteins under control of TDH3 
            promoter and terminator using methionine selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8999)
  AUTHORS   Park EJ, Grabinska KA, Guan Z, Stranecky V, Hartmannova H, Hodanova 
            K, Baresova V, Sovova J, Jozsef L, Ondruskova N, Hansikova H, Honzik
            T, Zeman J, Hulkova H, Wen R, Kmoch S, Sessa WC
  TITLE     Mutation of Nogo-B receptor, a subunit of cis-prenyltransferase, 
            causes a congenital disorder of glycosylation.
  JOURNAL   Cell Metab. 2014 Sep 2;20(3):448-57. doi: 
            10.1016/j.cmet.2014.06.016. Epub 2014 Jul 24.
  PUBMED    25066056
REFERENCE   2  (bases 1 to 8999)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cmet.2014.06.016"; journalName: "Cell Metab"; date: 
            "2014-09-2- 2"; volume: "20"; issue: "3"; pages: "448-57"
COMMENT     Sequence Label: pKG-GW2
FEATURES             Location/Qualifiers
     source          1..8999
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        54..720
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     590..613
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     protein_bind    723..847
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(872..877,878..895,896..902)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             956..1615
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1022..1041)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1957..2262
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2141..2160
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2303..2427)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2632..2649)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2803..3391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2883..2902)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3562..4353,4354..4422))
                     /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"
     primer_bind     4185..4204
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4423..4527)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4595..4613
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4651..4673)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4773..4792
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      4809..5791
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        5792..6197
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     promoter        6487..6836
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     CDS             6837..8171
                     /codon_start=1
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /product="O-acetylserine and O-acetylhomoserine 
                     sulfhydrylase, required for sulfur amino acid synthesis"
                     /label=MET17
                     /translation="MPSHFDTVQLHAGQENPGDNAHRSRAVPIYATTSYVFENSKHGSQ
                     LFGLEVPGYVYSRFQNPTSNVLEERIAALEGGAAALAVSSGQAAQTLAIQGLAHTGDNI
                     VSTSYLYGGTYNQFKISFKRFGIEARFVEGDNPEEFEKVFDERTKAVYLETIGNPKYNV
                     PDFEKIVAIAHKHGIPVVVDNTFGAGGYFCQPIKYGADIVTHSATKWIGGHGTTIGGII
                     VDSGKFPWKDYPEKFPQFSQPAEGYHSTIYNEAYGNLAYIVHVRTELLRDLGPLMNPFA
                     SFLLLQGVETLSLRAERHGENALKLAKWLEQSPYVSWVSYPGLASHSHHENARKYLSNG
                     FGGVLSFGVKDLPNADKETDPFKLSGAQVVDNLKLASNLANVGDAKTLVIAPYFTTHKQ
                     LNDKEKLASGVTKDLIRVSVGIEFIDDIIADFQQSFETVFAGQKP"
ORIGIN
        1 ggatccggta gaatcatttt gaataaaaaa cacgcttttt cagttcgagt ttatcattat
       61 caatactgcc atttcaaaga atacgtaaat aattaatagt agtgattttc ctaactttat
      121 ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt acatgcccaa aatagggggc
      181 gggttacaca gaatatataa catcgtaggt gtctgggtga acagtttatt cctggcatcc
      241 actaaatata atggagcccg ctttttaagc tggcatccag aaaaaaaaag aatcccagca
      301 ccaaaatatt gttttcttca ccaaccatca gttcataggt ccattctctt agcgcaacta
      361 cagagaacag gggcacaaac aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc
      421 tgcctggagt aaatgatgac acaaggcaat tgacccacgc atgtatctat ctcattttct
      481 tacaccttct attaccttct gctctctctg atttggaaaa agctgaaaaa aaaggttgaa
      541 accagttccc tgaaattatt cccctacttg actaataagt atataaagac ggtaggtatt
      601 gattgtaatt ctgtaaatct atttcttaaa cttcttaaat tctactttta tagttagtct
      661 tttttttagt tttaaaacac caagaactta gtttcgaata aacacacata aacagaatta
      721 tcacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat atcaatatat
      781 taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt
      841 cactatggcg gccgcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa
      901 tgtgtggatt ttgagttagg atccgtcgag attttcagga gctaaggaag ctaaaatgga
