LOCUS       sequence-225004-        5528 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  protein expression in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5528)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 5528)
  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-deltaC
FEATURES             Location/Qualifiers
     source          1..5528
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             1531..1551
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1558..1560,1561..1563,1564..2274)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1558..1560,1561..1563,1564..2274)
                     /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(1603..1624)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1864..1883)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2211..2232
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2281..2304
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2314..2331
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2411..2657
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2672..3085
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3092..3139
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3158..3532
                     /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      3598..3845
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3614..3632)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3920..4508)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4000..4019)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4590..5528
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
ORIGIN
        1 gagaggaaac agtcaaatgc tcattccact ttcgccgaca tcaatgaagt tgaagatgaa
       61 gcagagcaag aactgcaaca gcaagagaac aacaacaaca agagattctc tggaaacaga
      121 ggaccaaacc gagggaaaca aagaccattc agaggtttta gcagacaggt ttcgctagag
      181 acaggtttct cagtgctcaa cagagaatct agagaaagag atgacaaaaa gagtttaccg
      241 aggagtggtc gtagctttgg cgggtttgaa tcaggtggaa tcatcaacgg tggagatggt
      301 cggaaaacag attttagtat gtttaggact aaatcaacgc tcagcaaaca aaagtcttta
      361 ttgccttcta taataaggga gagagacatt gagaattctt taagaactga agatggtgag
      421 accaaagatg attctataaa tgaaaatgtt tctgcaggaa gatactttgc tgctcttaga
      481 ggacctgaat tggatgaagt caaggacaat gaagatattc tgcttccaaa agaggagcaa
      541 tggccgtttc tcctaaggtt cccaattgga tgcttcggta tctgtttggg acttagcagt
      601 caagctgtct tgtggcttgc cctagcgaaa agtcctgcaa caaacttttt acacatcaca
      661 ccattgatta acttagttgt atggctattt tcactggtag ttttagtctc tgtgtccttc
      721 acttacatac tcaaatgcat cttctacttt gaagctgtca aaagagagta ttttcatcct
      781 gttcgagtca acttcttctt cgctccttgg gtggtttgca tgttcctagc cattagcgta
      841 cctcccatgt tctcaccaaa ccgaaaatac ctccatcccg ccatttggtg tgttttcatg
      901 ggtccttatt tcttcctcga gctaaagatc tacgggcaat ggctttcggg agggaagagg
      961 cgactatgca aagtcgcaaa cccatcttct catctttctg ttgtagggaa tttcgtcggg
     1021 gccatcttag cttcaaaagt tggatgggat gaagtagcta agtttctatg ggcagtaggt
     1081 tttgcacatt acttggttgt gtttgtaaca ctttatcaga gacttcccac cagtgaagct
     1141 cttcctaaag agcttcatcc tgtttactct atgttcattg ctgccccatc agcagcaagt
     1201 attgcttgga acacgatata tggccaattc gacggatgtt caagaacttg cttcttcatt
     1261 gcactcttcc tatacatatc cctagttgct cggatcaatt tcttcactgg atttaagttc
     1321 tcagtggcgt ggtggtcata tacttttcct atgacaacag catccgtagc aacaataaag
     1381 tatgcagagg cagtacctgg ttacccgagc cgggctctag cactcactct ttccttcatt
     1441 tccacagcga tggtctgcgt tttgtttgtc tccacgcttc tccacgcctt tgtctggcaa
     1501 acgttgttcc caaacgatgt cgagggttcg gagaacctgt acttccaatc tggttcgatg
     1561 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1621 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1681 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1741 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1801 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1861 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1921 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1981 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2041 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2101 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2161 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2221 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtcgtcg
     2281 catcatcatc atcatcatca tcattaagtc gaccatcatc atcatcatca ttgagtttgt
     2341 agccttagac atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc
     2401 tagattctaa tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt
     2461 tgatactttt ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc
     2521 tcgtacgagc ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg
     2581 tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta
     2641 cagaagatta agtgagacct tcgtttgtgc ggatccccca cacaccatag cttcaaaatg
     2701 tttctactcc ttttttactc ttccagattt tctcggactc cgcgcatcgc cgtaccactt
     2761 caaaacaccc aagcacagca tactaaattt cccctctttc ttcctctagg gtgtcgttaa
     2821 ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc ctcgtttctt tttcttcgtc
     2881 gaaaaaggca ataaaaattt ttatcacgtt tctttttctt gaaaattttt ttttttgatt
     2941 tttttctctt tcgatgacct cccattgata tttaagttaa taaacggtct tcaatttctc
     3001 aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg ctcattagaa
     3061 agaaagcata gcaatctaat ctaagggcgg tgttgacaat taatcatcgg catagtatat
     3121 cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt
     3181 tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg
     3241 gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct
     3301 gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt
     3361 gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga
     3421 cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct
     3481 gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtccg
     3541 acgcggcccg acgggtccga ggcctcggag atccgtcccc cttttccttt gtcgatatca
     3601 tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa
     3661 aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata gttatgttag
     3721 tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg
     3781 catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg aaggctttaa
     3841 tttgcaagct ggagaccaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3901 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3961 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4021 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4081 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4141 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4201 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4261 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4321 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4381 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4441 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4501 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4561 cacgttaagg gattttggtc atgagatcag atctaacatc caaagacgaa aggttgaatg
     4621 aaaccttttt gccatccgac atccacaggt ccattctcac acataagtgc caaacgcaac
     4681 aggaggggat acactagcag cagaccgttg caaacgcagg acctccactc ctcttctcct
     4741 caacacccac ttttgccatc gaaaaaccag cccagttatt gggcttgatt ggagctcgct
     4801 cattccaatt ccttctatta ggctactaac accatgactt tattagcctg tctatcctgg
     4861 cccccctggc gaggttcatg tttgtttatt tccgaatgca acaagctccg cattacaccc
     4921 gaacatcact ccagatgagg gctttctgag tgtggggtca aatagtttca tgttccccaa
     4981 atggcccaaa actgacagtt taaacgctgt cttggaacct aatatgacaa aagcgtgatc
     5041 tcatccaaga tgaactaagt ttggttcgtt gaaatgctaa cggccagttg gtcaaaaaga
     5101 aacttccaaa agtcggcata ccgtttgtct tgtttggtat tgattgacga atgctcaaaa
     5161 ataatctcat taatgcttag cgcagtctct ctatcgcttc tgaaccccgg tgcacctgtg
     5221 ccgaaacgca aatggggaaa cacccgcttt ttggatgatt atgcattgtc tccacattgt
     5281 atgcttccaa gattctggtg ggaatactgc tgatagccta acgttcatga tcaaaattta
     5341 actgttctaa cccctacttg acagcaatat ataaacagaa ggaagctgcc ctgtcttaaa
     5401 cctttttttt tatcatcatt attagcttac tttcataatt gcgactggtt ccaattgaca
     5461 agcttttgat tttaacgact tttaacgaca acttgagaag atcaaaaaac aactaattat
     5521 tcgaaatg
//
