LOCUS       sequence-231208-        5684 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 5684)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 5684)
  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: OsNRAT-upGFP
FEATURES             Location/Qualifiers
     source          1..5684
                     /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             2627..2647
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(2654..2656,2657..2659,2660..3370)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2654..2656,2657..2659,2660..3370)
                     /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(2699..2720)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2960..2979)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3307..3328
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3377..3400
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3410..3427
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3507..3753
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3768..4181
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        4188..4235
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4254..4628
                     /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      4694..4941
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4710..4728)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(5016..5604)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5096..5115)
                     /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 tatggaaggg actggtgaga tgagagaggt tggacgagaa actctccatg gaggtgtggt
     1021 gcagagtgtt agtgagactg atgagtacaa agaaaagact attgacagtg aaaaagatgg
     1081 gcagtttcgg gtgcagccaa gatggaggaa attcttggct catgttgggc ctggagctct
     1141 ggtggccatt ggcttcctag atcctagcaa cttggaaact gacatgcaag ctggagctga
     1201 cttcaagtat gagcttctgt gggtcatctt agttgggatg gtctttgcac ttctgatcca
     1261 aacattagct gccaaccttg gagtgaaaac aggaaggcac cttgctgaac tgtgcaggga
     1321 agagtacccg cactatgtca acattttctt atggatcatt gcggagttgg cggtgatttc
     1381 tgatgacatt cctgaagtgt tgggcacagc ttttgcgttc aacatattgc ttaagattcc
     1441 agtgtgggct ggagttattc tcacagtgtt cagcaccctc ttgcttcttg gagtacagag
     1501 atttggggct cggaagctgg agttcatcat agcagcattc atgttcacta tggcagcttg
     1561 cttcttcgga gagctaagct atttgaggcc gtctgcagga gaggtggtca agggcatgtt
     1621 tgtcccttct ctccaaggga aaggcgctgc tgccaatgcg atcgcactct ttggcgctat
     1681 catcacacca tacaacttgt tcttgcactc tgctctggtc ctgtcaagga agaccccacg
     1741 atcagacaaa agcatcagag ctgcttgcag atacttcctt attgagtgta gtcttgcatt
     1801 catcgtggct ttcctcataa atgtttcagt cgttgttgtt gctggatcca tttgcaatgc
     1861 gaataatctt tctccagctg atgccaacac gtgtggtgac ctaactctgc aatccacacc
     1921 tcttttactc aggaatgttt tggggagatc aagctcggtg gtgtatgctg ttgcgctgct
     1981 agcttctggg caaagcacca ctatcagttg cacgttcgct gggcaggtca tcatgcaggg
     2041 gtttctggac atgaagatga agaattgggt aaggaacctc atcacccgag tcatcgccat
     2101 agctcctagc ctcatcgtct ccattgtcag tggtccctca ggcgccggca agctaatcat
     2161 cctgtcatca atgatcctgt catttgagct gccgttcgct ctcatccctc tactcaagtt
     2221 ctgcaacagc agcaagaaag ttggacccct caaggaatcc atctatacgg tggtgatcgc
     2281 gtggatcctg agcttcgcgc tcatcgtcgt caacacctac ttcctagtgt ggacatacgt
     2341 ggactggctc gtccacaaca acctccccaa gtatgccaat ggcctcatct ccgtggtggt
     2401 gttcgcgctc atggccgcct acctggtcgc cgtcgtctac ctgacgttca ggaaggacac
     2461 ggtggcgacg tacgtgccgg tgccggagcg ggcgcaggcg caggtggagg ccggcgggac
     2521 gccggtggtg gacgcctccg ccgccgacga ggaccagccg gctccgtaca ggaaggacct
     2581 tgccgatgct tccatggagg cattaaacct cagcctcgag ggttcggaga acctgtactt
     2641 ccaatctggt tcgatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     2701 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     2761 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     2821 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     2881 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     2941 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     3001 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     3061 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     3121 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     3181 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     3241 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     3301 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     3361 gctgtacaag tcgtcgcatc atcatcatca tcatcatcat taagtcgacc atcatcatca
     3421 tcatcattga gtttgtagcc ttagacatga ctgttcctca gttcaagttg ggcacttacg
     3481 agaagaccgg tcttgctaga ttctaatcaa gaggatgtca gaatgccatt tgcctgagag
     3541 atgcaggctt catttttgat acttttttat ttgtaaccta tatagtatag gatttttttt
     3601 gtcattttgt ttcttctcgt acgagcttgc tcctgatcag cctatctcgc agctgatgaa
     3661 tatcttgtgg taggggtttg ggaaaatcat tcgagtttga tgtttttctt ggtatttccc
     3721 actcctcttc agagtacaga agattaagtg agaccttcgt ttgtgcggat cccccacaca
     3781 ccatagcttc aaaatgtttc tactcctttt ttactcttcc agattttctc ggactccgcg
     3841 catcgccgta ccacttcaaa acacccaagc acagcatact aaatttcccc tctttcttcc
     3901 tctagggtgt cgttaattac ccgtactaaa ggtttggaaa agaaaaaaga gaccgcctcg
     3961 tttctttttc ttcgtcgaaa aaggcaataa aaatttttat cacgtttctt tttcttgaaa
     4021 attttttttt ttgatttttt tctctttcga tgacctccca ttgatattta agttaataaa
     4081 cggtcttcaa tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac
     4141 ttcttgctca ttagaaagaa agcatagcaa tctaatctaa gggcggtgtt gacaattaat
     4201 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca
     4261 agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct
     4321 ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc
     4381 gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc
     4441 tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt
     4501 ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg
     4561 ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc
     4621 aggactgaca cgtccgacgc ggcccgacgg gtccgaggcc tcggagatcc gtcccccttt
     4681 tcctttgtcg atatcatgta attagttatg tcacgcttac attcacgccc tccccccaca
     4741 tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt
     4801 tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg
     4861 tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga
     4921 cgctcgaagg ctttaatttg caagctggag accaacatgt gagcaaaagg ccagcaaaag
     4981 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5041 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5101 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5161 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5221 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5281 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5341 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5401 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5461 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5521 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5581 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5641 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gatc
//