      961 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
     1021 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
     1081 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat
     1141 tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct
     1201 ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt
     1261 ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca
     1321 agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat
     1381 gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa
     1441 tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa
     1501 ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg
     1561 cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaaacgcg
     1621 tggatccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg
     1681 tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggta tgctatgaag
     1741 cagcgtatta cagtgacagt tgacagcgac agctatcagt tgctcaaggc atatatgatg
     1801 tcaatatctc cggtctggta agcacaacca tgcagaatga agcccgtcgt ctgcgtgccg
     1861 aacgctggaa agcggaaaat caggaaggga tggctgaggt cgcccggttt attgaaatga
     1921 acggctcttt tgctgacgag aacaggggct ggtgaaatgc agtttaaggt ttacacctat
     1981 aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccc
     2041 gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt
     2101 gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg
     2161 gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat
     2221 gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg ctcccttata
     2281 cacagccagt ctgcaggtcg accatagtga ctggatatgt tgtgttttac agtattatgt
     2341 agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc
     2401 tcgttcagct ttcttgtaca aagtggtgat tcgacttggt tgaacacgtt gccaaggctt
     2461 aagtgaattt actttaaatc ttgcatttaa ataaattttc tttttatagc tttatgactt
     2521 agtttcaatt tatatactat tttaatgaca ttttcgattc ggatccgtgc attaatgaat
     2581 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     2641 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2701 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     2761 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2821 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2881 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2941 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3001 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3061 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3121 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3181 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3241 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3301 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3361 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3421 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3481 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3541 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3601 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     3661 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     3721 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3781 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     3841 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     3901 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3961 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4021 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4081 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4141 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4201 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4261 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4321 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4381 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4441 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4501 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     4561 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     4621 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     4681 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     4741 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     4801 gcaccatact agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
     4861 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
     4921 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
     4981 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
     5041 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
     5101 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
     5161 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
     5221 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
