LOCUS       sequence-231227-        5306 bp    DNA     circular SYN 15-APR-2025
DEFINITION  protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5306)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 5306)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: OsPQL upGFP
FEATURES             Location/Qualifiers
     source          1..5306
                     /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             2249..2269
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(2276..2278,2279..2281,2282..2992)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2276..2278,2279..2281,2282..2992)
                     /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(2321..2342)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2582..2601)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2929..2950
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2999..3022
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3032..3049
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3129..3375
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3390..3803
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3810..3857
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3876..4250
                     /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      4316..4563
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4332..4350)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4638..5226)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4718..4737)
                     /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 tatgggcatc ttcagtggag cgggagcaca tcgccctagc tgcccgtcgg ccgcgaattg
     1021 cgcaaaatgg gctcaaacct acctgaagta ctgcctttgt agcacgaggg atggcatggc
     1081 actgacgctt ggactgttaa gcgtcatcag ctggggagtt gctgaggtgc cacagatcat
     1141 aactaattac aagcataagt caacagaagg cctttccctt gcttttctaa tgacatggat
     1201 tgttggggac ttttttaacc ttattggctg cttcttggaa cctgagacgc tgccaacaca
     1261 attttacatg gctctgctat acaccatcac aactgttatt ctcactggac agacagtata
     1321 ctatagccac atatatcacc gtcttaaagc aaaaaaagcc agggcaacaa gcaagcctca
     1381 gaggcatcag cgtgctgatg cttcattgag agaaaagctt ttggggccca aggttattgg
     1441 ggaaatcaga aacaatagcc acctaggtgc aaccgtccct attccaacaa gttctccaat
     1501 tacagttaat acagaaatag tacggcaacg acatggccca agcagcctca gtgaatacta
     1561 ctacacgtca gcaagatctc tctcaagcag cccagtgcca atgtcaggca catggtcggc
     1621 taattatcat caaacaaatt ctccaccaga aatagatgac caaaaggaat ccttggtcag
     1681 tgagttttcc ccagcacaat atgcagcatc tcctttaata aagaattctc tctcagtggt
     1741 accatggatg tcccttctct tgggtatgag tgttttgcat tttttagttg gaacaaccca
     1801 tcaagaggtg ccaaatggta ttgtcatacc tgtcgggaga aggcttttac tgttggcgga
     1861 tgaccatgca gattcatctg tcagcaatgg cagtggaagt ggaattggaa gcttccttgg
     1921 ttgggctatg gcggtaatct atatgggtgg acgacttccc cagatctggt taaatatgaa
     1981 aagagggaat gctgagggac tcaatccact aatgtttact tttgcattag tgggaaatgt
     2041 gacatacgtt ggaagtatac ttgtgaaaag catggattgg tcaaaactta agccgaatct
     2101 accgtggctt gtagatgctg gaggatgtgt tttgcttgat acttttatta ttcttcagtt
     2161 tctttatttc cattatcgga agcgacacgt tcctgatgag cctgacagtg ctgataaagt
     2221 tgcattaaac ctcagcctcg agggttcgga gaacctgtac ttccaatctg gttcgatggt
     2281 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2341 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2401 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2461 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     2521 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2581 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2641 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2701 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     2761 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     2821 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2881 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2941 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtcgtcgca
     3001 tcatcatcat catcatcatc attaagtcga ccatcatcat catcatcatt gagtttgtag
     3061 ccttagacat gactgttcct cagttcaagt tgggcactta cgagaagacc ggtcttgcta
     3121 gattctaatc aagaggatgt cagaatgcca tttgcctgag agatgcaggc ttcatttttg
     3181 atactttttt atttgtaacc tatatagtat aggatttttt ttgtcatttt gtttcttctc
     3241 gtacgagctt gctcctgatc agcctatctc gcagctgatg aatatcttgt ggtaggggtt
     3301 tgggaaaatc attcgagttt gatgtttttc ttggtatttc ccactcctct tcagagtaca
     3361 gaagattaag tgagaccttc gtttgtgcgg atcccccaca caccatagct tcaaaatgtt
     3421 tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg taccacttca
     3481 aaacacccaa gcacagcata ctaaatttcc cctctttctt cctctagggt gtcgttaatt
     3541 acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga
     3601 aaaaggcaat aaaaattttt atcacgtttc tttttcttga aaattttttt ttttgatttt
     3661 tttctctttc gatgacctcc cattgatatt taagttaata aacggtcttc aatttctcaa
     3721 gtttcagttt catttttctt gttctattac aacttttttt acttcttgct cattagaaag
     3781 aaagcatagc aatctaatct aagggcggtg ttgacaatta atcatcggca tagtatatcg
     3841 gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc
     3901 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt
     3961 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt
     4021 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc
     4081 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg
     4141 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc
     4201 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtccgac
     4261 gcggcccgac gggtccgagg cctcggagat ccgtccccct tttcctttgt cgatatcatg
     4321 taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag
     4381 gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta
     4441 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca
     4501 tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt
     4561 tgcaagctgg agaccaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4621 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4681 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4741 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4801 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4861 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4921 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4981 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5041 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5101 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5161 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5221 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5281 cgttaaggga ttttggtcat gagatc
//
