LOCUS       sequence-66300-e        6981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEF1 and PGK1 promoter controlled expression cassettes with G418 
            resistance for yeast transformation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6981)
  AUTHORS   Vickers CE, Bydder SF, Zhou Y, Nielsen LK
  TITLE     Dual gene expression cassette vectors with antibiotic selection 
            markers for engineering in Saccharomyces cerevisiae.
  JOURNAL   Microb Cell Fact. 2013 Oct 25;12(1):96.
  PUBMED    24161108
REFERENCE   2  (bases 1 to 6981)
  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"; journalName: "Microb Cell
            Fact."; date: "2013-10-25"; volume: "12(1)"; pages: "96"
COMMENT     Sequence Label: pCEV-G2-Km
FEATURES             Location/Qualifiers
     source          1..6981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(2..167)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     88..112
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(315..338)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        1354..1760
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     1780..1799
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1780..1798
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1816..1845
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     terminator      1875..2064
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2136..2153)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2307..2895)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2387..2406)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3066..3857,3858..3926))
                     /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     3689..3708
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3927..4031)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4058..5400
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5021..5068)
                     /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)."
     protein_bind    5420..5453
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            5508..6864
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        5508..5851
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             5852..6661
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5919..5938)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      6667..6864
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    6927..6960
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ggagcgacct catgctatac ctgagaaagc aacctgacct acaggaaaga gttactcaag
       61 aataagaatt ttcgttttaa aacctaagag tcactttaaa atttgtatac acttattttt
      121 tttataactt atttaataat aaaaatcata aatcataaga aattcgctta tttagaagtg
      181 tcaacaacgt atctaccaac gatttgaccc ttttccatct tttcgtaaat ttctggcaag
      241 gtagacaagc cgacaacctt gattggagac ttgaccaaac ctctggcgaa gaattgttaa
      301 ttaagagctc agatcttatc gtcgtcatcc ttgtaatcca tcgatactag tgcggccgct
      361 tgttttatat ttgttgtaaa aagtagataa ttacttcctt gatgatctgt aaaaaagaga
      421 aaaagaaagc atctaagaac ttgaaaaact acgaattaga aaagaccaaa tatgtatttc
      481 ttgcattgac caatttatgc aagtttatat atatgtaaat gtaagtttca cgaggttcta
      541 ctaaactaaa ccaccccctt ggttagaaga aaagagtgtg tgagaacagg ctgttgttgt
      601 cacacgattc ggacaattct gtttgaaaga gagagagtaa cagtacgatc gaacgaactt
      661 tgctctggag atcacagtgg gcatcatagc atgtggtact aaaccctttc ccgccattcc
      721 agaaccttcg attgcttgtt acaaaacctg tgagccgtcg ctaggacctt gttgtgtgac
      781 gaaattggaa gctgcaatca ataggaagac aggaagtcga gcgtgtctgg gttttttcag
      841 ttttgttctt tttgcaaaca aatcacgagc gacggtaatt tctttctcga taagaggcca
      901 cgtgctttat gagggtaaca tcaattcaag aaggagggaa acacttcctt tttctggccc
      961 tgataatagt atgagggtga agccaaaata aaggattcgc gcccaaatcg gcatctttaa
     1021 atgcaggtat gcgatagttc ctcactcttt ccttactcac gagtaattct tgcaaatgcc
     1081 tattatgcag atgttataat atctgtgcgt cttgagttga agtcaggaat ctaaaataaa
     1141 aattaaggtt aataaaaaga ggaaagaaaa aaaaattaat cgatttacag aaacttgcac
     1201 actaaaaata cacaactaaa agcaattaca gtatgggaag tcatcgacgt tatctctact
     1261 atagtatatt atcatttcta ttattatcct gctcagtggt acttgcaaaa caagataaga
     1321 ccccattctt tgaaggtact tccaggccgg ccgcacacac catagcttca aaatgtttct
     1381 actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa
     1441 cacccaagca cagcatacta aatttcccct ctttcttcct ctagggtgtc gttaattacc
     1501 cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa
     1561 aggcaataaa aatttttatc acgtttcttt ttcttgaaaa tttttttttt tgattttttt
     1621 ctctttcgat gacctcccat tgatatttaa gttaataaac ggtcttcaat ttctcaagtt
     1681 tcagtttcat ttttcttgtt ctattacaac tttttttact tcttgctcat tagaaagaaa
     1741 gcatagcaat ctaatctaag ttttaattac aaggatccgt aatacgactc actatagggc
     1801 ccgggcgtcg acatggaaca gaagttgatt tccgaagaag acctcgagta agcttggtac
     1861 cgcggctagc taagatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag
     1921 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt
     1981 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt
     2041 gagaaggttt tgggacgctc gaagatccag ctgcattaat gaatcggcca acgcgcgggg
     2101 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2161 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2221 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2281 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2341 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2401 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2461 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2521 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2581 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2641 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2701 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     2761 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2821 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2881 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2941 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3001 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3061 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3121 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3181 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3241 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3301 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3361 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3421 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3481 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3541 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3601 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3661 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3721 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3781 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3841 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3901 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     3961 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4021 ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg tgcttcattt
     4081 tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat
     4141 ttttacagaa cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc
     4201 atttttgtaa aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc
     4261 tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat
     4321 acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct
     4381 tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca
     4441 ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa
     4501 aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt
     4561 caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa
     4621 cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta
     4681 ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac
     4741 tctatgaata gttcttacta caattttttt gtctaaagag taatactaga gataaacata
     4801 aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt
     4861 atatagggat atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga
     4921 agcggtattc gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa
     4981 agtgcgtctt cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag
     5041 agaataggaa cttcggaata ggaacttcaa agcgtttccg aaaacgagcg cttccgaaaa
     5101 tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc gcacctatat ctgcgtgttg
     5161 cctgtatata tatatacatg agaagaacgg catagtgcgt gtttatgctt aaatgcgtac
     5221 ttatatgcgt ctatttatgt aggatgaaag gtagtctagt acctcctgtg atattatccc
     5281 attccatgcg gggtatcgta tgcttccttc agcactaccc tttagctgtt ctatatgctg
     5341 ccactcctca attggattag tctcatcctt caatgctatc atttcctttg atattggatc
     5401 atggtagaca acccttaata taacttcgta taatgtatgc tatacgaagt tattaggtct
     5461 agagatctgt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac atggaggccc
     5521 agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt gactgtcgcc
     5581 cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat tttgatggcc
     5641 gcacggcgcg aagcaaaaat tacggctcct cgctgcagac ctgcgagcag ggaaacgctc
     5701 ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat ataaaaggtt
     5761 aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg ctaggataca
     5821 gttctcacat cacatccgaa cataaacaac catgggtaag gaaaagactc acgtttcgag
     5881 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     5941 tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt
     6001 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     6061 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     6121 tgatgcatgg ttactcacca ctgcgatccc cggcaaaaca gcattccagg tattagaaga
     6181 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     6241 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     6301 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     6361 ctggcctgtt gaacaagtct ggaaagaaat gcataagctt ttgccattct caccggattc
     6421 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     6481 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     6541 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     6601 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     6661 atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt atagtttttt
     6721 tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt ttttcgcctc
     6781 gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt
     6841 atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca tccagtgtcg
     6901 aaaacgagct ctcgagaacc cttaatataa cttcgtataa tgtatgctat acgaagttat
     6961 taggtgatat cagatccact a
//
