LOCUS       sequence-231062-        4481 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 4481)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 4481)
  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: AtCOPT6 upGFP
FEATURES             Location/Qualifiers
     source          1..4481
                     /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             981..998
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1424..1444
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1451..1453,1454..1456,1457..2167)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1451..1453,1454..1456,1457..2167)
                     /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(1496..1517)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1757..1776)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2104..2125
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2174..2197
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2207..2224
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2304..2550
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2565..2978
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2985..3032
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3051..3425
                     /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      3491..3738
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3507..3525)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3813..4401)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3893..3912)
                     /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 ggtaacatgc caccatcatc accatcatca atggtaaatc ataccaattc
     1021 caacatgatc atgatgcata tgactttctt ctggggaaag aacacggaga tactcttctc
     1081 cggctggccc gggacaagtc tcggcatgta cgttctatgt ctcatcgtcg tcttcctcct
     1141 tgctgttatc gtcgagtggc ttgcccattc ttcgatcctt cgtggacgtg gctccactag
     1201 ccgcgccaag gggctcgttc agaccgcagt ctacacactc aagacaggcc ttgcttatct
     1261 cgtgatgctc gccgttatgt cctttaacgg tggagttttc attgtcgcta tcgccggatt
     1321 cgccgtgggt ttcatgctct tcggaagcac tgctttcaag aacccctctg atgacgagaa
     1381 acccttcgaa caactagcat taaacctcag cctcgagggt tcggagaacc tgtacttcca
     1441 atctggttcg atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1501 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1561 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1621 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1681 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1741 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1801 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1861 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     1921 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     1981 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     2041 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     2101 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     2161 gtacaagtcg tcgcatcatc atcatcatca tcatcattaa gtcgaccatc atcatcatca
     2221 tcattgagtt tgtagcctta gacatgactg ttcctcagtt caagttgggc acttacgaga
     2281 agaccggtct tgctagattc taatcaagag gatgtcagaa tgccatttgc ctgagagatg
     2341 caggcttcat ttttgatact tttttatttg taacctatat agtataggat tttttttgtc
     2401 attttgtttc ttctcgtacg agcttgctcc tgatcagcct atctcgcagc tgatgaatat
     2461 cttgtggtag gggtttggga aaatcattcg agtttgatgt ttttcttggt atttcccact
     2521 cctcttcaga gtacagaaga ttaagtgaga ccttcgtttg tgcggatccc ccacacacca
     2581 tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat
     2641 cgccgtacca cttcaaaaca cccaagcaca gcatactaaa tttcccctct ttcttcctct
     2701 agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt
     2761 ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaaatt
     2821 tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt taataaacgg
     2881 tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc
     2941 ttgctcatta gaaagaaagc atagcaatct aatctaaggg cggtgttgac aattaatcat
     3001 cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt
     3061 tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga
     3121 ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg
     3181 acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg
     3241 cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca
     3301 cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc
     3361 gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg
     3421 actgacacgt ccgacgcggc ccgacgggtc cgaggcctcg gagatccgtc ccccttttcc
     3481 tttgtcgata tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc
     3541 gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt
     3601 atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac
     3661 agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc
     3721 tcgaaggctt taatttgcaa gctggagacc aacatgtgag caaaaggcca gcaaaaggcc
     3781 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3841 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3901 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3961 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4021 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4081 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4141 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4201 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4261 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4321 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4381 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4441 tggaacgaaa actcacgtta agggattttg gtcatgagat c
//
