LOCUS       sequence-445194-        4996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Pichia pastoris expression vector for generating mouse 
            TREK-1(21S-322T) + 3C cleavable site + eGFP + 10x His under the AOX1
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4996)
  AUTHORS   Deal PE, Lee H, Mondal A, Lolicato M, Mendonca PRF, Black H, Jang S,
            El-Hilali X, Bryant C, Isacoff EY, Renslo AR, Minor DL Jr
  TITLE     Development of covalent chemogenetic K(2P) channel activators.
  JOURNAL   Cell Chem Biol. 2024 Jul 18;31(7):1305-1323.e9. doi: 
            10.1016/j.chembiol.2024.06.006.
  PUBMED    39029456
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: 
            "10.1016/j.chembiol.2024.06"; journalName: "Cell Chem Biol"; date: 
            "2024-07-18- 18"; volume: "31"; issue: "7"; pages: "1305-1323.e9"
COMMENT     Sequence Label: pPICZ:K2P2.1(mTREK-1)Cryst S112C
FEATURES             Location/Qualifiers
     source          1..4996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..1017
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             1953..1976
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1992..1994,1995..2705)
                     /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(2034..2055)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2295..2314)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2642..2663
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2718..2747
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2758..2787
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2803..2820
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2900..3146
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3161..3573
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3580..3627
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3646..4020
                     /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      4086..4333
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4102..4120)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4408..4996)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4488..4507)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatc agatctaaca tccaaagacg aaaggttgaa tgaaaccttt
      121 ttgccatccg acatccacag gtccattctc acacataagt gccaaacgca acaggagggg
      181 atacactagc agcagaccgt tgcaaacgca ggacctccac tcctcttctc ctcaacaccc
      241 acttttgcca tcgaaaaacc agcccagtta ttgggcttga ttggagctcg ctcattccaa
      301 ttccttctat taggctacta acaccatgac tttattagcc tgtctatcct ggcccccctg
      361 gcgaggttca tgtttgttta tttccgaatg caacaagctc cgcattacac ccgaacatca
      421 ctccagatga gggctttctg agtgtggggt caaatagttt catgttcccc aaatggccca
      481 aaactgacag tttaaacgct gtcttggaac ctaatatgac aaaagcgtga tctcatccaa
      541 gatgaactaa gtttggttcg ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca
      601 aaagtcggca taccgtttgt cttgtttggt attgattgac gaatgctcaa aaataatctc
      661 attaatgctt agcgcagtct ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg
      721 caaatgggga aacacccgct ttttggatga ttatgcattg tctccacatt gtatgcttcc
      781 aagattctgg tgggaatact gctgatagcc taacgttcat gatcaaaatt taactgttct
      841 aacccctact tgacagcaat atataaacag aaggaagctg ccctgtctta aacctttttt
      901 tttatcatca ttattagctt actttcataa ttgcgactgg ttccaattga caagcttttg
      961 attttaacga cttttaacga caacttgaga agatcaaaaa acaactaatt attcgaagct
     1021 agcctcgagc caccatgtca ttctcctcaa aacccaccgt gcttgcttcc cgggtggaga
     1081 gtgactcggc cattaatgtt atgaaatgga agacggtctc cacgattttc ctggtggtcg
     1141 tcctctacct gatcatcgga gccacggtgt tcaaggcatt ggagcagcct caggagattt
     1201 cccagaggac caccattgtg atccagaggg agaagttctt acgcgcgcat ccctgcgtca
     1261 gcgaccagga gctggacgaa ctcatccagc aaatagtggc agcaataaac gcaggaatta
     1321 tccccttagg agccagctcc aatcaagtta gtcactggga cctcggatgc tctttcttct
     1381 ttgctggtac tgttatcaca accataggat ttggaaacat ctccccacga actgaaggtg
     1441 gaaaaatatt ctgcatcatc tatgccttgc tgggaattcc cctctttggc tttctactgg
     1501 ctggggttgg tgatcagcta ggaactatat ttggaaaagg aattgccaaa gtggaagaca
     1561 catttattaa gtggaatgtt agtcagacga agattcgtat catctccacc atcatcttca
     1621 tcctgtttgg ctgtgtcctc tttgtggctc tccctgcggt catattcaag cacatagaag
     1681 gctggagcgc cctggacgct atctattttg tggttatcac tctgacgacc attggatttg
     1741 gagactacgt ggcaggtgga tcagacattg aatatctgga cttctacaag cctgtggtgt
     1801 ggttctggat cctcgttggg ctggcctact ttgcagctgt tctgagcatg attggggact
     1861 ggctacgggt gatcgctaag aagacgaagg aagcggtggg agagttcaga gcgcatgccg
     1921 ctgagtggac agccaatgtc acttccaatt ctttggaagt tttgtttcaa ggtccaactg
     1981 ctgccgccgc tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2041 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     2101 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2161 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     2221 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2281 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2341 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2401 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     2461 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     2521 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2581 acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc
     2641 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2701 acaagtccgg aggtggtcac catcaccatc accaccatca ccatcactaa tagtctagaa
     2761 caaaaactca tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat
     2821 tgagtttgta gccttagaca tgactgttcc tcagttcaag ttgggcactt acgagaagac
     2881 cggtcttgct agattctaat caagaggatg tcagaatgcc atttgcctga gagatgcagg
     2941 cttcattttt gatacttttt tatttgtaac ctatatagta taggattttt tttgtcattt
     3001 tgtttcttct cgtacgagct tgctcctgat cagcctatct cgcagctgat gaatatcttg
     3061 tggtaggggt ttgggaaaat cattcgagtt tgatgttttt cttggtattt cccactcctc
     3121 ttcagagtac agaagattaa gtgagacctt cgtttgtgcg gatctcccac acaccatagc
     3181 ttcaaaatgt ttctactcct tttttactct tccagatttt ctcggactcc gcgcatcgcc
     3241 gtaccacttc aaaacaccca agcacagcat actaaatttc ccctctttct tcctctaggg
     3301 tgtcgttaat tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt
     3361 ttcttcgtcg aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg aaaatttttt
     3421 ttttgatttt tttctctttc gatgacctcc cattgatatt taagttaata aacggtcttc
     3481 aatttctcaa gtttcagttt catttttctt gttctattac aacttttttt acttcttgct
     3541 cattagaaag aaagcatagc aatctaatct aagggcggtg ttgacaatta atcatcggca
     3601 tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc
     3661 agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac
     3721 cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac
     3781 gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg
     3841 gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac
     3901 ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag
     3961 ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga
     4021 cacgtccgac gcggcccgac gggtccgagg cctcggagat ccgtccccct tttcctttgt
     4081 cgatatcatg taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct
     4141 aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt
     4201 tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg
     4261 cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa
     4321 ggctttaatt tgcaagctgg agaccaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4381 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgccccccta acgagcatca
     4441 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4501 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4561 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4621 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4681 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4741 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4801 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4861 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttagtagct cttgatccgg
     4921 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4981 aaaaaaagga tctcaa
//
