LOCUS       sequence-224984-        4994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein expression in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4994)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 4994)
  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"
COMMENT     Sequence Label: SLAC-deltaCdeltaN
FEATURES             Location/Qualifiers
     source          1..4994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             997..1017
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1024..1026,1027..1029,1030..1740)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1024..1026,1027..1029,1030..1740)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1069..1090)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1330..1349)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1677..1698
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1747..1770
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1780..1797
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1877..2123
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2138..2551
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2558..2605
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2624..2998
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      3064..3311
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3080..3098)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3386..3974)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3466..3485)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4056..4992
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
ORIGIN
        1 gagcaatggc cgtttctcct aaggttccca attggatgct tcggtatctg tttgggactt
       61 agcagtcaag ctgtcttgtg gcttgcccta gcgaaaagtc ctgcaacaaa ctttttacac
      121 atcacaccat tgattaactt agttgtatgg ctattttcac tggtagtttt agtctctgtg
      181 tccttcactt acatactcaa atgcatcttc tactttgaag ctgtcaaaag agagtatttt
      241 catcctgttc gagtcaactt cttcttcgct ccttgggtgg tttgcatgtt cctagccatt
      301 agcgtacctc ccatgttctc accaaaccga aaatacctcc atcccgccat ttggtgtgtt
      361 ttcatgggtc cttatttctt cctcgagcta aagatctacg ggcaatggct ttcgggaggg
      421 aagaggcgac tatgcaaagt cgcaaaccca tcttctcatc tttctgttgt agggaatttc
      481 gtcggggcca tcttagcttc aaaagttgga tgggatgaag tagctaagtt tctatgggca
      541 gtaggttttg cacattactt ggttgtgttt gtaacacttt atcagagact tcccaccagt
      601 gaagctcttc ctaaagagct tcatcctgtt tactctatgt tcattgctgc cccatcagca
      661 gcaagtattg cttggaacac gatatatggc caattcgacg gatgttcaag aacttgcttc
      721 ttcattgcac tcttcctata catatcccta gttgctcgga tcaatttctt cactggattt
      781 aagttctcag tggcgtggtg gtcatatact tttcctatga caacagcatc cgtagcaaca
      841 ataaagtatg cagaggcagt acctggttac ccgagccggg ctctagcact cactctttcc
      901 ttcatttcca cagcgatggt ctgcgttttg tttgtctcca cgcttctcca cgcctttgtc
      961 tggcaaacgt tgttcccaaa cgatgtcgag ggttcggaga acctgtactt ccaatctggt
     1021 tcgatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1081 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1141 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1201 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1261 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1321 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1381 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1441 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     1501 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     1561 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1621 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     1681 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     1741 tcgtcgcatc atcatcatca tcatcatcat taagtcgacc atcatcatca tcatcattga
     1801 gtttgtagcc ttagacatga ctgttcctca gttcaagttg ggcacttacg agaagaccgg
     1861 tcttgctaga ttctaatcaa gaggatgtca gaatgccatt tgcctgagag atgcaggctt
     1921 catttttgat acttttttat ttgtaaccta tatagtatag gatttttttt gtcattttgt
     1981 ttcttctcgt acgagcttgc tcctgatcag cctatctcgc agctgatgaa tatcttgtgg
     2041 taggggtttg ggaaaatcat tcgagtttga tgtttttctt ggtatttccc actcctcttc
     2101 agagtacaga agattaagtg agaccttcgt ttgtgcggat cccccacaca ccatagcttc
     2161 aaaatgtttc tactcctttt ttactcttcc agattttctc ggactccgcg catcgccgta
     2221 ccacttcaaa acacccaagc acagcatact aaatttcccc tctttcttcc tctagggtgt
     2281 cgttaattac ccgtactaaa ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc
     2341 ttcgtcgaaa aaggcaataa aaatttttat cacgtttctt tttcttgaaa attttttttt
     2401 ttgatttttt tctctttcga tgacctccca ttgatattta agttaataaa cggtcttcaa
     2461 tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac ttcttgctca
     2521 ttagaaagaa agcatagcaa tctaatctaa gggcggtgtt gacaattaat catcggcata
     2581 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag
     2641 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
     2701 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
     2761 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
     2821 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
     2881 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
     2941 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
     3001 cgtccgacgc ggcccgacgg gtccgaggcc tcggagatcc gtcccccttt tcctttgtcg
     3061 atatcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
     3121 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     3181 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     3241 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
     3301 ctttaatttg caagctggag accaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3361 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3421 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3481 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3541 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3601 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3661 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3721 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3781 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3841 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3901 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3961 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4021 aaaactcacg ttaagggatt ttggtcatga gatcagatct aacatccaaa gacgaaaggt
     4081 tgaatgaaac ctttttgcca tccgacatcc acaggtccat tctcacacat aagtgccaaa
     4141 cgcaacagga ggggatacac tagcagcaga ccgttgcaaa cgcaggacct ccactcctct
     4201 tctcctcaac acccactttt gccatcgaaa aaccagccca gttattgggc ttgattggag
     4261 ctcgctcatt ccaattcctt ctattaggct actaacacca tgactttatt agcctgtcta
     4321 tcctggcccc cctggcgagg ttcatgtttg tttatttccg aatgcaacaa gctccgcatt
     4381 acacccgaac atcactccag atgagggctt tctgagtgtg gggtcaaata gtttcatgtt
     4441 ccccaaatgg cccaaaactg acagtttaaa cgctgtcttg gaacctaata tgacaaaagc
     4501 gtgatctcat ccaagatgaa ctaagtttgg ttcgttgaaa tgctaacggc cagttggtca
     4561 aaaagaaact tccaaaagtc ggcataccgt ttgtcttgtt tggtattgat tgacgaatgc
     4621 tcaaaaataa tctcattaat gcttagcgca gtctctctat cgcttctgaa ccccggtgca
     4681 cctgtgccga aacgcaaatg gggaaacacc cgctttttgg atgattatgc attgtctcca
     4741 cattgtatgc ttccaagatt ctggtgggaa tactgctgat agcctaacgt tcatgatcaa
     4801 aatttaactg ttctaacccc tacttgacag caatatataa acagaaggaa gctgccctgt
     4861 cttaaacctt tttttttatc atcattatta gcttactttc ataattgcga ctggttccaa
     4921 ttgacaagct tttgatttta acgactttta acgacaactt gagaagatca aaaaacaact
     4981 aattattcga aatg
//
