LOCUS       sequence-225011-        5675 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 5675)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 5675)
  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: SLAC1-FL
FEATURES             Location/Qualifiers
     source          1..5675
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..939
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             2617..2637
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(2644..2646,2647..2649,2650..3360)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2644..2646,2647..2649,2650..3360)
                     /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(2689..2710)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2950..2969)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3297..3318
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3367..3390
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3400..3417
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3497..3743
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3758..4171
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        4178..4225
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4244..4618
                     /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      4684..4931
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4700..4718)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(5006..5594)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5086..5105)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gatctaacat ccaaagacga aaggttgaat gaaacctttt tgccatccga catccacagg
       61 tccattctca cacataagtg ccaaacgcaa caggagggga tacactagca gcagaccgtt
      121 gcaaacgcag gacctccact cctcttctcc tcaacaccca cttttgccat cgaaaaacca
      181 gcccagttat tgggcttgat tggagctcgc tcattccaat tccttctatt aggctactaa
      241 caccatgact ttattagcct gtctatcctg gcccccctgg cgaggttcat gtttgtttat
      301 ttccgaatgc aacaagctcc gcattacacc cgaacatcac tccagatgag ggctttctga
      361 gtgtggggtc aaatagtttc atgttcccca aatggcccaa aactgacagt ttaaacgctg
      421 tcttggaacc taatatgaca aaagcgtgat ctcatccaag atgaactaag tttggttcgt
      481 tgaaatgcta acggccagtt ggtcaaaaag aaacttccaa aagtcggcat accgtttgtc
      541 ttgtttggta ttgattgacg aatgctcaaa aataatctca ttaatgctta gcgcagtctc
      601 tctatcgctt ctgaaccccg gtgcacctgt gccgaaacgc aaatggggaa acacccgctt
      661 tttggatgat tatgcattgt ctccacattg tatgcttcca agattctggt gggaatactg
      721 ctgatagcct aacgttcatg atcaaaattt aactgttcta acccctactt gacagcaata
      781 tataaacaga aggaagctgc cctgtcttaa accttttttt ttatcatcat tattagctta
      841 ctttcataat tgcgactggt tccaattgac aagcttttga ttttaacgac ttttaacgac
      901 aacttgagaa gatcaaaaaa caactaatta ttcgaaatgg agaggaaaca gtcaaatgct
      961 cattccactt tcgccgacat caatgaagtt gaagatgaag cagagcaaga actgcaacag
     1021 caagagaaca acaacaacaa gagattctct ggaaacagag gaccaaaccg agggaaacaa
     1081 agaccattca gaggttttag cagacaggtt tcgctagaga caggtttctc agtgctcaac
     1141 agagaatcta gagaaagaga tgacaaaaag agtttaccga ggagtggtcg tagctttggc
     1201 gggtttgaat caggtggaat catcaacggt ggagatggtc ggaaaacaga ttttagtatg
     1261 tttaggacta aatcaacgct cagcaaacaa aagtctttat tgccttctat aataagggag
     1321 agagacattg agaattcttt aagaactgaa gatggtgaga ccaaagatga ttctataaat
     1381 gaaaatgttt ctgcaggaag atactttgct gctcttagag gacctgaatt ggatgaagtc
     1441 aaggacaatg aagatattct gcttccaaaa gaggagcaat ggccgtttct cctaaggttc
     1501 ccaattggat gcttcggtat ctgtttggga cttagcagtc aagctgtctt gtggcttgcc
     1561 ctagcgaaaa gtcctgcaac aaacttttta cacatcacac cattgattaa cttagttgta
     1621 tggctatttt cactggtagt tttagtctct gtgtccttca cttacatact caaatgcatc
     1681 ttctactttg aagctgtcaa aagagagtat tttcatcctg ttcgagtcaa cttcttcttc
     1741 gctccttggg tggtttgcat gttcctagcc attagcgtac ctcccatgtt ctcaccaaac
     1801 cgaaaatacc tccatcccgc catttggtgt gttttcatgg gtccttattt cttcctcgag
     1861 ctaaagatct acgggcaatg gctttcggga gggaagaggc gactatgcaa agtcgcaaac
     1921 ccatcttctc atctttctgt tgtagggaat ttcgtcgggg ccatcttagc ttcaaaagtt
     1981 ggatgggatg aagtagctaa gtttctatgg gcagtaggtt ttgcacatta cttggttgtg
     2041 tttgtaacac tttatcagag acttcccacc agtgaagctc ttcctaaaga gcttcatcct
     2101 gtttactcta tgttcattgc tgccccatca gcagcaagta ttgcttggaa cacgatatat
     2161 ggccaattcg acggatgttc aagaacttgc ttcttcattg cactcttcct atacatatcc
     2221 ctagttgctc ggatcaattt cttcactgga tttaagttct cagtggcgtg gtggtcatat
     2281 acttttccta tgacaacagc atccgtagca acaataaagt atgcagaggc agtacctggt
     2341 tacccgagcc gggctctagc actcactctt tccttcattt ccacagcgat ggtctgcgtt
     2401 ttgtttgtct ccacgcttct ccacgccttt gtctggcaaa cgttgttccc aaacgatctt
     2461 gcaattgcca ttacaaagag gaaacttacc agggagaaga agccttttaa aagagcttat
     2521 gacttgaagc gctggaccaa gcaggctctg gcgaagaaaa tatctgcaga aaaagatttt
     2581 gaagcagagg aagagtcgca tcacgtcgag ggttcggaga acctgtactt ccaatctggt
     2641 tcgatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2701 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2761 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     2821 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     2881 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2941 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     3001 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     3061 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     3121 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     3181 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     3241 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     3301 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3361 tcgtcgcatc atcatcatca tcatcatcat taagtcgacc atcatcatca tcatcattga
     3421 gtttgtagcc ttagacatga ctgttcctca gttcaagttg ggcacttacg agaagaccgg
     3481 tcttgctaga ttctaatcaa gaggatgtca gaatgccatt tgcctgagag atgcaggctt
     3541 catttttgat acttttttat ttgtaaccta tatagtatag gatttttttt gtcattttgt
     3601 ttcttctcgt acgagcttgc tcctgatcag cctatctcgc agctgatgaa tatcttgtgg
     3661 taggggtttg ggaaaatcat tcgagtttga tgtttttctt ggtatttccc actcctcttc
     3721 agagtacaga agattaagtg agaccttcgt ttgtgcggat cccccacaca ccatagcttc
     3781 aaaatgtttc tactcctttt ttactcttcc agattttctc ggactccgcg catcgccgta
     3841 ccacttcaaa acacccaagc acagcatact aaatttcccc tctttcttcc tctagggtgt
     3901 cgttaattac ccgtactaaa ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc
     3961 ttcgtcgaaa aaggcaataa aaatttttat cacgtttctt tttcttgaaa attttttttt
     4021 ttgatttttt tctctttcga tgacctccca ttgatattta agttaataaa cggtcttcaa
     4081 tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac ttcttgctca
     4141 ttagaaagaa agcatagcaa tctaatctaa gggcggtgtt gacaattaat catcggcata
     4201 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag
     4261 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
     4321 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
     4381 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
     4441 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
     4501 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
     4561 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
     4621 cgtccgacgc ggcccgacgg gtccgaggcc tcggagatcc gtcccccttt tcctttgtcg
     4681 atatcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
     4741 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     4801 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     4861 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
     4921 ctttaatttg caagctggag accaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4981 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5041 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5101 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5161 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5221 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5281 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5341 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5401 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5461 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5521 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5581 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5641 aaaactcacg ttaagggatt ttggtcatga gatca
//
