LOCUS       sequence-224993-        5602 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 5602)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 5602)
  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: MtPT4-eGFP
FEATURES             Location/Qualifiers
     source          1..5602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1605..1625
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1632..1634,1635..1637,1638..2348)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1632..1634,1635..1637,1638..2348)
                     /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(1677..1698)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1938..1957)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2285..2306
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2355..2378
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2388..2405
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2485..2731
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2746..3159
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3166..3213
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3232..3606
                     /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      3672..3919
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3688..3706)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3994..4582)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4074..4093)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4664..5602
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
ORIGIN
        1 aggaattcat gggattagaa gtccttgagg ctcttgattc agctcgtaca caatggtacc
       61 atgtaacagc tatagtaatt gctggcatgg gattcttcac tgatgcctat gatctgttct
      121 gcatttcaac cgtttcgaag ctcttgggac gattatatta ctttgatcca agcactaaca
      181 aaccagggaa gcttccacca tcagttaata atgtagtcac cggtgtggcc cttgtcggaa
      241 cactttctgg ccaattagtc ttcggctggc tcggagacaa acttggacgc aagaaagttt
      301 atggtgtcac actcattatc atggttgctt gtgccatttg ctctggtctt tcttttggtt
      361 cttccgcgaa atctgttatg ataacccttt gttttttcag gttttggctt ggatttggta
      421 ttggtggaga ctacccttta tcagcaacca tcatgtctga atacgccaac aaaaggacac
      481 gaggcgcttt catagcagca gttttcgcaa tgcaaggcgt gggaatcatc tttgccggat
      541 tggtttcaat ggtattttca ggaattttta aagcatacta ccaggctccc cgtttcaatg
      601 aagacccaat tttatcaaca cagcccgaag gggatttact ctggcgatta attctcatga
      661 tcggtgctgt tccagctgcg atgacttact actggagaat gaaaatgcct gaaaccggtc
      721 gttacactgc aatcgtagaa gggaacgcga aacaagccgc tgcagacatg gcaagagttt
      781 tggacatcga aatcatagcg gagcaagata agttggctga atttaaagca gcaaatgatt
      841 accctctttg gtctagcgag tttttcaaca ggcacggtcg tcatttaatc ggtacaatga
      901 gttgttggtt cttgttagat attgctttct acagtcagaa tttgacacag aaagatattt
      961 accctgcaat gggacttatt cgtcaagaca aagaaatgaa tgctattgat gaagttttcc
     1021 aaacttcgcg cgccatgttt gttgtcgcct tgtttggaac attccccggt tattggttca
     1081 ctgttttctt cattgaaaag cttggaagat tcaagataca gttggttggt ttcttcatga
     1141 tgtctttctt catgtttgtc attggtgtta aatatgaata cttgaaagac gaaaacaaaa
     1201 acctcttcgc tcttttatac gggctaacat tcttcttcgc gaactttggg cctaatagta
     1261 caacttttgt gttgcccgct gagctttttc ctacgcgggt gaggtcaaca tgccatgctt
     1321 tcagtgctgc atctgggaag gcgggtgcta tggttggtgc gttcgggata caatactaca
     1381 cgttggatgg tacgccaagg aagattagac gcgcaatgat gattctggct tttacaaatt
     1441 tgataggatt cttttgcacg ttcttggtga cggaaactaa gggacgatct ttggaagaga
     1501 tttcgggtga ggatggaaga gagagtgaac tcactgcaac accaaatgac agggctccgg
     1561 ggattcgcca ggactcaaga actgagaaga tgctcgaggg ttcggagaac ctgtacttcc
     1621 aatctggttc gatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1681 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1741 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1801 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     1861 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1921 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1981 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     2041 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     2101 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     2161 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2221 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     2281 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2341 tgtacaagtc gtcgcatcat catcatcatc atcatcatta agtcgaccat catcatcatc
     2401 atcattgagt ttgtagcctt agacatgact gttcctcagt tcaagttggg cacttacgag
     2461 aagaccggtc ttgctagatt ctaatcaaga ggatgtcaga atgccatttg cctgagagat
     2521 gcaggcttca tttttgatac ttttttattt gtaacctata tagtatagga ttttttttgt
     2581 cattttgttt cttctcgtac gagcttgctc ctgatcagcc tatctcgcag ctgatgaata
     2641 tcttgtggta ggggtttggg aaaatcattc gagtttgatg tttttcttgg tatttcccac
     2701 tcctcttcag agtacagaag attaagtgag accttcgttt gtgcggatcc cccacacacc
     2761 atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca
     2821 tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc
     2881 tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt
     2941 tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaaat
     3001 tttttttttt gatttttttc tctttcgatg acctcccatt gatatttaag ttaataaacg
     3061 gtcttcaatt tctcaagttt cagtttcatt tttcttgttc tattacaact ttttttactt
     3121 cttgctcatt agaaagaaag catagcaatc taatctaagg gcggtgttga caattaatca
     3181 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag
     3241 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
     3301 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
     3361 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
     3421 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
     3481 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
     3541 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
     3601 gactgacacg tccgacgcgg cccgacgggt ccgaggcctc ggagatccgt cccccttttc
     3661 ctttgtcgat atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc
     3721 cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt
     3781 tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta
     3841 cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg
     3901 ctcgaaggct ttaatttgca agctggagac caacatgtga gcaaaaggcc agcaaaaggc
     3961 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4021 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4081 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4141 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4201 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4261 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4321 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4381 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4441 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4501 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4561 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4621 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga tcagatctaa catccaaaga
     4681 cgaaaggttg aatgaaacct ttttgccatc cgacatccac aggtccattc tcacacataa
     4741 gtgccaaacg caacaggagg ggatacacta gcagcagacc gttgcaaacg caggacctcc
     4801 actcctcttc tcctcaacac ccacttttgc catcgaaaaa ccagcccagt tattgggctt
     4861 gattggagct cgctcattcc aattccttct attaggctac taacaccatg actttattag
     4921 cctgtctatc ctggcccccc tggcgaggtt catgtttgtt tatttccgaa tgcaacaagc
     4981 tccgcattac acccgaacat cactccagat gagggctttc tgagtgtggg gtcaaatagt
     5041 ttcatgttcc ccaaatggcc caaaactgac agtttaaacg ctgtcttgga acctaatatg
     5101 acaaaagcgt gatctcatcc aagatgaact aagtttggtt cgttgaaatg ctaacggcca
     5161 gttggtcaaa aagaaacttc caaaagtcgg cataccgttt gtcttgtttg gtattgattg
     5221 acgaatgctc aaaaataatc tcattaatgc ttagcgcagt ctctctatcg cttctgaacc
     5281 ccggtgcacc tgtgccgaaa cgcaaatggg gaaacacccg ctttttggat gattatgcat
     5341 tgtctccaca ttgtatgctt ccaagattct ggtgggaata ctgctgatag cctaacgttc
     5401 atgatcaaaa tttaactgtt ctaaccccta cttgacagca atatataaac agaaggaagc
     5461 tgccctgtct taaacctttt tttttatcat cattattagc ttactttcat aattgcgact
     5521 ggttccaatt gacaagcttt tgattttaac gacttttaac gacaacttga gaagatcaaa
     5581 aaacaactaa ttattcgaaa cg
//