     5281 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
     5341 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
     5401 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
     5461 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
     5521 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
     5581 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
     5641 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
     5701 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     5761 gttctacaaa atgaagcaca gatgcttcgt taactgtggg aatactcagg tatcgtaaga
     5821 tgcaagagtt cgaatctctt agcaaccatt atttttttcc tcaacataac gagaacacac
     5881 aggggcgcta tcgcacagaa tcaaattcga tgactggaaa ttttttgtta atttcagagg
     5941 tcgcctgacg catatacctt tttcaactga aaaattggga gaaaaaggaa aggtgagagc
     6001 gccggaaccg gcttttcata tagaatagag aagcgttcat gactaaatgc ttgcatcaca
     6061 atacttgaag ttgacaatat tatttaagga cctattgttt tttccaatag gtggttagca
     6121 atcgtcttac tttctaactt ttcttacctt ttacatttca gcaatatata tatatatttc
     6181 aaggatatac cattctaatg tctgccccta agaagatcgt cgttttgcca ggtgaccata
     6241 atatactagt aacgtaaata ctagttagta gatgatagtt gatttttatt ccaacactaa
     6301 gaaataattt cgccatttct tgaatgtatt taaagatatt taatgctata atagacattt
     6361 aaatccaatt cttccaacat acaatgggag tttggccgag tggtttaagg cgtcagattt
     6421 aggtggattt aacctctaaa atctctgata tcttcggatg caagggttcg aatcccttag
     6481 ctctcattat tttttgcttt ttctcttgag gtcacatgat cgcaaaatgg caaatggcac
     6541 gtgaagctgt cgatattggg gaactgtggt ggttggcaaa tgactaatta agttagtcaa
     6601 ggcgccatcc tcatgaaaac tgtgtaacat aataaccgaa gtgtcgaaaa ggtggcacct
     6661 tgtccaattg aacacgctcg atgaaaaaat aagatatata taaggttaag taaagcgtct
     6721 gttagaaagg aagtttttcc tttttcttgc tctcttgtct tttcatctac tatttccttc
     6781 gtgtaataca gggtcgtcag atacatagat acaattctat tacccccatc catacaatgc
     6841 catctcattt cgatactgtt caactacacg ccggccaaga gaaccctggt gacaatgctc
     6901 acagatccag agctgtacca atttacgcca ccacttctta tgttttcgaa aactctaagc
     6961 atggttcgca attgtttggt ctagaagttc caggttacgt ctattcccgt ttccaaaacc
     7021 caaccagtaa tgttttggaa gaaagaattg ctgctttaga aggtggtgct gctgctttgg
     7081 ctgtttcctc cggtcaagcc gctcaaaccc ttgccatcca aggtttggca cacactggtg
     7141 acaacatcgt ttccacttct tacttatacg gtggtactta taaccagttc aaaatctcgt
     7201 tcaaaagatt tggtatcgag gctagatttg ttgaaggtga caatccagaa gaattcgaaa
     7261 aggtctttga tgaaagaacc aaggctgttt atttggaaac cattggtaat ccaaagtaca
     7321 atgttccgga ttttgaaaaa attgttgcaa ttgctcacaa acacggtatt ccagttgtcg
     7381 ttgacaacac atttggtgcc ggtggttact tctgtcagcc aattaaatac ggtgctgata
     7441 ttgtaacaca ttctgctacc aaatggattg gtggtcatgg tactactatc ggtggtatta
     7501 ttgttgactc tggtaagttc ccatggaagg actacccaga aaagttccct caattctctc
     7561 aacctgccga aggatatcac agtactatct acaatgaagc ctacggtaac ttggcataca
     7621 tcgttcatgt tagaactgaa ctattaagag atttgggtcc attgatgaac ccatttgcct
     7681 ctttcttgct actacaaggt gttgaaacat tatctttgag agctgaaaga cacggtgaaa
     7741 atgcattgaa gttagccaaa tggttagaac aatccccata cgtatcttgg gtttcatacc
     7801 ctggtttagc atctcattct catcatgaaa atgctaggaa gtatctatct aacggtttcg
     7861 gtggtgtctt atctttcggt gtaaaagact taccaaatgc cgacaaggaa actgacccat
     7921 tcaaactttc tggtgctcaa gttgttgaca atttaaagct tgcctctaac ttggccaatg
     7981 ttggtgatgc caagacctta gtcattgctc catacttcac tacccacaaa caattaaatg
     8041 acaaagaaaa gttggcatct ggtgttacca aggacttaat tcgtgtctct gttggtatcg
     8101 aatttattga tgacattatt gcagacttcc agcaatcttt tgaaactgtt ttcgctggcc
     8161 aaaaaccatg agtgtgcgta atgagttgta aaattatgta taaacctact ttctctcaca
     8221 agtactatac ttttataaaa cgaactttat tgaaatgaat atcctttttt tcccttgtta
     8281 catgtcgtga ctcgtacttt gaacctaaat tgttctaaca tcaaagaaca gtgttaattc
     8341 gcagtcgaga agaaaaatat ggtgaacaag actcatctac ttcatgagac tactttacgc
     8401 ctcctataaa gctgtcacac tgtacataaa ctttataaat gaaattcata atagaaacga
     8461 cacgaaatta caaaatggaa tatgttcata gggtagacga aactatatac gcaatctaca
     8521 tacatttatc aagaaggaga aaaaggagga tgtaaaggaa tacaggtaag caaattgata
     8581 ctaatggctc aacgtgataa ggaaaaagaa ttgcacttta acattaatat tgacaaggag
     8641 gagggcacca cacaaaaagt taggtgtaac agaaaatcat gaaactatga ttcctaattt
     8701 atatattgga ggattttctc taaaaaaaaa aaaatacaac aaataaaaaa cactcaatga
     8761 cctgaccatt tgatggagtt taagtcaata ccttcttgaa ccatttccca taatggtgaa
     8821 agttccctca agaattttac tctgtcagaa acggccttaa cgacgtaccg gatctatgcg
     8881 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
     8941 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagc
//
