LOCUS       sequence-231039-        4751 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 4751)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 4751)
  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: TaALMT1-upGFP 3'
FEATURES             Location/Qualifiers
     source          1..4751
                     /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             1694..1714
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1721..1723,1724..1726,1727..2437)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1721..1723,1724..1726,1727..2437)
                     /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(1766..1787)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2027..2046)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2374..2395
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2444..2467
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2477..2494
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2574..2820
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2835..3248
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3255..3302
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3321..3695
                     /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      3761..4008
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3777..3795)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4083..4671)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4163..4182)
                     /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 tatgctcgcc tccggcaacc tcgacaaact cgctcagttc attgaaggaa tggaattcaa
     1021 ctgctttggc gaaaacagtg ttgcaaataa ttttggggga aaagatttcc cccaaatgca
     1081 caagagcgtc cttaattcga aggccactga ggactctttg tgcacctttg ccaaatggga
     1141 gcctcgtcat ggccagttca gatttcgaca cccatggagt caataccaga agctgggaac
     1201 tctttgtcgc caatgtgcgt cttctatgga ggctcttgct tcatatgtca tcacaacctc
     1261 aaaaacccag tgccctgctg cagccaaccc tgagctatca tgtaaggttc gaaaaacatg
     1321 tggcgaaatg agcttgcatt cctccaaggt gcttagggat ctcgcaatgg caactcgaac
     1381 aatgactgtg ccgtctccag tgaatatcac catggctaca gccgtgaaag cagcggaaag
     1441 cctcagaagc gagcttgcag agaacacggc tctgttgcaa gtgatgcatg tggccgtcac
     1501 cgcaacactt cttgcggact tggttgatag ggtgaaggaa atcgcggaat gtgttgatgt
     1561 cctagcaaga ctggcgcact ttaagaaccc cgaggacaca aaaaatgtcg ttgttagtac
     1621 cgtgagtcga gggatagacg aacctttgcc tgacgtggtt attttgccat taaacctcag
     1681 cctcgagggt tcggagaacc tgtacttcca atctggttcg atggtgagca agggcgagga
     1741 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1801 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     1861 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     1921 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1981 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2041 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2101 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     2161 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     2221 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     2281 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     2341 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2401 cgccgggatc actctcggca tggacgagct gtacaagtcg tcgcatcatc atcatcatca
     2461 tcatcattaa gtcgaccatc atcatcatca tcattgagtt tgtagcctta gacatgactg
     2521 ttcctcagtt caagttgggc acttacgaga agaccggtct tgctagattc taatcaagag
     2581 gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg
     2641 taacctatat agtataggat tttttttgtc attttgtttc ttctcgtacg agcttgctcc
     2701 tgatcagcct atctcgcagc tgatgaatat cttgtggtag gggtttggga aaatcattcg
     2761 agtttgatgt ttttcttggt atttcccact cctcttcaga gtacagaaga ttaagtgaga
     2821 ccttcgtttg tgcggatccc ccacacacca tagcttcaaa atgtttctac tcctttttta
     2881 ctcttccaga ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca cccaagcaca
     2941 gcatactaaa tttcccctct ttcttcctct agggtgtcgt taattacccg tactaaaggt
     3001 ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc gtcgaaaaag gcaataaaaa
     3061 tttttatcac gtttcttttt cttgaaaatt tttttttttg atttttttct ctttcgatga
     3121 cctcccattg atatttaagt taataaacgg tcttcaattt ctcaagtttc agtttcattt
     3181 ttcttgttct attacaactt tttttacttc ttgctcatta gaaagaaagc atagcaatct
     3241 aatctaaggg cggtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
     3301 acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
     3361 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
     3421 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
     3481 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
     3541 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
     3601 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
     3661 actgcgtgca cttcgtggcc gaggagcagg actgacacgt ccgacgcggc ccgacgggtc
     3721 cgaggcctcg gagatccgtc ccccttttcc tttgtcgata tcatgtaatt agttatgtca
     3781 cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa
     3841 cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt
     3901 atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg
     3961 aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa gctggagacc
     4021 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4081 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4141 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4201 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4261 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4321 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4381 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4441 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4501 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4561 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4621 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4681 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4741 gtcatgagat c
//
