LOCUS       sequence-224996-        5266 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 5266)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 5266)
  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: MCA2-eGFP
FEATURES             Location/Qualifiers
     source          1..5266
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1269..1289
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1296..1298,1299..1301,1302..2012)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1296..1298,1299..1301,1302..2012)
                     /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(1341..1362)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1602..1621)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1949..1970
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2019..2042
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2052..2069
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2149..2395
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2410..2823
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2830..2877
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2896..3270
                     /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      3336..3583
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3352..3370)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3658..4246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3738..3757)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4328..5266
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
ORIGIN
        1 aggaattcat ggctaattca tgggatcagt taggagaaat tgcgtcagtg gctcagctta
       61 caggcataga cgctctgaaa ctgattggaa tgatagtaaa cgctgcgaat acagctcgaa
      121 tgcacaagaa gaattgtcgt caattcgctc accatcttaa gctcatccgt aatttgctag
      181 agcagattaa aaactctgag atgaatcaac gttcagagat tcttgagccg cttcaaggac
      241 ttgatgatgc tcttagaaga tcttacattt tagtcaaaag ttgtcaagaa aaaagttatc
      301 tttacttgtt ggctatgggc tggaacattg ttaaccagtt tgaaaaagct cagaatgaga
      361 ttgatctgtt tctcaagatc gttccactaa taaatatggc ggataatgct cggattcggg
      421 agaggctaga agctattgaa cgtgatcagc gtgagtatac tcttgatgaa gaggatagaa
      481 aggtgcaaga tgttattttg aaacaagaat ctactagaga agctgctaca tcggttttaa
      541 agaagacgct ctctcgttca tacccgaaca tggggttctg cgaagcgctt aaaaccgagg
      601 aggagaaact gcaacttgag ttgcaacgtt cgcgggcgcg gtatgatgct gatcaatgtg
      661 aagttataca acgtttgatt gatgttacac aaactgctgc tactgttgaa cctaatcttg
      721 aaaaggtgtt gactaagaaa gaagagctta cctctagcaa gaagagagat gatttgtatg
      781 acacggatag tagtagtatt cgagcagatt caagatcaac gtcttatgtt tcatccggac
      841 atgaactact ttcgggaaga tcattgcaac accgtggaaa ttggcacgcc gacttgctgg
      901 attgttgttc agaaccttgt ctttgcttga agacactctt tttcccctgt ggtacattgg
      961 caaagatttc cactgtggca actagtaggc aaatctcttc aactgaagtg tgtaagaacc
     1021 taatcgtgta ttctttgata ctctcatgct gttgctatac ttgctgcata aggaagaagc
     1081 ttcgaaagac actgaacata acgggagggt gcattgacga ctttttgtca catctaatgt
     1141 gttgttgttg tgcccttgtc caagaactga gagaagtcga gattcatcga gcttcttacg
     1201 gtacagagaa gagtaataag gaaatgagtc caccaacacc tcagtttatg gaagaactcg
     1261 agggttcgga gaacctgtac ttccaatctg gttcgatggt gagcaagggc gaggagctgt
     1321 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1381 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1441 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1501 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1561 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1621 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1681 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1741 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     1801 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1861 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     1921 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     1981 ggatcactct cggcatggac gagctgtaca agtcgtcgca tcatcatcat catcatcatc
     2041 attaagtcga ccatcatcat catcatcatt gagtttgtag ccttagacat gactgttcct
     2101 cagttcaagt tgggcactta cgagaagacc ggtcttgcta gattctaatc aagaggatgt
     2161 cagaatgcca tttgcctgag agatgcaggc ttcatttttg atactttttt atttgtaacc
     2221 tatatagtat aggatttttt ttgtcatttt gtttcttctc gtacgagctt gctcctgatc
     2281 agcctatctc gcagctgatg aatatcttgt ggtaggggtt tgggaaaatc attcgagttt
     2341 gatgtttttc ttggtatttc ccactcctct tcagagtaca gaagattaag tgagaccttc
     2401 gtttgtgcgg atcccccaca caccatagct tcaaaatgtt tctactcctt ttttactctt
     2461 ccagattttc tcggactccg cgcatcgccg taccacttca aaacacccaa gcacagcata
     2521 ctaaatttcc cctctttctt cctctagggt gtcgttaatt acccgtacta aaggtttgga
     2581 aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt
     2641 atcacgtttc tttttcttga aaattttttt ttttgatttt tttctctttc gatgacctcc
     2701 cattgatatt taagttaata aacggtcttc aatttctcaa gtttcagttt catttttctt
     2761 gttctattac aacttttttt acttcttgct cattagaaag aaagcatagc aatctaatct
     2821 aagggcggtg ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag
     2881 gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac
     2941 gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag
     3001 gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac
     3061 caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac
     3121 gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc
     3181 gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc
     3241 gtgcacttcg tggccgagga gcaggactga cacgtccgac gcggcccgac gggtccgagg
     3301 cctcggagat ccgtccccct tttcctttgt cgatatcatg taattagtta tgtcacgctt
     3361 acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga
     3421 agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt
     3481 tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac
     3541 cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcaagctgg agaccaacat
     3601 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3661 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3721 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3781 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3841 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3901 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3961 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4021 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4081 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4141 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4201 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4261 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4321 gagatcagat ctaacatcca aagacgaaag gttgaatgaa acctttttgc catccgacat
     4381 ccacaggtcc attctcacac ataagtgcca aacgcaacag gaggggatac actagcagca
     4441 gaccgttgca aacgcaggac ctccactcct cttctcctca acacccactt ttgccatcga
     4501 aaaaccagcc cagttattgg gcttgattgg agctcgctca ttccaattcc ttctattagg
     4561 ctactaacac catgacttta ttagcctgtc tatcctggcc cccctggcga ggttcatgtt
     4621 tgtttatttc cgaatgcaac aagctccgca ttacacccga acatcactcc agatgagggc
     4681 tttctgagtg tggggtcaaa tagtttcatg ttccccaaat ggcccaaaac tgacagttta
     4741 aacgctgtct tggaacctaa tatgacaaaa gcgtgatctc atccaagatg aactaagttt
     4801 ggttcgttga aatgctaacg gccagttggt caaaaagaaa cttccaaaag tcggcatacc
     4861 gtttgtcttg tttggtattg attgacgaat gctcaaaaat aatctcatta atgcttagcg
     4921 cagtctctct atcgcttctg aaccccggtg cacctgtgcc gaaacgcaaa tggggaaaca
     4981 cccgcttttt ggatgattat gcattgtctc cacattgtat gcttccaaga ttctggtggg
     5041 aatactgctg atagcctaac gttcatgatc aaaatttaac tgttctaacc cctacttgac
     5101 agcaatatat aaacagaagg aagctgccct gtcttaaacc ttttttttta tcatcattat
     5161 tagcttactt tcataattgc gactggttcc aattgacaag cttttgattt taacgacttt
     5221 taacgacaac ttgagaagat caaaaaacaa ctaattattc gaaacg
//
