LOCUS       sequence-291942-        4996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  P. pastoris expression vector. It will generate the mouse TREK-1 
            channel (21-322) fused to a C-terminal GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4996)
  AUTHORS   Lolicato M, Arrigoni C, Mori T, Sekioka Y, Bryant C, Clark KA, Minor
            DL Jr
  TITLE     K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity 
            filter binding site.
  JOURNAL   Nature. 2017 Jul 20;547(7663):364-368. doi: 10.1038/nature22988. 
            Epub 2017 Jul 10.
  PUBMED    28693035
REFERENCE   2  (bases 1 to 4996)
  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"; doi: 
            "10.1038/nature22988"; journalName: "Nature"; date: "2017-07-20- 
            20"; volume: "547"; issue: "7663"; pages: "364-368"
COMMENT     Sequence Label: pPICZ:mTREK-1_cryst
FEATURES             Location/Qualifiers
     source          1..4996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..937
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             1873..1896
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1912..1914,1915..2625)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1954..1975)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2215..2234)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2562..2583
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2638..2667
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2678..2707
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2723..2740
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2820..3066
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3081..3493
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3500..3547
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3566..3940
                     /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      4006..4253
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4022..4040)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4328..4916)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4408..4427)
                     /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 attcgaagct agcctcgagc caccatgtca
      961 ttctcctcaa aacccaccgt gcttgcttcc cgggtggaga gtgactcggc cattaatgtt
     1021 atgaaatgga agacggtctc cacgattttc ctggtggtcg tcctctacct gatcatcgga
     1081 gccacggtgt tcaaggcatt ggagcagcct caggagattt cccagaggac caccattgtg
     1141 atccagaggg agaagttctt acgcgcgcat ccctgcgtca gcgaccagga gctggacgaa
     1201 ctcatccagc aaatagtggc agcaataaac gcaggaatta tccccttagg agccagctcc
     1261 aatcaagtta gtcactggga cctcggaagc tctttcttct ttgctggtac tgttatcaca
     1321 accataggat ttggaaacat ctccccacga actgaaggtg gaaaaatatt ctgcatcatc
     1381 tatgccttgc tgggaattcc cctctttggc tttctactgg ctggggttgg tgatcagcta
     1441 ggaactatat ttggaaaagg aattgccaaa gtggaagaca catttattaa gtggaatgtt
     1501 agtcagacga agattcgtat catctccacc atcatcttca tcctgtttgg ctgtgtcctc
     1561 tttgtggctc tccctgcggt catattcaag cacatagaag gctggagcgc cctggacgct
     1621 atctattttg tggttatcac tctgacgacc attggatttg gagactacgt ggcaggtgga
     1681 tcagacattg aatatctgga cttctacaag cctgtggtgt ggttctggat cctcgttggg
     1741 ctggcctact ttgcagctgt tctgagcatg attggggact ggctacgggt gatcgctaag
     1801 aagacgaagg aagcggtggg agagttcaga gcgcatgccg ctgagtggac agccaatgtc
     1861 acttccaatt ctttggaagt tttgtttcaa ggtccaactg ctgccgccgc tgtgagcaag
     1921 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1981 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2041 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2101 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     2161 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2221 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2281 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2341 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     2401 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     2461 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     2521 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2581 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtccgg aggtggtcac
     2641 catcaccatc accaccatca ccatcactaa tagtctagaa caaaaactca tctcagaaga
     2701 ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttgta gccttagaca
     2761 tgactgttcc tcagttcaag ttgggcactt acgagaagac cggtcttgct agattctaat
     2821 caagaggatg tcagaatgcc atttgcctga gagatgcagg cttcattttt gatacttttt
     2881 tatttgtaac ctatatagta taggattttt tttgtcattt tgtttcttct cgtacgagct
     2941 tgctcctgat cagcctatct cgcagctgat gaatatcttg tggtaggggt ttgggaaaat
     3001 cattcgagtt tgatgttttt cttggtattt cccactcctc ttcagagtac agaagattaa
     3061 gtgagacctt cgtttgtgcg gatctcccac acaccatagc ttcaaaatgt ttctactcct
     3121 tttttactct tccagatttt ctcggactcc gcgcatcgcc gtaccacttc aaaacaccca
     3181 agcacagcat actaaatttc ccctctttct tcctctaggg tgtcgttaat tacccgtact
     3241 aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt ttcttcgtcg aaaaaggcaa
     3301 taaaaatttt tatcacgttt ctttttcttg aaaatttttt ttttgatttt tttctctttc
     3361 gatgacctcc cattgatatt taagttaata aacggtcttc aatttctcaa gtttcagttt
     3421 catttttctt gttctattac aacttttttt acttcttgct cattagaaag aaagcatagc
     3481 aatctaatct aagggcggtg ttgacaatta atcatcggca tagtatatcg gcatagtata
     3541 atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac
     3601 cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga
     3661 cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc
     3721 ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga
     3781 cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc
     3841 ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc
     3901 cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtccgac gcggcccgac
     3961 gggtccgagg cctcggagat ccgtccccct tttcctttgt cgatatcatg taattagtta
     4021 tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta
     4081 gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt
     4141 tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta
     4201 tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcaagctgg
     4261 agaccaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4321 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4381 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4441 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4501 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4561 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4621 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4681 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4741 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4801 cagttacctt cggaaaaaga gttagtagct cttgatccgg caaacaaacc accgctggta
     4861 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4921 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4981 ttttggtcat gagatc
//
