LOCUS       sequence-231229-        4655 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 4655)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 4655)
  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: CBL5-upGFP
FEATURES             Location/Qualifiers
     source          1..4655
                     /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             1598..1618
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1625..1627,1628..1630,1631..2341)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1625..1627,1628..1630,1631..2341)
                     /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(1670..1691)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1931..1950)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2278..2299
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2348..2371
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2381..2398
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2478..2724
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2739..3152
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3159..3206
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3225..3599
                     /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      3665..3912
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3681..3699)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3987..4575)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4067..4086)
                     /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 tatgggatgt gtttgcagca agcaattaga aggaagaagg caagaagaca tttcgcttct
     1021 tgcttctcaa acctttttta gcgaggctga ggttgaagtt ttgcatggat tgttcatcaa
     1081 actcacttct tgtcttagca acgacaatct tttgaccaaa gaaaaatttc aatttatctt
     1141 gatcaagaat accaaaaagc gcagtctttc tgcagaacgg atttttggat tattcgatat
     1201 gagaaacgat ggggctatag attttggaga gtttgtacac acactcaaca tttttcaccc
     1261 aaactcatca ccaagagata aggccatatt tgcatttaga ttatacgata ctcgtgagac
     1321 tggattcatt gaaccggagg aggtgaaaga gatgataata gacgttctag aggaatcaga
     1381 acttatgcta tccgagagta tcattgattc tatcgtgtct aagacatttg aagaggccga
     1441 ttggaaaaaa gatgggataa tagatttgga agaatgggag aattttgttg ccacctatcc
     1501 tttgacacta aagaacatga ctatcccttt cttgaaggac attccgagga ttttcccaac
     1561 ctttctccgg ggattaaacc tcagcctcga gggttcggag aacctgtact tccaatctgg
     1621 ttcgatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1681 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1741 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1801 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1861 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1921 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1981 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     2041 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     2101 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     2161 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     2221 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     2281 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     2341 gtcgtcgcat catcatcatc atcatcatca ttaagtcgac catcatcatc atcatcattg
     2401 agtttgtagc cttagacatg actgttcctc agttcaagtt gggcacttac gagaagaccg
     2461 gtcttgctag attctaatca agaggatgtc agaatgccat ttgcctgaga gatgcaggct
     2521 tcatttttga tactttttta tttgtaacct atatagtata ggattttttt tgtcattttg
     2581 tttcttctcg tacgagcttg ctcctgatca gcctatctcg cagctgatga atatcttgtg
     2641 gtaggggttt gggaaaatca ttcgagtttg atgtttttct tggtatttcc cactcctctt
     2701 cagagtacag aagattaagt gagaccttcg tttgtgcgga tcccccacac accatagctt
     2761 caaaatgttt ctactccttt tttactcttc cagattttct cggactccgc gcatcgccgt
     2821 accacttcaa aacacccaag cacagcatac taaatttccc ctctttcttc ctctagggtg
     2881 tcgttaatta cccgtactaa aggtttggaa aagaaaaaag agaccgcctc gtttcttttt
     2941 cttcgtcgaa aaaggcaata aaaattttta tcacgtttct ttttcttgaa aatttttttt
     3001 tttgattttt ttctctttcg atgacctccc attgatattt aagttaataa acggtcttca
     3061 atttctcaag tttcagtttc atttttcttg ttctattaca acttttttta cttcttgctc
     3121 attagaaaga aagcatagca atctaatcta agggcggtgt tgacaattaa tcatcggcat
     3181 agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca
     3241 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc
     3301 ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg
     3361 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg
     3421 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact
     3481 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt
     3541 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac
     3601 acgtccgacg cggcccgacg ggtccgaggc ctcggagatc cgtccccctt ttcctttgtc
     3661 gatatcatgt aattagttat gtcacgctta cattcacgcc ctccccccac atccgctcta
     3721 accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt
     3781 atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc
     3841 gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag
     3901 gctttaattt gcaagctgga gaccaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3961 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4021 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4081 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4141 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4201 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4261 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4321 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4381 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4441 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4501 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4561 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4621 gaaaactcac gttaagggat tttggtcatg agatc
//
