LOCUS       sequence-230235-        4628 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 4628)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 4628)
  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 5'
FEATURES             Location/Qualifiers
     source          1..4628
                     /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             complement(1067..1078)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1571..1591
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1598..1600,1601..1603,1604..2314)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1598..1600,1601..1603,1604..2314)
                     /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(1643..1664)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1904..1923)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2251..2272
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2321..2344
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2354..2371
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2451..2697
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2712..3125
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3132..3179
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3198..3572
                     /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      3638..3885
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3654..3672)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3960..4548)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4040..4059)
                     /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 tatggagaaa gtgagagaga tagtgagaga agggattaga gtagggaatg aagacccgag
     1021 aagaattatt catgctttca aagtgggact tgctcttgtt ttggtctctt ccttctatta
     1081 ttaccaacct ttcggtcctt tcaccgatta cttcggtatc aatgcaatgt gggctgtcat
     1141 gaccgtcgtt gttgtctttg aattctccgt aggggctaca cttgggaaag gactaaatag
     1201 aggagtggca acattagtag caggaggact aggaattgga gctcatcaac tagcaagatt
     1261 gtcgggtgca acagtggaac ctattctttt ggtcatgttg gtctttgtgc aagctgcgtt
     1321 gtcgacgttc gtaaggtttt tcccatgggt aaagacaaag ttcgattatg ggatattgat
     1381 attcatattg acgttcgcgc tgatatcgtt gtcggggttt agagacgaag agataatgga
     1441 tttggcagaa agcagactat cgacagtggt gattggagga gtcagttgca tccttatctc
     1501 tatctttgtc tgtcctgttt gggctggaca agatctacat tctgcattaa acctcagcct
     1561 cgagggttcg gagaacctgt acttccaatc tggttcgatg gtgagcaagg gcgaggagct
     1621 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1681 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1741 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     1801 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1861 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1921 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1981 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     2041 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     2101 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     2161 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     2221 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2281 cgggatcact ctcggcatgg acgagctgta caagtcgtcg catcatcatc atcatcatca
     2341 tcattaagtc gaccatcatc atcatcatca ttgagtttgt agccttagac atgactgttc
     2401 ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa tcaagaggat
     2461 gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt ttatttgtaa
     2521 cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc ttgctcctga
     2581 tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa tcattcgagt
     2641 ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta agtgagacct
     2701 tcgtttgtgc ggatccccca cacaccatag cttcaaaatg tttctactcc ttttttactc
     2761 ttccagattt tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca
     2821 tactaaattt cccctctttc ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg
     2881 gaaaagaaaa aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt
     2941 ttatcacgtt tctttttctt gaaaattttt ttttttgatt tttttctctt tcgatgacct
     3001 cccattgata tttaagttaa taaacggtct tcaatttctc aagtttcagt ttcatttttc
     3061 ttgttctatt acaacttttt ttacttcttg ctcattagaa agaaagcata gcaatctaat
     3121 ctaagggcgg tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca
     3181 aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg
     3241 acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg
     3301 aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg
     3361 accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt
     3421 acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga
     3481 ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact
     3541 gcgtgcactt cgtggccgag gagcaggact gacacgtccg acgcggcccg acgggtccga
     3601 ggcctcggag atccgtcccc cttttccttt gtcgatatca tgtaattagt tatgtcacgc
     3661 ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct
     3721 gaagtctagg tccctattta tttttttata gttatgttag tattaagaac gttatttata
     3781 tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa
     3841 accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcaagct ggagaccaac
     3901 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3961 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4021 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4081 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4141 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4201 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4261 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4321 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4381 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4441 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4501 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4561 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4621 atgagatc
//
