LOCUS       sequence-231144-        4556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4556)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 4556)
  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: AtCOPT1 upGFP
FEATURES             Location/Qualifiers
     source          1..4556
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..940
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             996..1025
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1499..1519
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1526..1528,1529..1531,1532..2242)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1526..1528,1529..1531,1532..2242)
                     /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(1571..1592)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1832..1851)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2179..2200
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2249..2272
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2282..2299
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2379..2625
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2640..3053
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3060..3107
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3126..3500
                     /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      3566..3813
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3582..3600)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3888..4476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3968..3987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag
       61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt
      121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc
      181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta
      241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta
      301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg
      361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct
      421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg
      481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt
      541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct
      601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct
      661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact
      721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat
      781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt
      841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga
      901 caacttgaga agatcaaaaa acaactaatt attcgaaacg aggaattcgc tgaggcttaa
      961 tatggatcat gatcacatgc atggaatgcc tcgaccatca tcatcatcat catcatcacc
     1021 atcatccatg atgaacaatg gatccatgaa cgaaggagga ggacatcacc acatgaagat
     1081 gatgatgcac atgaccttct tttggggcaa gaacacggag gttctcttct ccggttggcc
     1141 cggaacaagc tccggcatgt atgctctttg tcttatcttt gtcttctttc tcgccgtcct
     1201 caccgaatgg cttgctcatt cctctctcct ccgtggctcc accggagatt cggctaatcg
     1261 tgccgccggg ttaatccaaa ccgccgtgta taccctccgt atcggcctcg cctatctagt
     1321 catgctcgcc gtcatgtcgt ttaacgccgg tgtgtttctc gtcgctctgg ccggtcatgc
     1381 cgttggtttc atgttgttcg gaagccaaac tttccggaat acctccgatg accggaaaac
     1441 taactatgtt cctccctcag gttgtgcttg tgcattaaac ctcagcctcg agggttcgga
     1501 gaacctgtac ttccaatctg gttcgatggt gagcaagggc gaggagctgt tcaccggggt
     1561 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     1621 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     1681 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     1741 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     1801 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     1861 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1921 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     1981 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     2041 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2101 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     2161 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     2221 cggcatggac gagctgtaca agtcgtcgca tcatcatcat catcatcatc attaagtcga
     2281 ccatcatcat catcatcatt gagtttgtag ccttagacat gactgttcct cagttcaagt
     2341 tgggcactta cgagaagacc ggtcttgcta gattctaatc aagaggatgt cagaatgcca
     2401 tttgcctgag agatgcaggc ttcatttttg atactttttt atttgtaacc tatatagtat
     2461 aggatttttt ttgtcatttt gtttcttctc gtacgagctt gctcctgatc agcctatctc
     2521 gcagctgatg aatatcttgt ggtaggggtt tgggaaaatc attcgagttt gatgtttttc
     2581 ttggtatttc ccactcctct tcagagtaca gaagattaag tgagaccttc gtttgtgcgg
     2641 atcccccaca caccatagct tcaaaatgtt tctactcctt ttttactctt ccagattttc
     2701 tcggactccg cgcatcgccg taccacttca aaacacccaa gcacagcata ctaaatttcc
     2761 cctctttctt cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa
     2821 gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc
     2881 tttttcttga aaattttttt ttttgatttt tttctctttc gatgacctcc cattgatatt
     2941 taagttaata aacggtcttc aatttctcaa gtttcagttt catttttctt gttctattac
     3001 aacttttttt acttcttgct cattagaaag aaagcatagc aatctaatct aagggcggtg
     3061 ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact
     3121 aaaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag
     3181 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg
     3241 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc
     3301 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt
     3361 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg
     3421 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
     3481 tggccgagga gcaggactga cacgtccgac gcggcccgac gggtccgagg cctcggagat
     3541 ccgtccccct tttcctttgt cgatatcatg taattagtta tgtcacgctt acattcacgc
     3601 cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc
     3661 cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt
     3721 cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag
     3781 aaggttttgg gacgctcgaa ggctttaatt tgcaagctgg agaccaacat gtgagcaaaa
     3841 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3901 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3961 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4021 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4081 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4141 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4201 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4261 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4321 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4381 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4441 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4501 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagatc
//
