LOCUS       sequence-261313-        4987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  P. pastoris expression vector. It will generate the human TRAAK 
            channel (1-300) fused to a C-terminal GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4987)
  AUTHORS   Lolicato M, Riegelhaupt PM, Arrigoni C, Clark KA, Minor DL Jr
  TITLE     Transmembrane helix straightening and buckling underlies activation 
            of mechanosensitive and thermosensitive K(2P) channels.
  JOURNAL   Neuron. 2014 Dec 17;84(6):1198-212. doi: 
            10.1016/j.neuron.2014.11.017. Epub 2014 Dec 11.
  PUBMED    25500157
REFERENCE   2  (bases 1 to 4987)
  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.neuron.2014.11.017"; journalName: "Neuron"; date: 
            "2014-12-17- 17"; volume: "84"; issue: "6"; pages: "1198-212"
COMMENT     Sequence Label: pPICZ:hTRAAK(G124I)
FEATURES             Location/Qualifiers
     source          1..4987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        296..1231
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             2158..2181
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(2197..2199,2200..2910)
                     /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(2239..2260)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2500..2519)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2847..2868
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2923..2952
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2963..2992
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3008..3025
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3105..3351
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3366..3778
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3785..3832
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3851..4225
                     /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      4291..4538
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4307..4325)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(join(4613..4987,1..214))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4693..4712)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
       61 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
      121 tagtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
      181 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
      241 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gatcagatct
      301 aacatccaaa gacgaaaggt tgaatgaaac ctttttgcca tccgacatcc acaggtccat
      361 tctcacacat aagtgccaaa cgcaacagga ggggatacac tagcagcaga ccgttgcaaa
      421 cgcaggacct ccactcctct tctcctcaac acccactttt gccatcgaaa aaccagccca
      481 gttattgggc ttgattggag ctcgctcatt ccaattcctt ctattaggct actaacacca
      541 tgactttatt agcctgtcta tcctggcccc cctggcgagg ttcatgtttg tttatttccg
      601 aatgcaacaa gctccgcatt acacccgaac atcactccag atgagggctt tctgagtgtg
      661 gggtcaaata gtttcatgtt ccccaaatgg cccaaaactg acagtttaaa cgctgtcttg
      721 gaacctaata tgacaaaagc gtgatctcat ccaagatgaa ctaagtttgg ttcgttgaaa
      781 tgctaacggc cagttggtca aaaagaaact tccaaaagtc ggcataccgt ttgtcttgtt
      841 tggtattgat tgacgaatgc tcaaaaataa tctcattaat gcttagcgca gtctctctat
      901 cgcttctgaa ccccggtgca cctgtgccga aacgcaaatg gggaaacacc cgctttttgg
      961 atgattatgc attgtctcca cattgtatgc ttccaagatt ctggtgggaa tactgctgat
     1021 agcctaacgt tcatgatcaa aatttaactg ttctaacccc tacttgacag caatatataa
     1081 acagaaggaa gctgccctgt cttaaacctt tttttttatc atcattatta gcttactttc
     1141 ataattgcga ctggttccaa ttgacaagct tttgatttta acgactttta acgacaactt
     1201 gagaagatca aaaaacaact aattattcga agctagcctc gagccaccat gactaccgct
     1261 ccacaagaac cacccgctcg cccactccaa gctggctccg gtgctggtcc tgctcctgga
     1321 cgtgctatga gatcaacaac gctcctcgct ctgctggccc tcgtattgct gtacctcgtg
     1381 tctggtgctc tggtattccg tgctctcgaa caaccacatg aacaacaggc tcaacgtgaa
     1441 ctgggagaag tccgtgaaaa attcctccgt gcccatccgt gtgtttccga tcaagagttg
     1501 ggattgctca tcaaggaagt cgccgacgct ttgggaggcg gtgccgaccc tgagacacaa
     1561 tccacctcac agtcctctca ctctgcctgg gatttgggat ctgccttctt cttctctata
     1621 accatcatca ccactatcgg ttacggtaac gtggcactcc gtaccgacgc cggtagactg
     1681 ttctgtattt tctacgccct cgttggtatc cctctcttcg gaatcttgtt ggctggagtc
     1741 ggagatcgcc tcggttcatc attgaggcat ggcatcggtc atattgaggc cattttcttg
     1801 aaatggcacg tgccacctga attggttcgt gtactgtccg ctatgctgtt cctcctcatc
     1861 ggttgcttgc tcttcgttct cacccccact ttcgtattct gctacatgga ggactggtct
     1921 aaactggagg ccatctactt cgtgatcgtg accctcacta ctgttggatt cggagactac
     1981 gttgctggag ccgatcctag acaggattct cctgcctacc aacctttggt gtggttctgg
     2041 attctgttgg gactggcata cttcgctagt gtgctcacca ccattggcaa ctggttgaga
     2101 gtggtatcac gtcgtactcg tgctgagatg ggaggactca ctgctcaatc gaattctttg
     2161 gaagttttgt ttcaaggtcc aactgctgcc gccgctgtga gcaagggcga ggagctgttc
     2221 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2281 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     2341 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     2401 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2461 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2521 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2581 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     2641 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     2701 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     2761 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc
     2821 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2881 atcactctcg gcatggacga gctgtacaag tccggaggtg gtcaccatca ccatcaccac
     2941 catcaccatc actaatagtc tagaacaaaa actcatctca gaagaggatc tgaatagcgc
     3001 cgtcgaccat catcatcatc atcattgagt ttgtagcctt agacatgact gttcctcagt
     3061 tcaagttggg cacttacgag aagaccggtc ttgctagatt ctaatcaaga ggatgtcaga
     3121 atgccatttg cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata
     3181 tagtatagga ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc
     3241 tatctcgcag ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg
     3301 tttttcttgg tatttcccac tcctcttcag agtacagaag attaagtgag accttcgttt
     3361 gtgcggatct cccacacacc atagcttcaa aatgtttcta ctcctttttt actcttccag
     3421 attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac agcatactaa
     3481 atttcccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg tttggaaaag
     3541 aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa atttttatca
     3601 cgtttctttt tcttgaaaat tttttttttg atttttttct ctttcgatga cctcccattg
     3661 atatttaagt taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct
     3721 attacaactt tttttacttc ttgctcatta gaaagaaagc atagcaatct aatctaaggg
     3781 cggtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
     3841 gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
     3901 cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga
     3961 cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt
     4021 ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga
     4081 gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat
     4141 cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca
     4201 cttcgtggcc gaggagcagg actgacacgt ccgacgcggc ccgacgggtc cgaggcctcg
     4261 gagatccgtc ccccttttcc tttgtcgata tcatgtaatt agttatgtca cgcttacatt
     4321 cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct
     4381 aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa
     4441 tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc
     4501 ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa gctggagacc aacatgtgag
     4561 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4621 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4681 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4741 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4801 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4861 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4921 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4981 ttagcag
//
