LOCUS       sequence-230223-        7123 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 7123)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 7123)
  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: CHL1 T101A-eGFP
FEATURES             Location/Qualifiers
     source          1..7123
                     /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             2728..2748
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(2755..2757,2758..2760,2761..3471)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2755..2757,2758..2760,2761..3471)
                     /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(2800..2821)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3061..3080)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3408..3429
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3478..3501
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3511..3528
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3608..3854
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3869..4282
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        4289..4336
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4355..4729
                     /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      4795..5042
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4811..4829)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(6455..7043)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6535..6554)
                     /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 aggaattcat gtctcttcct
      961 gaaactaaat ctgatgatat ccttcttgat gcttgggact tccaaggccg tcccgccgat
     1021 cgctcaaaaa ccggcggctg ggccagcgcc gccatgattc tttgtattga ggccgtggag
     1081 aggctgacga cgttaggtat cggagttaat ctggtgacgt atttgacggg aactatgcat
     1141 ttaggcaatg caactgcggc taacaccgtt accaatttcc tcggaacttc tttcatgctc
     1201 tgtctcctcg gtggcttcat cgccgatacc tttctcggca ggtacctagc gattgctata
     1261 ttcgccgcaa tccaagccac gggtgtttca atcttaactc tatcaacaat cataccggga
     1321 cttcgaccac caagatgcaa tccaacaacg tcgtctcact gcgaacaagc aagtggaata
     1381 caactgacgg tcctatactt agccttatac ctcaccgctc taggaacggg aggcgtgaag
     1441 gctagtgtct cgggtttcgg gtcggaccaa ttcgatgaga ccgaaccaaa agaacgatcg
     1501 aaaatgacat atttcttcaa ccgtttcttc ttttgtatca acgttggctc tcttttagct
     1561 gtgacggtcc ttgtctacgt acaagacgat gttggacgca aatggggcta tggaatttgc
     1621 gcgtttgcga tcgtgcttgc actcagcgtt ttcttggccg gaacaaaccg ctaccgtttc
     1681 aagaagttga tcggtagccc gatgacgcag gttgctgcgg ttatcgtggc ggcgtggagg
     1741 aataggaagc tcgagctgcc ggcagatccg tcctatctct acgatgtgga tgatattatt
     1801 gcggcggaag gttcgatgaa gggtaaacaa aagctgccac acactgaaca attccgttca
     1861 ttagataagg cagcaataag ggatcaggaa gcgggagtta cctcgaatgt attcaacaag
     1921 tggacactct caacactaac agatgttgag gaagtgaaac aaatcgtgcg aatgttacca
     1981 atttgggcaa catgcatcct cttctggacc gtccacgctc aattaacgac attatcagtc
     2041 gcacaatccg agacattgga ccgttccatc gggagcttcg agatccctcc agcatcgatg
     2101 gcagtcttct acgtcggtgg cctcctccta accaccgccg tctatgaccg cgtcgccatt
     2161 cgtctatgca aaaagctatt caactacccc catggtctaa gaccgcttca acggatcggt
     2221 ttggggcttt tcttcggatc aatggctatg gctgtggctg ctttggtcga gctcaaacgt
     2281 cttagaactg cacacgctca tggtccaaca gtcaaaacgc ttcctctagg gttttatcta
     2341 ctcatcccac aatatcttat tgtcggtatc ggcgaagcgt taatctacac aggacagtta
     2401 gatttcttct tgagagagtg ccctaaaggt atgaaaggga tgagcacggg tctattgttg
     2461 agcacattgg cattaggctt tttcttcagc tcggttctcg tgacaatcgt cgagaaattc
     2521 accgggaaag ctcatccatg gattgccgat gatctcaaca agggccgtct ttacaatttc
     2581 tactggcttg tggccgtact tgttgccttg aacttcctca ttttcctagt tttctccaag
     2641 tggtacgttt acaaggaaaa aagactagct gaggtgggga ttgagttgga tgatgagccg
     2701 agtattccaa tgggtcatag cggccgcgag aacctgtact tccaatctgg ttcgatggtg
     2761 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     2821 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     2881 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     2941 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     3001 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     3061 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     3121 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     3181 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     3241 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     3301 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     3361 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     3421 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtcgtcgcat
