LOCUS       sequence-230312-        4946 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 4946)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 4946)
  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: AtALMT1-upGFP 3'
FEATURES             Location/Qualifiers
     source          1..4946
                     /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             1889..1909
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1916..1918,1919..1921,1922..2632)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1916..1918,1919..1921,1922..2632)
                     /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(1961..1982)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2222..2241)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2569..2590
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2639..2662
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2672..2689
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2769..3015
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3030..3443
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3450..3497
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3516..3890
                     /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      3956..4203
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3972..3990)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4278..4866)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4358..4377)
                     /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 tatgctcctt gcctctaact ttgacactct ctcccacttc cttcaagatt ttggggacga
     1021 atattttgaa gcgagagaga agggggacta caaagtagtg gagaagagga aaaagaatct
     1081 tgagagatat aaaagtgttc ttgactccaa aagcgatgaa gaagctttgg ctaattacgc
     1141 agaatgggaa ccgcctcatg gacaatttag gtttaggcat ccatggaaac aatatgttgc
     1201 cgttggtgca ttactccggc agtgtgccta caggattgat gccttgaatt catacatcaa
     1261 ctcagatttt cagatcccag tggacataaa aaagaaatta gaaacaccat taagaagaat
     1321 gagctcggaa tcgggaaact caatgaaaga aatgtccatt tcactaaaac aaatgataaa
     1381 atcatcttct tctgatatcc atgtctcaaa ctcacaagct gcatgcaaat ctctttctac
     1441 tttacttaaa tcaggcatct tgaacgatgt tgagcctctt caaatgatct cattgatgac
     1501 gactgtctcc atgctcatag atattgttaa cttaaccgaa aagatctcgg aatctgtaca
     1561 cgagcttgca tccgctgcaa gatttaagaa caagatgagg ccgaccgtgc tatacgagaa
     1621 gtcggattcc ggaagcattg gtcgtgccat gccaatagat tctcatgaag atcatcatgt
     1681 tgtcacagtt ttacatgacg ttgataatga tagatcaaac aatgttgatg attcacgtgg
     1741 agggagttca caggactcat gtcatcatgt tgccataaag attgttgatg ataactcaaa
     1801 ccatgaaaaa catgaagatg gtgaaataca tgtacataca ttaagtaatg ggcatcttca
     1861 ggcattaaac ctcagcctcg agggttcgga gaacctgtac ttccaatctg gttcgatggt
     1921 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1981 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2041 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2101 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     2161 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2221 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2281 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2341 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     2401 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     2461 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2521 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2581 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtcgtcgca
     2641 tcatcatcat catcatcatc attaagtcga ccatcatcat catcatcatt gagtttgtag
     2701 ccttagacat gactgttcct cagttcaagt tgggcactta cgagaagacc ggtcttgcta
     2761 gattctaatc aagaggatgt cagaatgcca tttgcctgag agatgcaggc ttcatttttg
     2821 atactttttt atttgtaacc tatatagtat aggatttttt ttgtcatttt gtttcttctc
     2881 gtacgagctt gctcctgatc agcctatctc gcagctgatg aatatcttgt ggtaggggtt
     2941 tgggaaaatc attcgagttt gatgtttttc ttggtatttc ccactcctct tcagagtaca
     3001 gaagattaag tgagaccttc gtttgtgcgg atcccccaca caccatagct tcaaaatgtt
     3061 tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg taccacttca
     3121 aaacacccaa gcacagcata ctaaatttcc cctctttctt cctctagggt gtcgttaatt
     3181 acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga
     3241 aaaaggcaat aaaaattttt atcacgtttc tttttcttga aaattttttt ttttgatttt
     3301 tttctctttc gatgacctcc cattgatatt taagttaata aacggtcttc aatttctcaa
     3361 gtttcagttt catttttctt gttctattac aacttttttt acttcttgct cattagaaag
     3421 aaagcatagc aatctaatct aagggcggtg ttgacaatta atcatcggca tagtatatcg
     3481 gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc
     3541 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt
     3601 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt
     3661 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc
     3721 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg
     3781 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc
     3841 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtccgac
     3901 gcggcccgac gggtccgagg cctcggagat ccgtccccct tttcctttgt cgatatcatg
     3961 taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag
     4021 gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta
     4081 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca
     4141 tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt
     4201 tgcaagctgg agaccaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4261 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4321 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4381 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4441 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4501 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4561 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4621 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4681 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4741 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4801 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4861 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4921 cgttaaggga ttttggtcat gagatc
//
