LOCUS       sequence-309639-        4910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses K2P4.1 isoform B with TEV protease cleavable N- and 
            C-terminal affinity tags..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4910)
  AUTHORS   Schrecke S, Zhu Y, McCabe JW, Bartz M, Packianathan C, Zhao M, Zhou 
            M, Russell D, Laganowsky A
  TITLE     Selective regulation of human TRAAK channels by biologically active 
            phospholipids.
  JOURNAL   Nat Chem Biol. 2021 Jan;17(1):89-95. doi: 
            10.1038/s41589-020-00659-5. Epub 2020 Sep 28.
  PUBMED    32989299
REFERENCE   2  (bases 1 to 4910)
  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/s41589-020-00659-5"; journalName: "Nat Chem Biol"; date: 
            "2021-01"; volume: "17"; issue: "1"; pages: "89-95"
COMMENT     Sequence Label: pPICZ-His6-K2P4.1B-mCherry-StreptagII
FEATURES             Location/Qualifiers
     source          1..4910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        225..1160
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     regulatory      1165..1174
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1177..1194
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1201..1221
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2107..2127
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2854..2877
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     terminator      2957..3203
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3218..3630
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3637..3684
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3703..4077
                     /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      4143..4390
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4159..4177)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(join(4465..4910,1..143))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4545..4564)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
       61 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
      121 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
      181 agtggaacga aaactcacgt taagggattt tggtcatgag atcagatcta acatccaaag
      241 acgaaaggtt gaatgaaacc tttttgccat ccgacatcca caggtccatt ctcacacata
      301 agtgccaaac gcaacaggag gggatacact agcagcagac cgttgcaaac gcaggacctc
      361 cactcctctt ctcctcaaca cccacttttg ccatcgaaaa accagcccag ttattgggct
      421 tgattggagc tcgctcattc caattccttc tattaggcta ctaacaccat gactttatta
      481 gcctgtctat cctggccccc ctggcgaggt tcatgtttgt ttatttccga atgcaacaag
      541 ctccgcatta cacccgaaca tcactccaga tgagggcttt ctgagtgtgg ggtcaaatag
      601 tttcatgttc cccaaatggc ccaaaactga cagtttaaac gctgtcttgg aacctaatat
      661 gacaaaagcg tgatctcatc caagatgaac taagtttggt tcgttgaaat gctaacggcc
      721 agttggtcaa aaagaaactt ccaaaagtcg gcataccgtt tgtcttgttt ggtattgatt
      781 gacgaatgct caaaaataat ctcattaatg cttagcgcag tctctctatc gcttctgaac
      841 cccggtgcac ctgtgccgaa acgcaaatgg ggaaacaccc gctttttgga tgattatgca
      901 ttgtctccac attgtatgct tccaagattc tggtgggaat actgctgata gcctaacgtt
      961 catgatcaaa atttaactgt tctaacccct acttgacagc aatatataaa cagaaggaag
     1021 ctgccctgtc ttaaaccttt ttttttatca tcattattag cttactttca taattgcgac
     1081 tggttccaat tgacaagctt ttgattttaa cgacttttaa cgacaacttg agaagatcaa
     1141 aaaacaacta attattcgaa gctagccacc atggctcacc atcatcatca tcacggttcc
     1201 gagaatctgt acttccaaag cggtaccatg actaccgctc ctcaagagcc accagcaaga
     1261 ccccttcaag caggctcagg cgcaggtccc gctcctggcc gtgccatgag gtcaaccact
     1321 ttgttggctc tgcttgcttt agtgctgctt tacctagtca gtggtgccct ggtgtttagg
     1381 gccttggaac aaccccacga gcaacaagct cagcgtgagc tgggtgaggt cagagaaaaa
     1441 tttttaagag cacacccatg tgtatccgac caagaacttg gactgctaat caaagaggtg
     1501 gccgacgccc ttggcggcgg cgccgaccct gaaacccaat ccacctctca gtcaagtcac
     1561 agtgcctggg atctgggaag tgccttcttt ttcagtggta caattatcac gacaataggc