     3481 catcatcatc atcatcatca ttaagtcgac catcatcatc atcatcattg agtttgtagc
     3541 cttagacatg actgttcctc agttcaagtt gggcacttac gagaagaccg gtcttgctag
     3601 attctaatca agaggatgtc agaatgccat ttgcctgaga gatgcaggct tcatttttga
     3661 tactttttta tttgtaacct atatagtata ggattttttt tgtcattttg tttcttctcg
     3721 tacgagcttg ctcctgatca gcctatctcg cagctgatga atatcttgtg gtaggggttt
     3781 gggaaaatca ttcgagtttg atgtttttct tggtatttcc cactcctctt cagagtacag
     3841 aagattaagt gagaccttcg tttgtgcgga tcccccacac accatagctt caaaatgttt
     3901 ctactccttt tttactcttc cagattttct cggactccgc gcatcgccgt accacttcaa
     3961 aacacccaag cacagcatac taaatttccc ctctttcttc ctctagggtg tcgttaatta
     4021 cccgtactaa aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa
     4081 aaaggcaata aaaattttta tcacgtttct ttttcttgaa aatttttttt tttgattttt
     4141 ttctctttcg atgacctccc attgatattt aagttaataa acggtcttca atttctcaag
     4201 tttcagtttc atttttcttg ttctattaca acttttttta cttcttgctc attagaaaga
     4261 aagcatagca atctaatcta agggcggtgt tgacaattaa tcatcggcat agtatatcgg
     4321 catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc
     4381 ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt
     4441 ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt
     4501 catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg
     4561 cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc
     4621 ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg
     4681 cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtccgacg
     4741 cggcccgacg ggtccgaggc ctcggagatc cgtccccctt ttcctttgtc gatatcatgt
     4801 aattagttat gtcacgctta cattcacgcc ctccccccac atccgctcta accgaaaagg
     4861 aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt atgttagtat
     4921 taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc gtgtacgcat
     4981 gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag gctttaattt
     5041 gcaagctgga gaccaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5101 ccgcgttgct ctgatgaatc ccctaatgat tttggtaaaa atcattaagt taaggtggat
     5161 acacatcttg tcatatgatc aaatggtttc gcgaaaaatc aataatcaga caacaagatg
     5221 tgcgaactcg atattttaca cgactctctt taccaattct gccccgaatt acacttaaaa
     5281 cgactcaaca gcttaacgtt ggcttgccac gcattacttg actgtaaaac tctcactctt
     5341 accgaacttg gccgtaacct gccaaccaaa gcgagaacaa aacataacat caaacgaatc
     5401 gaccgattgt taggtaatcg tcacctccac aaagagcgac tcgctgtata ccgttggcat
     5461 gctagcttta tctgttcggg caatacgatg cccattgtac ttgttgactg gtctgatatt
     5521 cgtgagcaaa aacgacttat ggtattgcga gcttcagtcg cactacacgg tcgttctgtt
     5581 actctttatg agaaagcgtt cccgctttca gagcaatgtt caaagaaagc tcatgaccaa
     5641 tttctagccg accttgcgag cattctaccg agtaacacca caccgctcat tgtcagtgat
     5701 gctggcttta aagtgccatg gtataaatcc gttgagaagc tgggttggta ctggttaagt
     5761 cgagtaagag gaaaagtaca atatgcagac ctaggagcgg aaaactggaa acctatcagc
     5821 aacttacatg atatgtcatc tagtcactca aagactttag gctataagag gctgactaaa
     5881 agcaatccaa tctcatgcca aattctattg tataaatctc gctctaaagg ccgaaaaaat
     5941 cagcgctcga cacggactca ttgtcaccac ccgtcaccta aaatctactc agcgtcggca
     6001 aaggagccat gggttctagc aactaactta cctgttgaaa ttcgaacacc caaacaactt
     6061 gttaatatct attcgaagcg aatgcagatt gaagaaacct tccgagactt gaaaagtcct
     6121 gcctacggac taggcctacg ccatagccga acgagcagct cagagcgttt tgatatcatg
     6181 ctgctaatcg ccctgatgct tcaactaaca tgttggcttg cgggcgttca tgctcagaaa
     6241 caaggttggg acaagcactt ccaggctaac acagtcagaa atcgaaacgt actctcaaca
     6301 gttcgcttag gcatggaagt tttgcggcat tctggctaca caataacaag ggaagactta
     6361 ctcgtggctg caaccctact agctcaaaat ttattcacac atggttacgc tttggggaaa
     6421 ttatgagggg atctctcagc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6481 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6541 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6601 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6661 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6721 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6781 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6841 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6901 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6961 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     7021 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     7081 agtggaacga aaactcacgt taagggattt tggtcatgag atc
//
