LOCUS       sequence-241189-        5110 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mouse GIRK2 R201A as a TEV protease cleavable C-terminal 
            fusion to eGFP, strep tag II, and 6x His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5110)
  AUTHORS   Liu Y, LoCaste CE, Liu W, Poltash ML, Russell DH, Laganowsky A
  TITLE     Selective binding of a toxin and phosphatidylinositides to a 
            mammalian potassium channel.
  JOURNAL   Nat Commun. 2019 Mar 22;10(1):1352. doi: 10.1038/s41467-019-09333-4.
  PUBMED    30902995
REFERENCE   2  (bases 1 to 5110)
  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"; journalName: "Nat 
            Commun."; date: "2019-03-22"; volume: "10(1):1352. doi"; pages: " 
            10.1038/s41467-019-09333-4"
COMMENT     Sequence Label: pPICZ-mouseGIRK2R201A-eGFP-StII-6xHis
FEATURES             Location/Qualifiers
     source          1..5110
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..936
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     regulatory      948..957
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1956..1976
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2001..2003,2004..2714)
                     /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(2043..2064)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2304..2323)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2651..2672
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2733..2756
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2763..2780
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2791..2820
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2836..2853
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2933..3179
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3194..3606
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3613..3660
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3679..4053
                     /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      4119..4366
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4135..4153)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(4441..5029)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4521..4540)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gatctaacat ccaaagacga aaggttgaat gaaacctttt tgccatccga catccacagg
       61 tccattctca cacataagtg ccaaacgcaa caggagggga tacactagca gcagaccgtt
      121 gcaaacgcag gacctccact cctcttctcc tcaacaccca cttttgccat cgaaaaacca
      181 gcccagttat tgggcttgat tggagctcgc tcattccaat tccttctatt aggctactaa
      241 caccatgact ttattagcct gtctatcctg gcccccctgg cgaggttcat gtttgtttat
      301 ttccgaatgc aacaagctcc gcattacacc cgaacatcac tccagatgag ggctttctga
      361 gtgtggggtc aaatagtttc atgttcccca aatggcccaa aactgacagt ttaaacgctg
      421 tcttggaacc taatatgaca aaagcgtgat ctcatccaag atgaactaag tttggttcgt
      481 tgaaatgcta acggccagtt ggtcaaaaag aaacttccaa aagtcggcat accgtttgtc
      541 ttgtttggta ttgattgacg aatgctcaaa aataatctca ttaatgctta gcgcagtctc
      601 tctatcgctt ctgaaccccg gtgcacctgt gccgaaacgc aaatggggaa acacccgctt
      661 tttggatgat tatgcattgt ctccacattg tatgcttcca agattctggt gggaatactg
      721 ctgatagcct aacgttcatg atcaaaattt aactgttcta acccctactt gacagcaata
      781 tataaacaga aggaagctgc cctgtcttaa accttttttt ttatcatcat tattagctta
      841 ctttcataat tgcgactggt tccaattgac aagcttttga ttttaacgac ttttaacgac
      901 aacttgagaa gatcaaaaaa caactaatta ttcgaagcta gcctcgagcc accatggcaa
      961 aacgtaagat ccaacgttac gttcgtaagg acggtaagtg caatgtgcat catggtaatg
     1021 taagagaaac ctacagatac ctaacggaca ttttcaccac tttggtggat ttgaaatggc
     1081 gtttcaactt attgatattt gttatggtat acaccgttac gtggctattt ttcggtatga
     1141 tttggtggct aattgcatat ataagaggtg acatggatca catcgaagac ccctcctgga
     1201 ctccatgtgt taccaatcta aacggctttg tttccgcatt cttgttttcc atcgaaactg
     1261 agaccacaat tggttacggt taccgtgtaa ttacagataa atgcccagag ggtatcatct
     1321 tgttactaat tcaatccgtt ttaggctcca ttgttaatgc tttcatggtc ggttgcatgt
     1381 ttgtgaaaat ttctcagccc aaaaaggcgg ctgaaactct tgttttctcc acacatgcag
     1441 ttatatcaat gagggatggc aagttatgcc tgatgtttcg tgtcggagac ctaagaaatt
     1501 cacatatagt ggaagcaagt attagagcta aactgataaa aagtaagcaa acatccgagg
     1561 gcgagttcat cccattgaat cagtctgaca ttaacgtggg atactatact ggcgacgacc
     1621 gtcttttttt agttagtcca cttatcattt cccacgaaat taatcagcag tccccttttt
     1681 gggagatttc caaggctcaa ttgcctaagg aagaactaga gattgtcgtg attcttgaag
     1741 gcatcgtcga agccactggc atgacgtgcc aagcacgttc ctcttacatc acatccgaga
     1801 tactatgggg atatagattc acgcccgtct taaccatgga agatggattc tacgaagtag
     1861 attataacag tttccatgag acctatgaga cgagtactcc ttccctatct gcaaaggaat
     1921 tggctgaact ggctaataga gccgagtcga attctgaaaa cctgtatttt cagggttccg
     1981 gtagcactgc tgccgccgct gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     2041 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     2101 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     2161 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     2221 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     2281 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     2341 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     2401 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     2461 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     2521 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     2581 tgctgcccga caaccactac ctgagcaccc agtccaagct gagcaaagac cccaacgaga
     2641 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2701 acgagctgta caagtccgga ggtggtgcaa gctggagcca cccgcagttc gaaaagggta
     2761 gccaccacca ccatcatcac taatagtcta gaacaaaaac tcatctcaga agaggatctg
     2821 aatagcgccg tcgaccatca tcatcatcat cattgagttt gtagccttag acatgactgt
     2881 tcctcagttc aagttgggca cttacgagaa gaccggtctt gctagattct aatcaagagg
     2941 atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt ttttatttgt
     3001 aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga gcttgctcct
     3061 gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa aatcattcga
     3121 gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat taagtgagac
     3181 cttcgtttgt gcggatctcc cacacaccat agcttcaaaa tgtttctact ccttttttac
     3241 tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag
     3301 catactaaat ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt
     3361 tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat
     3421 ttttatcacg tttctttttc ttgaaaattt tttttttgat ttttttctct ttcgatgacc
     3481 tcccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag tttcattttt
     3541 cttgttctat tacaactttt tttacttctt gctcattaga aagaaagcat agcaatctaa
     3601 tctaagggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     3661 aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc
     3721 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg
     3781 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag
     3841 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg
     3901 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg
     3961 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
     4021 tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacgcggccc gacgggtccg
     4081 aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag ttatgtcacg
     4141 cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc
     4201 tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat
     4261 atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa
     4321 aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcaagc tggagaccaa
     4381 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4441 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4501 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4561 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4621 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4681 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4741 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4801 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4861 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     4921 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4981 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5041 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5101 catgagatca
//
