LOCUS       sequence-231110-        4487 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 4487)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 4487)
  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: AtCOPT2 upGFP
FEATURES             Location/Qualifiers
     source          1..4487
                     /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             990..1013
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1463..1483
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1490..1492,1493..1495,1496..2206)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1490..1492,1493..1495,1496..2206)
                     /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(1535..1556)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1796..1815)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2143..2164
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2213..2230
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2310..2556
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2571..2984
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2991..3038
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3057..3431
                     /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      3497..3744
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3513..3531)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3819..4407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3899..3918)
                     /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 tatggatcat gatcacatgc atgacatgcc accaccatca ccatcatcat catcgatgtc
     1021 gaatcataca acaccacaca tgatgatgat gcacatgacc ttcttttggg gtaagaacac
     1081 ggaggttctc ttctccggat ggcccgggac aagctccggc atgtacgctc tctgtctcat
     1141 tgttatattc ctcctcgccg taattgccga gtggctcgct cattcaccga tcctacgtgt
     1201 cagtggctca accaatcgag ccgccgggct cgctcaaacc gctgtgtaca cactcaagac
     1261 tggcctttcg tatttggtga tgctcgctgt tatgtccttt aacgcaggtg ttttcatcgt
     1321 cgctatcgcc ggctatggcg ttggtttctt tctctttggg agcactactt tcaagaaacc
     1381 ctccgatgac cagaaaaccg ccgagcttct tccgccgtct tcaggctgcg tttgtgcatt
     1441 aaacctcagc ctcgagggtt cggagaacct gtacttccaa tctggttcga tggtgagcaa
     1501 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1561 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1621 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1681 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     1741 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     1801 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     1861 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     1921 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     1981 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     2041 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     2101 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     2161 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtcgt cgcatcatca
     2221 tcatcatcat tgagtttgta gccttagaca tgactgttcc tcagttcaag ttgggcactt
     2281 acgagaagac cggtcttgct agattctaat caagaggatg tcagaatgcc atttgcctga
     2341 gagatgcagg cttcattttt gatacttttt tatttgtaac ctatatagta taggattttt
     2401 tttgtcattt tgtttcttct cgtacgagct tgctcctgat cagcctatct cgcagctgat
     2461 gaatatcttg tggtaggggt ttgggaaaat cattcgagtt tgatgttttt cttggtattt
     2521 cccactcctc ttcagagtac agaagattaa gtgagacctt cgtttgtgcg gatcccccac
     2581 acaccatagc ttcaaaatgt ttctactcct tttttactct tccagatttt ctcggactcc
     2641 gcgcatcgcc gtaccacttc aaaacaccca agcacagcat actaaatttc ccctctttct
     2701 tcctctaggg tgtcgttaat tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc
     2761 tcgtttcttt ttcttcgtcg aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg
     2821 aaaatttttt tttttgattt ttttctcttt cgatgacctc ccattgatat ttaagttaat
     2881 aaacggtctt caatttctca agtttcagtt tcatttttct tgttctatta caactttttt
     2941 tacttcttgc tcattagaaa gaaagcatag caatctaatc taagggcggt gttgacaatt
     3001 aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg
     3061 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt
     3121 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg
     3181 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca
     3241 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg
     3301 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt
     3361 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg
     3421 agcaggactg acacgtccga cgcggcccga cgggtccgag gcctcggaga tccgtccccc
     3481 ttttcctttg tcgatatcat gtaattagtt atgtcacgct tacattcacg ccctcccccc
     3541 acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat
     3601 ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt
     3661 ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg
     3721 ggacgctcga aggctttaat ttgcaagctg gagaccaaca tgtgagcaaa aggccagcaa
     3781 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3841 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3901 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3961 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4021 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4081 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4141 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4201 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4261 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4321 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4381 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4441 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagatc
//
