LOCUS       sequence-231299-        5414 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 5414)
  TITLE     Chang lab yeast expression plasmids
REFERENCE   2  (bases 1 to 5414)
  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: CIPK2-upGFP
FEATURES             Location/Qualifiers
     source          1..5414
                     /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             2357..2377
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(2384..2386,2387..2389,2390..3100)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2384..2386,2387..2389,2390..3100)
                     /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(2429..2450)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2690..2709)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3037..3058
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3107..3130
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3140..3157
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3237..3483
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3498..3911
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3918..3965
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3984..4358
                     /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      4424..4671
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4440..4458)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4746..5334)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4826..4845)
                     /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 tatggagaac aaaccaagtg tattaactga aagatatgaa gttgggaggt tattgggtca
     1021 aggtactttt gctaaagtgt attttggaag gagtaatcat accaacgaga gtgtagctat
     1081 caagatgatt gacaaggaca aagttatgag agtcgggctt agccagcaaa tcaagcgaga
     1141 gatctctgta atgaggatcg ctaaacaccc gaatgtcgtt gagttatacg aggttatggc
     1201 tacaaagtca aggatttact ttgttattga gtattgtaaa ggtggtgagc ttttcaacaa
     1261 ggttgcaaaa ggaaaactta aagaagatgt tgcttggaag tatttttatc agcttattag
     1321 tgcggttgat ttttgtcaca gccgcggagt ttatcaccgc gacattaagc cggaaaatct
     1381 tttgttggat gacaatgata atcttaaggt atctgatttt ggtttaagtg cgcttgctga
     1441 ttgcaagcgg caagatggtc ttctacatac aacttgtggt acacctgctt atgttgcgcc
     1501 cgaggttatt aaccgaaaag gatacgaggg tacgaaagcg gatatttggt cttgtggtgt
     1561 tgttttgttt gttcttttgg ctggttatct tcctttccat gacactaatc ttatggagat
     1621 gtataggaag ataggtaaag cagacttcaa gtgtcccagc tggtttgctc ctgaggtaaa
     1681 gagactattg tgtaagatgt tggaccctaa ccatgagact agaatcacta ttgcaaaaat
     1741 caaggagagt tcttggttca gaaagggttt gcatttgaag caaaaaaaga tggaaaagat
     1801 ggagaaacaa caagtcagag aggctactaa tcccatggaa gctggaggtt caggccaaaa
     1861 tgaaaacgga gaaaaccacg agccgcctcg acttgctacc ttgaatgctt tcgatatcat
     1921 cgccttgtct acggggtttg gtctggcagg actttttggg gatgtatatg acaagcgaga
     1981 atctaggttc gcgtcgcaaa aacctgcttc agagatcatt tctaagctag tagaggttgc
     2041 caagtgcctg aagctgaaga taagaaagca aggcgcaggc ttgttcaaac tggaaagagt
     2101 aaaggaagga aaaaacggaa ttttgacgat ggatgcagag atattccaag tgacgccgac
     2161 gtttcacctg gtggaagtga agaaatgtaa tggagataca atggagtatc agaagttagt
     2221 ggaggaggat cttaggcctg ctttggcaga tattgtttgg gtttggcaag gcgagaagga
     2281 gaaagaggag cagttactgc aggatgaaca aggagagcaa gaaccatcag cattaaacct
     2341 cagcctcgag ggttcggaga acctgtactt ccaatctggt tcgatggtga gcaagggcga
     2401 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     2461 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     2521 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     2581 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     2641 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     2701 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     2761 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     2821 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     2881 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     2941 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     3001 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     3061 cgccgccggg atcactctcg gcatggacga gctgtacaag tcgtcgcatc atcatcatca
     3121 tcatcatcat taagtcgacc atcatcatca tcatcattga gtttgtagcc ttagacatga
     3181 ctgttcctca gttcaagttg ggcacttacg agaagaccgg tcttgctaga ttctaatcaa
     3241 gaggatgtca gaatgccatt tgcctgagag atgcaggctt catttttgat acttttttat
     3301 ttgtaaccta tatagtatag gatttttttt gtcattttgt ttcttctcgt acgagcttgc
     3361 tcctgatcag cctatctcgc agctgatgaa tatcttgtgg taggggtttg ggaaaatcat
     3421 tcgagtttga tgtttttctt ggtatttccc actcctcttc agagtacaga agattaagtg
     3481 agaccttcgt ttgtgcggat cccccacaca ccatagcttc aaaatgtttc tactcctttt
     3541 ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc
     3601 acagcatact aaatttcccc tctttcttcc tctagggtgt cgttaattac ccgtactaaa
     3661 ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa
     3721 aaatttttat cacgtttctt tttcttgaaa attttttttt ttgatttttt tctctttcga
     3781 tgacctccca ttgatattta agttaataaa cggtcttcaa tttctcaagt ttcagtttca
     3841 tttttcttgt tctattacaa ctttttttac ttcttgctca ttagaaagaa agcatagcaa
     3901 tctaatctaa gggcggtgtt gacaattaat catcggcata gtatatcggc atagtataat
     3961 acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg
     4021 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
     4081 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
     4141 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
     4201 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
     4261 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
     4321 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtccgacgc ggcccgacgg
     4381 gtccgaggcc tcggagatcc gtcccccttt tcctttgtcg atatcatgta attagttatg
     4441 tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga
     4501 caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta
     4561 tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata
     4621 ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg caagctggag
     4681 accaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4741 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4801 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4861 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4921 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4981 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5041 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5101 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5161 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5221 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5281 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5341 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5401 ttggtcatga gatc
//
