LOCUS       sequence-309640-        4832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses K2P4.1 isoform A 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 4832)
  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 4832)
  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-K2P4.1A-mCherry-StreptagII
FEATURES             Location/Qualifiers
     source          1..4832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      5..14
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             17..34
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             41..61
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             869..889
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1616..1639
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     terminator      1719..1965
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        1980..2392
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2399..2446
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2465..2839
                     /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      2905..3152
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2921..2939)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3227..3815)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3307..3326)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3897..4832
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
ORIGIN
        1 gctagccacc atggctcacc atcatcatca tcacggttcc gagaatctgt acttccaaag
       61 cggtaccatg aggtcaacca ctttgttggc tctgcttgct ttagtgctgc tttacctagt
      121 cagtggtgcc ctggtgttta gggccttgga acaaccccac gagcaacaag ctcagcgtga
      181 gctgggtgag gtcagagaaa aatttttaag agcacaccca tgtgtatccg accaagaact
      241 tggactgcta atcaaagagg tggccgacgc ccttggcggc ggcgccgacc ctgaaaccca
      301 atccacctct cagtcaagtc acagtgcctg ggatctggga agtgccttct ttttcagtgg
      361 tacaattatc acgacaatag gctacggaaa cgtggccttg aggacggatg ccggtagact
      421 gttttgcata ttctacgcat tagtaggaat ccccctgttt ggtatcttgc tagctggtgt
      481 gggtgatcgt ctgggatcat cacttagaca tggtataggc catatagagg ccatcttcct
      541 gaaatggcat gtaccaccag aattggtcag ggttttatca gctatgttat ttttacttat
      601 cggatgccta ttgtttgtac tgaccccaac attcgttttc tgctacatgg aagactggtc
      661 caaacttgaa gctatttatt ttgtgatagt gaccttaact acggtcggct ttggcgatta
      721 cgtcgcaggc gcagatccac gtcaagattc tccagcatat caaccactag tgtggttctg
      781 gatcctactg ggattagcct acttcgcttc tgtcttgact actataggca actggttgag
      841 agtcgtttcc cgtaggaccg gtaccagcga aaacctgtat tttcagggta ctagtgtctc
      901 taagggagaa gaggacaata tggctattat caaggagttc atgagattta aagtccatat
      961 ggagggctct gttaatggtc atgaatttga aattgaaggt gaaggagaag gaagacctta
     1021 tgagggaacc caaacggcta agctgaaagt aactaaaggc ggcccacttc ctttcgcatg
     1081 ggatatactg tctccacaat tcatgtatgg ctctaaagct tatgtaaagc accccgccga
     1141 cattcccgat tatctaaagt tatcctttcc cgagggattt aaatgggaaa gggttatgaa
     1201 cttcgaggat ggcggcgtgg taactgttac gcaggactcc tcccttcaag atggtgaatt
     1261 tatttacaaa gtgaagcttc gtggtactaa tttcccaagt gacggtcctg tgatgcagaa
     1321 gaagactatg ggttgggaag ctagttccga gaggatgtac cccgaagatg gtgccctgaa
     1381 gggtgagatt aagcaacgtc taaaattaaa agacggcggt cactatgacg ctgaagtaaa
     1441 gaccacatat aaggcaaaaa agcccgtcca gcttcctggc gcctacaatg ttaatatcaa
     1501 gttggacatc acctcccata acgaagatta cactatcgtc gagcaatacg agagggcaga
     1561 gggaagacat tccacaggtg gtatggacga gctatataaa ctcgagggcg cttcctggtc
     1621 acatccacag tttgagaaat gagtttgtag ccttagacat gactgttcct cagttcaagt
     1681 tgggcactta cgagaagacc ggtcttgcta gattctaatc aagaggatgt cagaatgcca
     1741 tttgcctgag agatgcaggc ttcatttttg atactttttt atttgtaacc tatatagtat
     1801 aggatttttt ttgtcatttt gtttcttctc gtacgagctt gctcctgatc agcctatctc
     1861 gcagctgatg aatatcttgt ggtaggggtt tgggaaaatc attcgagttt gatgtttttc
     1921 ttggtatttc ccactcctct tcagagtaca gaagattaag tgagaccttc gtttgtgcgg
     1981 atctcccaca caccatagct tcaaaatgtt tctactcctt ttttactctt ccagattttc
     2041 tcggactccg cgcatcgccg taccacttca aaacacccaa gcacagcata ctaaatttcc
     2101 cctctttctt cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa
     2161 gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc
     2221 tttttcttga aaattttttt tttgattttt ttctctttcg atgacctccc attgatattt
     2281 aagttaataa acggtcttca atttctcaag tttcagtttc atttttcttg ttctattaca
     2341 acttttttta cttcttgctc attagaaaga aagcatagca atctaatcta agggcggtgt
     2401 tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta
     2461 aaccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc
     2521 ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc
     2581 cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc
     2641 ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc
     2701 ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga
     2761 gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt
     2821 ggccgaggag caggactgac acgtccgacg cggcccgacg ggtccgaggc ctcggagatc
     2881 cgtccccctt ttcctttgtc gatatcatgt aattagttat gtcacgctta cattcacgcc
     2941 ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc
     3001 ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc
     3061 ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga
     3121 aggttttggg acgctcgaag gctttaattt gcaagctgga gaccaacatg tgagcaaaag
     3181 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3241 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3301 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3361 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3421 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3481 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3541 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3601 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3661 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3721 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3781 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3841 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agatcagatc
     3901 taacatccaa agacgaaagg ttgaatgaaa cctttttgcc atccgacatc cacaggtcca
     3961 ttctcacaca taagtgccaa acgcaacagg aggggataca ctagcagcag accgttgcaa
     4021 acgcaggacc tccactcctc ttctcctcaa cacccacttt tgccatcgaa aaaccagccc
     4081 agttattggg cttgattgga gctcgctcat tccaattcct tctattaggc tactaacacc
     4141 atgactttat tagcctgtct atcctggccc ccctggcgag gttcatgttt gtttatttcc
     4201 gaatgcaaca agctccgcat tacacccgaa catcactcca gatgagggct ttctgagtgt
     4261 ggggtcaaat agtttcatgt tccccaaatg gcccaaaact gacagtttaa acgctgtctt
     4321 ggaacctaat atgacaaaag cgtgatctca tccaagatga actaagtttg gttcgttgaa
     4381 atgctaacgg ccagttggtc aaaaagaaac ttccaaaagt cggcataccg tttgtcttgt
     4441 ttggtattga ttgacgaatg ctcaaaaata atctcattaa tgcttagcgc agtctctcta
     4501 tcgcttctga accccggtgc acctgtgccg aaacgcaaat ggggaaacac ccgctttttg
     4561 gatgattatg cattgtctcc acattgtatg cttccaagat tctggtggga atactgctga
     4621 tagcctaacg ttcatgatca aaatttaact gttctaaccc ctacttgaca gcaatatata
     4681 aacagaagga agctgccctg tcttaaacct ttttttttat catcattatt agcttacttt
     4741 cataattgcg actggttcca attgacaagc ttttgatttt aacgactttt aacgacaact
     4801 tgagaagatc aaaaaacaac taattattcg aa
//
