LOCUS       sequence-231285-        4910 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 4910)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 4910)
  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: AtPQL upGFP
FEATURES             Location/Qualifiers
     source          1..4910
                     /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             1853..1873
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1880..1882,1883..1885,1886..2596)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1880..1882,1883..1885,1886..2596)
                     /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(1925..1946)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2186..2205)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2533..2554
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2603..2626
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2636..2653
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2733..2979
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2994..3407
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3414..3461
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3480..3854
                     /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      3920..4167
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3936..3954)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4242..4830)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4322..4341)
                     /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 tatgattcga gatgatttgt cactatctct tggtatcatc agtgttatca gttggagtgt
     1021 tgccgagata ccgcagatta tgactaacta caatcaaaag tccattgaag gagtctctat
     1081 aaccttctta acaacatgga tgcttggtga tatctttaat gttgttggct gcttgatgga
     1141 accagctagt cttccagttc aattctacac ggcagtgttg tatactctgg ctacacttgt
     1201 tctctatgtt caatctatat actacgggca tatttacccg cggttgatga aaaaccgaag
     1261 gaatcatcag atggttgatg tagaagagcc tttgcttcgt gaagaagcta agcgtccttc
     1321 taccaaatcg ttgctatgtg tggtctcggt gttcttgttt cttggatcat tcaacgtatt
     1381 gagtggttca agaagtatgg atttgagagg gaaagataga gtgtttgtag ttggaggagc
     1441 aggagcaaga aagcttttgg aggtctctag cggtaactta ggagaaaaca acaatattgg
     1501 aatgtggttg ggttgggcaa tggcggcgat ttacatgggt ggaaggcttc ctcaaatatg
     1561 tatgaatgta aggagaggaa atgttgaggg attgaatcca ttaatgttct tctttgcatt
     1621 cattggcaat gtgacttatg ttgcaagcat actagtgaac agcgtcgaat ggtcgaaaat
     1681 cgaaccgaat ctaccatggc tagttgattc aggaggatgt gctgttcttg attttcttat
     1741 tctgcttcag tttttctact ttcactgtcg caaagtcgag gcagattccg tcaagaagaa
     1801 gcatgaaacc ggtgaagaag ctgtcgcatt aaacctcagc ctcgagggtt cggagaacct
     1861 gtacttccaa tctggttcga tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1921 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1981 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2041 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2101 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2161 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2221 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2281 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2341 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2401 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2461 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     2521 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     2581 ggacgagctg tacaagtcgt cgcatcatca tcatcatcat catcattaag tcgaccatca
     2641 tcatcatcat cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca
     2701 cttacgagaa gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc
     2761 tgagagatgc aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt
     2821 ttttttgtca ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct
     2881 gatgaatatc ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta
     2941 tttcccactc ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatcccc
     3001 cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac
     3061 tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt
     3121 tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc
     3181 gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc
     3241 ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt
     3301 aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt
     3361 ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagggc ggtgttgaca
     3421 attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca
     3481 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
     3541 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
     3601 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
     3661 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
     3721 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
     3781 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     3841 aggagcagga ctgacacgtc cgacgcggcc cgacgggtcc gaggcctcgg agatccgtcc
     3901 cccttttcct ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc
     3961 cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt
     4021 tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt
     4081 tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt
     4141 ttgggacgct cgaaggcttt aatttgcaag ctggagacca acatgtgagc aaaaggccag
     4201 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4261 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4321 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4381 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4441 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4501 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4561 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4621 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4681 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4741 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4801 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4861 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc
//
