LOCUS       sequence-231138-        5423 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 5423)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 5423)
  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: TaALMT-upGFP
FEATURES             Location/Qualifiers
     source          1..5423
                     /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             2366..2386
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(2393..2395,2396..2398,2399..3109)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2393..2395,2396..2398,2399..3109)
                     /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(2438..2459)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2699..2718)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3046..3067
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3116..3139
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3149..3166
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3246..3492
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3507..3920
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3927..3974
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3993..4367
                     /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      4433..4680
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4449..4467)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4755..5343)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4835..4854)
                     /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 tatggatatt gatcacggca gagagagcga cggcgagatg gtgggcacca tcgccagctg
     1021 cgggctgctg ctccactcgc ttctcgccgg gctcgggcgt cgcgccgccg ggttcgcccg
     1081 gaaggtgggc ggcgccgcgc gggaggaccc gaggcgggtg gcgcactcgc tcaaagtcgg
     1141 cctggcgctc gcgctggtgt ccgtcgtcta cttcgtcacg ccgctcttca acggcctcgg
     1201 ggtgtccgcg atatgggccg tgctcaccgt cgtcgtcgtc atggagtaca ccgtcggtgc
     1261 cacgctgagt aaaggcttga acagagcctt ggcgacgttg gtggctggct gcatcgccgt
     1321 cggagctcat cagttagctg aattagctga acgctgtggt gatcagggag agcccataat
     1381 gcttaccgtg ctcgtcttct tcgtagcgtc agcggcgacg ttcttgcgct tcatcccgga
     1441 gatcaaggcc aagtacgact acggcgtgac catcttcata ctgaccttcg gtctggtggc
     1501 cgtgtcgagc tacagagtgg aggagctcat ccagctcgcg caccagcggt tctacaccat
     1561 agccgtcggc gtcttcatct gcctctgcac caccgtcttc ctcttccccg tctgggccgg
     1621 agaggacgtc cacaagctcg cctccggcaa cctcgacaaa ctcgctcagt tcattgaagg
     1681 aatggaattc aactgctttg gcgaaaacag tgttgcaaat aattttgggg gaaaagattt
     1741 cccccaaatg cacaagagcg tccttaattc gaaggccact gaggactctt tgtgcacctt
     1801 tgccaaatgg gagcctcgtc atggccagtt cagatttcga cacccatgga gtcaatacca
     1861 gaagctggga actctttgtc gccaatgtgc gtcttctatg gaggctcttg cttcatatgt
     1921 catcacaacc tcaaaaaccc agtgccctgc tgcagccaac cctgagctat catgtaaggt
     1981 tcgaaaaaca tgtggcgaaa tgagcttgca ttcctccaag gtgcttaggg atctcgcaat
     2041 ggcaactcga acaatgactg tgccgtctcc agtgaatatc accatggcta cagccgtgaa
     2101 agcagcggaa agcctcagaa gcgagcttgc agagaacacg gctctgttgc aagtgatgca
     2161 tgtggccgtc accgcaacac ttcttgcgga cttggttgat agggtgaagg aaatcgcgga
     2221 atgtgttgat gtcctagcaa gactggcgca ctttaagaac cccgaggaca caaaaaatgt
     2281 cgttgttagt accgtgagtc gagggataga cgaacctttg cctgacgtgg ttattttgcc
     2341 attaaacctc agcctcgagg gttcggagaa cctgtacttc caatctggtt cgatggtgag
     2401 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     2461 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2521 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2581 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2641 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2701 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2761 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2821 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2881 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2941 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     3001 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3061 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt cgtcgcatca
     3121 tcatcatcat catcatcatt aagtcgacca tcatcatcat catcattgag tttgtagcct
     3181 tagacatgac tgttcctcag ttcaagttgg gcacttacga gaagaccggt cttgctagat
     3241 tctaatcaag aggatgtcag aatgccattt gcctgagaga tgcaggcttc atttttgata
     3301 cttttttatt tgtaacctat atagtatagg attttttttg tcattttgtt tcttctcgta
     3361 cgagcttgct cctgatcagc ctatctcgca gctgatgaat atcttgtggt aggggtttgg
     3421 gaaaatcatt cgagtttgat gtttttcttg gtatttccca ctcctcttca gagtacagaa
     3481 gattaagtga gaccttcgtt tgtgcggatc ccccacacac catagcttca aaatgtttct
     3541 actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa
     3601 cacccaagca cagcatacta aatttcccct ctttcttcct ctagggtgtc gttaattacc
     3661 cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa
     3721 aggcaataaa aatttttatc acgtttcttt ttcttgaaaa tttttttttt tgattttttt
     3781 ctctttcgat gacctcccat tgatatttaa gttaataaac ggtcttcaat ttctcaagtt
     3841 tcagtttcat ttttcttgtt ctattacaac tttttttact tcttgctcat tagaaagaaa
     3901 gcatagcaat ctaatctaag ggcggtgttg acaattaatc atcggcatag tatatcggca
     3961 tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg
     4021 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct
     4081 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca
     4141 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg
     4201 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct
     4261 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg
     4321 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtccgacgcg
     4381 gcccgacggg tccgaggcct cggagatccg tccccctttt cctttgtcga tatcatgtaa
     4441 ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa
     4501 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta
     4561 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt
     4621 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc
     4681 aagctggaga ccaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4741 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4801 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4861 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4921 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4981 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5041 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5101 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5161 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5221 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5281 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5341 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5401 taagggattt tggtcatgag atc
//
