LOCUS       sequence-224992-        5141 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 5141)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 5141)
  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-deltaN
FEATURES             Location/Qualifiers
     source          1..5141
                     /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             2083..2103
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(2110..2112,2113..2115,2116..2826)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2110..2112,2113..2115,2116..2826)
                     /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(2155..2176)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2416..2435)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2763..2784
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2833..2856
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2866..2883
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2963..3209
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3224..3637
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3644..3691
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3710..4084
                     /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      4150..4397
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4166..4184)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4472..5060)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4552..4571)
                     /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 agcaatggcc gtttctccta
      961 aggttcccaa ttggatgctt cggtatctgt ttgggactta gcagtcaagc tgtcttgtgg
     1021 cttgccctag cgaaaagtcc tgcaacaaac tttttacaca tcacaccatt gattaactta
     1081 gttgtatggc tattttcact ggtagtttta gtctctgtgt ccttcactta catactcaaa
     1141 tgcatcttct actttgaagc tgtcaaaaga gagtattttc atcctgttcg agtcaacttc
     1201 ttcttcgctc cttgggtggt ttgcatgttc ctagccatta gcgtacctcc catgttctca
     1261 ccaaaccgaa aatacctcca tcccgccatt tggtgtgttt tcatgggtcc ttatttcttc
     1321 ctcgagctaa agatctacgg gcaatggctt tcgggaggga agaggcgact atgcaaagtc
     1381 gcaaacccat cttctcatct ttctgttgta gggaatttcg tcggggccat cttagcttca
     1441 aaagttggat gggatgaagt agctaagttt ctatgggcag taggttttgc acattacttg
     1501 gttgtgtttg taacacttta tcagagactt cccaccagtg aagctcttcc taaagagctt
     1561 catcctgttt actctatgtt cattgctgcc ccatcagcag caagtattgc ttggaacacg
     1621 atatatggcc aattcgacgg atgttcaaga acttgcttct tcattgcact cttcctatac
     1681 atatccctag ttgctcggat caatttcttc actggattta agttctcagt ggcgtggtgg
     1741 tcatatactt ttcctatgac aacagcatcc gtagcaacaa taaagtatgc agaggcagta
     1801 cctggttacc cgagccgggc tctagcactc actctttcct tcatttccac agcgatggtc
     1861 tgcgttttgt ttgtctccac gcttctccac gcctttgtct ggcaaacgtt gttcccaaac
     1921 gatcttgcaa ttgccattac aaagaggaaa cttaccaggg agaagaagcc ttttaaaaga
     1981 gcttatgact tgaagcgctg gaccaagcag gctctggcga agaaaatatc tgcagaaaaa
     2041 gattttgaag cagaggaaga gtcgcatcac gtcgagggtt cggagaacct gtacttccaa
     2101 tctggttcga tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     2161 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     2221 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     2281 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     2341 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2401 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2461 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2521 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     2581 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     2641 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2701 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     2761 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2821 tacaagtcgt cgcatcatca tcatcatcat catcattaag tcgaccatca tcatcatcat
     2881 cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa
     2941 gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc
     3001 aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca
     3061 ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc
     3121 ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc
     3181 ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatcccc cacacaccat
     3241 agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc
     3301 gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt tcttcctcta
     3361 gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc
     3421 tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaaattt
     3481 ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt aataaacggt
     3541 cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt ttttacttct
     3601 tgctcattag aaagaaagca tagcaatcta atctaagggc ggtgttgaca attaatcatc
     3661 ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt
     3721 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac
     3781 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga
     3841 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc
     3901 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac
     3961 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg
     4021 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga
     4081 ctgacacgtc cgacgcggcc cgacgggtcc gaggcctcgg agatccgtcc cccttttcct
     4141 ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg
     4201 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta
     4261 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca
     4321 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct
     4381 cgaaggcttt aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca
     4441 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4501 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4561 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4621 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4681 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4741 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4801 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4861 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4921 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4981 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5041 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5101 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc a
//
