LOCUS       sequence-261311-        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(W262S)
FEATURES             Location/Qualifiers
     source          1..4987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             927..950
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(966..968,969..1679)
                     /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(1008..1029)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1269..1288)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1616..1637
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1692..1721
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1732..1761
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1777..1794
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1874..2120
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2135..2547
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2554..2601
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2620..2994
                     /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      3060..3307
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3076..3094)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3382..3970)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3462..3481)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4052..4987
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
ORIGIN
        1 gctagcctcg agccaccatg actaccgctc cacaagaacc acccgctcgc ccactccaag
       61 ctggctccgg tgctggtcct gctcctggac gtgctatgag atcaacaacg ctcctcgctc
      121 tgctggccct cgtattgctg tacctcgtgt ctggtgctct ggtattccgt gctctcgaac
      181 aaccacatga acaacaggct caacgtgaac tgggagaagt ccgtgaaaaa ttcctccgtg
      241 cccatccgtg tgtttccgat caagagttgg gattgctcat caaggaagtc gccgacgctt
      301 tgggaggcgg tgccgaccct gagacacaat ccacctcaca gtcctctcac tctgcctggg
      361 atttgggatc tgccttcttc ttctctggaa ccatcatcac cactatcggt tacggtaacg
      421 tggcactccg taccgacgcc ggtagactgt tctgtatttt ctacgccctc gttggtatcc
      481 ctctcttcgg aatcttgttg gctggagtcg gagatcgcct cggttcatca ttgaggcatg
      541 gcatcggtca tattgaggcc attttcttga aatggcacgt gccacctgaa ttggttcgtg
      601 tactgtccgc tatgctgttc ctcctcatcg gttgcttgct cttcgttctc acccccactt
      661 tcgtattctg ctacatggag gactggtcta aactggaggc catctacttc gtgatcgtga
      721 ccctcactac tgttggattc ggagactacg ttgctggagc cgatcctaga caggattctc
      781 ctgcctacca acctttggtg tcgttctgga ttctgttggg actggcatac ttcgctagtg
      841 tgctcaccac cattggcaac tggttgagag tggtatcacg tcgtactcgt gctgagatgg
      901 gaggactcac tgctcaatcg aattctttgg aagttttgtt tcaaggtcca actgctgccg
      961 ccgctgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1021 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1081 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1141 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1201 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1261 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1321 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1381 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1441 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1501 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1561 actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
     1621 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1681 ccggaggtgg tcaccatcac catcaccacc atcaccatca ctaatagtct agaacaaaaa
     1741 ctcatctcag aagaggatct gaatagcgcc gtcgaccatc atcatcatca tcattgagtt
     1801 tgtagcctta gacatgactg ttcctcagtt caagttgggc acttacgaga agaccggtct
     1861 tgctagattc taatcaagag gatgtcagaa tgccatttgc ctgagagatg caggcttcat
     1921 ttttgatact tttttatttg taacctatat agtataggat tttttttgtc attttgtttc
     1981 ttctcgtacg agcttgctcc tgatcagcct atctcgcagc tgatgaatat cttgtggtag
     2041 gggtttggga aaatcattcg agtttgatgt ttttcttggt atttcccact cctcttcaga
     2101 gtacagaaga ttaagtgaga ccttcgtttg tgcggatctc ccacacacca tagcttcaaa
     2161 atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat cgccgtacca
     2221 cttcaaaaca cccaagcaca gcatactaaa tttcccctct ttcttcctct agggtgtcgt
     2281 taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc
     2341 gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaaatt ttttttttga
     2401 tttttttctc tttcgatgac ctcccattga tatttaagtt aataaacggt cttcaatttc
     2461 tcaagtttca gtttcatttt tcttgttcta ttacaacttt ttttacttct tgctcattag
     2521 aaagaaagca tagcaatcta atctaagggc ggtgttgaca attaatcatc ggcatagtat
     2581 atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt gaccagtgcc
     2641 gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc
     2701 gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc
     2761 ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg
     2821 gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg
     2881 gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc
     2941 ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtc
     3001 cgacgcggcc cgacgggtcc gaggcctcgg agatccgtcc cccttttcct ttgtcgatat
     3061 catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga
     3121 aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt
     3181 agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta
     3241 cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt
     3301 aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3361 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3421 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3481 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3541 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     3601 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3661 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     3721 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     3781 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     3841 cgctctgctg aagccagtta ccttcggaaa aagagttagt agctcttgat ccggcaaaca
     3901 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     3961 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4021 ctcacgttaa gggattttgg tcatgagatc agatctaaca tccaaagacg aaaggttgaa
     4081 tgaaaccttt ttgccatccg acatccacag gtccattctc acacataagt gccaaacgca
     4141 acaggagggg atacactagc agcagaccgt tgcaaacgca ggacctccac tcctcttctc
     4201 ctcaacaccc acttttgcca tcgaaaaacc agcccagtta ttgggcttga ttggagctcg
     4261 ctcattccaa ttccttctat taggctacta acaccatgac tttattagcc tgtctatcct
     4321 ggcccccctg gcgaggttca tgtttgttta tttccgaatg caacaagctc cgcattacac
     4381 ccgaacatca ctccagatga gggctttctg agtgtggggt caaatagttt catgttcccc
     4441 aaatggccca aaactgacag tttaaacgct gtcttggaac ctaatatgac aaaagcgtga
     4501 tctcatccaa gatgaactaa gtttggttcg ttgaaatgct aacggccagt tggtcaaaaa
     4561 gaaacttcca aaagtcggca taccgtttgt cttgtttggt attgattgac gaatgctcaa
     4621 aaataatctc attaatgctt agcgcagtct ctctatcgct tctgaacccc ggtgcacctg
     4681 tgccgaaacg caaatgggga aacacccgct ttttggatga ttatgcattg tctccacatt
     4741 gtatgcttcc aagattctgg tgggaatact gctgatagcc taacgttcat gatcaaaatt
     4801 taactgttct aacccctact tgacagcaat atataaacag aaggaagctg ccctgtctta
     4861 aacctttttt tttatcatca ttattagctt actttcataa ttgcgactgg ttccaattga
     4921 caagcttttg attttaacga cttttaacga caacttgaga agatcaaaaa acaactaatt
     4981 attcgaa
//