     1621 tacggaaacg tggccttgag gacggatgcc ggtagactgt tttgcatatt ctacgcatta
     1681 gtaggaatcc ccctgtttgg tatcttgcta gctggtgtgg gtgatcgtct gggatcatca
     1741 cttagacatg gtataggcca tatagaggcc atcttcctga aatggcatgt accaccagaa
     1801 ttggtcaggg ttttatcagc tatgttattt ttacttatcg gatgcctatt gtttgtactg
     1861 accccaacat tcgttttctg ctacatggaa gactggtcca aacttgaagc tatttatttt
     1921 gtgatagtga ccttaactac ggtcggcttt ggcgattacg tcgcaggcgc agatccacgt
     1981 caagattctc cagcatatca accactagtg tggttctgga tcctactggg attagcctac
     2041 ttcgcttctg tcttgactac tataggcaac tggttgagag tcgtttcccg taggaccggt
     2101 accagcgaaa acctgtattt tcagggtact agtgtctcta agggagaaga ggacaatatg
     2161 gctattatca aggagttcat gagatttaaa gtccatatgg agggctctgt taatggtcat
     2221 gaatttgaaa ttgaaggtga aggagaagga agaccttatg agggaaccca aacggctaag
     2281 ctgaaagtaa ctaaaggcgg cccacttcct ttcgcatggg atatactgtc tccacaattc
     2341 atgtatggct ctaaagctta tgtaaagcac cccgccgaca ttcccgatta tctaaagtta
     2401 tcctttcccg agggatttaa atgggaaagg gttatgaact tcgaggatgg cggcgtggta
     2461 actgttacgc aggactcctc ccttcaagat ggtgaattta tttacaaagt gaagcttcgt
     2521 ggtactaatt tcccaagtga cggtcctgtg atgcagaaga agactatggg ttgggaagct
     2581 agttccgaga ggatgtaccc cgaagatggt gccctgaagg gtgagattaa gcaacgtcta
     2641 aaattaaaag acggcggtca ctatgacgct gaagtaaaga ccacatataa ggcaaaaaag
     2701 cccgtccagc ttcctggcgc ctacaatgtt aatatcaagt tggacatcac ctcccataac
     2761 gaagattaca ctatcgtcga gcaatacgag agggcagagg gaagacattc cacaggtggt
     2821 atggacgagc tatataaact cgagggcgct tcctggtcac atccacagtt tgagaaatga
     2881 gtttgtagcc ttagacatga ctgttcctca gttcaagttg ggcacttacg agaagaccgg
     2941 tcttgctaga ttctaatcaa gaggatgtca gaatgccatt tgcctgagag atgcaggctt
     3001 catttttgat acttttttat ttgtaaccta tatagtatag gatttttttt gtcattttgt
     3061 ttcttctcgt acgagcttgc tcctgatcag cctatctcgc agctgatgaa tatcttgtgg
     3121 taggggtttg ggaaaatcat tcgagtttga tgtttttctt ggtatttccc actcctcttc
     3181 agagtacaga agattaagtg agaccttcgt ttgtgcggat ctcccacaca ccatagcttc
     3241 aaaatgtttc tactcctttt ttactcttcc agattttctc ggactccgcg catcgccgta
     3301 ccacttcaaa acacccaagc acagcatact aaatttcccc tctttcttcc tctagggtgt
     3361 cgttaattac ccgtactaaa ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc
     3421 ttcgtcgaaa aaggcaataa aaatttttat cacgtttctt tttcttgaaa attttttttt
     3481 tgattttttt ctctttcgat gacctcccat tgatatttaa gttaataaac ggtcttcaat
     3541 ttctcaagtt tcagtttcat ttttcttgtt ctattacaac tttttttact tcttgctcat
     3601 tagaaagaaa gcatagcaat ctaatctaag ggcggtgttg acaattaatc atcggcatag
     3661 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt
     3721 gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg
     3781 ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg
     3841 accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg
     3901 tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc
     3961 cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc
     4021 gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac
     4081 gtccgacgcg gcccgacggg tccgaggcct cggagatccg tccccctttt cctttgtcga
     4141 tatcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac
     4201 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat
     4261 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt
     4321 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc
     4381 tttaatttgc aagctggaga ccaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4441 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4501 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4561 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4621 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4681 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4741 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4801 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4861 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
//
