LOCUS       sequence-224953-        5281 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 5281)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 5281)
  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: MCA1-eGFP
FEATURES             Location/Qualifiers
     source          1..5281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1284..1304
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1311..1313,1314..1316,1317..2027)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1311..1313,1314..1316,1317..2027)
                     /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(1356..1377)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1617..1636)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1964..1985
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2034..2057
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2067..2084
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2164..2410
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2425..2838
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2845..2892
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2911..3285
                     /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      3351..3598
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3367..3385)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3673..4261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3753..3772)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4343..5281
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
ORIGIN
        1 aggaattcat gtcacattca tgggatggtc ttggggagat tgcgtcagtg gctcagctga
       61 ctggtttaga tgcggtcaag ttgattggtc tcatagtcaa agctgcgaat actgcttgga
      121 tgcataagaa gaattgtcgt caattcgctc aacatctcaa actcattggg aatttgcttg
      181 aacagctgaa gatctctgag atgaagaagt atcctgagac tcgagagcct ctcgaagggc
      241 ttgaggatgc tttgagaagg tcttaccttc tggtcaatag ctgtagagac cggagctatc
      301 tttacttgtt ggctatgggt tggaacattg tttaccagtt caggaaacat caggatgaga
      361 ttgatcgttt tcttaagatc atacctctca tcactttggt cgataacgct cgcattcggg
      421 agaggtttga gtatattgac cgtgatcagc gtgagtacac tctggatgaa gaggatagac
      481 atgtgcaaga tgttattttg aaacaagaat ccaccagaga agcagcatca gtactgaaga
      541 agactctctc ttgctcatac cctaacttgc gtttctgtga agctctcaaa acagaaaacg
      601 agaaactgca gatcgaactt cagcggtcgc aggagcatta tgatgtggct cagtgtgaag
      661 tcattcagcg tttaattggt gttacacaag ctgctgctgc tgttgaacct gactctgaga
      721 aggagctgac aaagaaagct tccaaaaaat ctgaacgtag ctcaagcatg aagacggaat
      781 attcgtatga cgaagattct cccaagaaaa gcagtactcg tgcagcttcg agatccactt
      841 ctaatgtttc atctgggcat gatctgcttt cacgaagagc atcacaggca caacaccatg
      901 aagaatggca taccgactta ctagcttgtt gctcggaacc ttctctttgc ttcaagacat
      961 tctttttccc gtgtggtact ttggcaaaga ttgccactgc agcatccaac aggcacatct
     1021 cttcagctga agcatgtaat gagctaatgg cgtattcttt gatactttct tgttgctgct
     1081 atacttgttg cgtgaggagg aaactccgga agacactgaa catcacggga ggattcattg
     1141 acgattttct atctcatgta atgtgttgtt gctgtgccct tgtacaagaa ctgagagaag
     1201 ttgagattcg tggggcatat ggtacggaga agacgaaaat aagcccgcct tcgtcgcagt
     1261 tcatggaaca tgtcgagggt tcggagaacc tgtacttcca atctggttcg atggtgagca
     1321 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1381 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     1441 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1501 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     1561 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     1621 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1681 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1741 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     1801 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     1861 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     1921 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1981 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcg tcgcatcatc
     2041 atcatcatca tcatcattaa gtcgaccatc atcatcatca tcattgagtt tgtagcctta
     2101 gacatgactg ttcctcagtt caagttgggc acttacgaga agaccggtct tgctagattc
     2161 taatcaagag gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact
     2221 tttttatttg taacctatat agtataggat tttttttgtc attttgtttc ttctcgtacg
     2281 agcttgctcc tgatcagcct atctcgcagc tgatgaatat cttgtggtag gggtttggga
     2341 aaatcattcg agtttgatgt ttttcttggt atttcccact cctcttcaga gtacagaaga
     2401 ttaagtgaga ccttcgtttg tgcggatccc ccacacacca tagcttcaaa atgtttctac
     2461 tcctttttta ctcttccaga ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca
     2521 cccaagcaca gcatactaaa tttcccctct ttcttcctct agggtgtcgt taattacccg
     2581 tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc gtcgaaaaag
     2641 gcaataaaaa tttttatcac gtttcttttt cttgaaaatt tttttttttg atttttttct
     2701 ctttcgatga cctcccattg atatttaagt taataaacgg tcttcaattt ctcaagtttc
     2761 agtttcattt ttcttgttct attacaactt tttttacttc ttgctcatta gaaagaaagc
     2821 atagcaatct aatctaaggg cggtgttgac aattaatcat cggcatagta tatcggcata
     2881 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg
     2941 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
     3001 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
     3061 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
     3121 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
     3181 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
     3241 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt ccgacgcggc
     3301 ccgacgggtc cgaggcctcg gagatccgtc ccccttttcc tttgtcgata tcatgtaatt
     3361 agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg
     3421 agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag
     3481 aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa
     3541 cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa
     3601 gctggagacc aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3661 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3721 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3781 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3841 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3901 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3961 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4021 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4081 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4141 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4201 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4261 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4321 agggattttg gtcatgagat cagatctaac atccaaagac gaaaggttga atgaaacctt
     4381 tttgccatcc gacatccaca ggtccattct cacacataag tgccaaacgc aacaggaggg
     4441 gatacactag cagcagaccg ttgcaaacgc aggacctcca ctcctcttct cctcaacacc
     4501 cacttttgcc atcgaaaaac cagcccagtt attgggcttg attggagctc gctcattcca
     4561 attccttcta ttaggctact aacaccatga ctttattagc ctgtctatcc tggcccccct
     4621 ggcgaggttc atgtttgttt atttccgaat gcaacaagct ccgcattaca cccgaacatc
     4681 actccagatg agggctttct gagtgtgggg tcaaatagtt tcatgttccc caaatggccc
     4741 aaaactgaca gtttaaacgc tgtcttggaa cctaatatga caaaagcgtg atctcatcca
     4801 agatgaacta agtttggttc gttgaaatgc taacggccag ttggtcaaaa agaaacttcc
     4861 aaaagtcggc ataccgtttg tcttgtttgg tattgattga cgaatgctca aaaataatct
     4921 cattaatgct tagcgcagtc tctctatcgc ttctgaaccc cggtgcacct gtgccgaaac
     4981 gcaaatgggg aaacacccgc tttttggatg attatgcatt gtctccacat tgtatgcttc
     5041 caagattctg gtgggaatac tgctgatagc ctaacgttca tgatcaaaat ttaactgttc
     5101 taacccctac ttgacagcaa tatataaaca gaaggaagct gccctgtctt aaaccttttt
     5161 ttttatcatc attattagct tactttcata attgcgactg gttccaattg acaagctttt
     5221 gattttaacg acttttaacg acaacttgag aagatcaaaa aacaactaat tattcgaaac
     5281 g
//
