LOCUS       sequence-375900-        7638 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEF1 promoter controlled expression plasmid with G418 resistance for
            expressing ymNeongreen in budding yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7638)
  TITLE     Expression plasmids with KanMX/G418 marker
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7638)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCEV-G2-Km ymNeongreen
FEATURES             Location/Qualifiers
     source          1..7638
                     /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"
     regulatory      2061..2070
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     terminator      2532..2721
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2793..2810)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2964..3552)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3044..3063)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3723..4514,4515..4583))
                     /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     4346..4365
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4584..4688)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4715..6057
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5678..5725)
                     /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    6077..6110
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            6165..7521
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        6165..6508
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             6509..7318
                     /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(6576..6595)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      7324..7521
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    7584..7617
                     /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 ccgggatggt ctctaagggt gaagaagata acatggcttc tttgccagcc acccacgagt
     1861 tgcacatctt cggttccatt aacggtgtcg acttcgatat ggtcggtcaa ggtaccggta
     1921 acccaaacga cggttacgaa gaattgaact tgaagtctac caagggcgac ttgcagttct
     1981 ccccttggat tttggttcca cacatcggtt acggtttcca ccaatacttg ccatacccag
     2041 acggtatgtc cccattccaa gccgccatgg ttgacggttc tggttaccaa gtccacagaa
     2101 ccatgcaatt cgaagacggt gcttccttga ccgtgaacta cagatacacc tacgaaggtt
     2161 ctcacatcaa gggcgaagcc caagttaagg gtaccggttt ccctgctgac ggtccagtca
     2221 tgactaactc cttgaccgct gctgactggt gtcgttccaa gaagacctac ccaaacgata
     2281 agaccatcat ttccactttc aagtggtcct acaccaccgg taacggcaag agataccgct
     2341 ctaccgctcg taccacctac accttcgcca agccaatggc tgccaactac ttgaagaacc
     2401 agcctatgta cgtcttccgt aagaccgagc tgaagcattc caagactgaa ttgaacttca
     2461 aggaatggca aaaggctttc accgacgtca tgggtatgga cgaattgtac aagtaaccgc
     2521 ggctagctaa gatccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc
     2581 cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt
     2641 cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag
     2701 aaggttttgg gacgctcgaa gatccagctg cattaatgaa tcggccaacg cgcggggaga
     2761 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     2821 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     2881 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     2941 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3001 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3061 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3121 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3181 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3241 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3301 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3361 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     3421 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3481 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3541 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3601 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     3661 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     3721 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     3781 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     3841 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     3901 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     3961 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4021 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4081 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4141 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     4201 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4261 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     4321 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     4381 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     4441 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     4501 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     4561 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     4621 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     4681 gttccgcgca catttccccg aaaagtgcca cctgaacgaa gcatctgtgc ttcattttgt
     4741 agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt
     4801 tacagaacag aaatgcaacg cgaaagcgct attttaccaa cgaagaatct gtgcttcatt
     4861 tttgtaaaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc
     4921 atttttacag aacagaaatg caacgcgaga gcgctatttt accaacaaag aatctatact
     4981 tcttttttgt tctacaaaaa tgcatcccga gagcgctatt tttctaacaa agcatcttag
     5041 attacttttt ttctcctttg tgcgctctat aatgcagtct cttgataact ttttgcactg
     5101 taggtccgtt aaggttagaa gaaggctact ttggtgtcta ttttctcttc cataaaaaaa
     5161 gcctgactcc acttcccgcg tttactgatt actagcgaag ctgcgggtgc attttttcaa
     5221 gataaaggca tccccgatta tattctatac cgatgtggat tgcgcatact ttgtgaacag
     5281 aaagtgatag cgttgatgat tcttcattgg tcagaaaatt atgaacggtt tcttctattt
     5341 tgtctctata tactacgtat aggaaatgtt tacattttcg tattgttttc gattcactct
     5401 atgaatagtt cttactacaa tttttttgtc taaagagtaa tactagagat aaacataaaa
     5461 aatgtagagg tcgagtttag atgcaagttc aaggagcgaa aggtggatgg gtaggttata
     5521 tagggatata gcacagagat atatagcaaa gagatacttt tgagcaatgt ttgtggaagc
     5581 ggtattcgca atattttagt agctcgttac agtccggtgc gtttttggtt ttttgaaagt
     5641 gcgtcttcag agcgcttttg gttttcaaaa gcgctctgaa gttcctatac tttctagaga
     5701 ataggaactt cggaatagga acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc
     5761 aacgcgagct gcgcacatac agctcactgt tcacgtcgca cctatatctg cgtgttgcct
     5821 gtatatatat atacatgaga agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta
     5881 tatgcgtcta tttatgtagg atgaaaggta gtctagtacc tcctgtgata ttatcccatt
     5941 ccatgcgggg tatcgtatgc ttccttcagc actacccttt agctgttcta tatgctgcca
     6001 ctcctcaatt ggattagtct catccttcaa tgctatcatt tcctttgata ttggatcatg
     6061 gtagacaacc cttaatataa cttcgtataa tgtatgctat acgaagttat taggtctaga
     6121 gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg gaggcccaga
     6181 ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt
     6241 acatttagcc catacatccc catgtataat catttgcatc catacatttt gatggccgca
     6301 cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga aacgctcccc
     6361 tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata aaaggttagg
     6421 atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta ggatacagtt
     6481 ctcacatcac atccgaacat aaacaaccat gggtaaggaa aagactcacg tttcgaggcc
     6541 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     6601 cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt
     6661 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     6721 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     6781 tgcatggtta ctcaccactg cgatccccgg caaaacagca ttccaggtat tagaagaata
     6841 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     6901 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     6961 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     7021 gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac cggattcagt
     7081 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     7141 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     7201 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     7261 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc
     7321 agtactgaca ataaaaagat tcttgttttc aagaacttgt catttgtata gtttttttat
     7381 attgtagttg ttctatttta atcaaatgtt agcgtgattt atattttttt tcgcctcgac
     7441 atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt caatcgtatg
     7501 tgaatgctgg tcgctatact gctgtcgatt cgatactaac gccgccatcc agtgtcgaaa
     7561 acgagctctc gagaaccctt aatataactt cgtataatgt atgctatacg aagttattag
     7621 gtgatatcag atccacta
//
