LOCUS       sequence-241188-        5110 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mouse GIRK2 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-mouseGIRK2-eGFP-StII-6xHis
FEATURES             Location/Qualifiers
     source          1..5110
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      9..18
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1017..1037
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1062..1064,1065..1775)
                     /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(1104..1125)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1365..1384)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1712..1733
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1794..1817
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1824..1841
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1852..1881
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1897..1914
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1994..2240
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2255..2667
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2674..2721
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2740..3114
                     /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      3180..3427
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3196..3214)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3502..4090)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3582..3601)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4172..5107
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
ORIGIN
        1 agcctcgagc caccatggca aaacgtaaga tccaacgtta cgttcgtaag gacggtaagt
       61 gcaatgtgca tcatggtaat gtaagagaaa cctacagata cctaacggac attttcacca
      121 ctttggtgga tttgaaatgg cgtttcaact tattgatatt tgttatggta tacaccgtta
      181 cgtggctatt tttcggtatg atttggtggc taattgcata tataagaggt gacatggatc
      241 acatcgaaga cccctcctgg actccatgtg ttaccaatct aaacggcttt gtttccgcat
      301 tcttgttttc catcgaaact gagaccacaa ttggttacgg ttaccgtgta attacagata
      361 aatgcccaga gggtatcatc ttgttactaa ttcaatccgt tttaggctcc attgttaatg
      421 ctttcatggt cggttgcatg tttgtgaaaa tttctcagcc caaaaagagg gctgaaactc
      481 ttgttttctc cacacatgca gttatatcaa tgagggatgg caagttatgc ctgatgtttc
      541 gtgtcggaga cctaagaaat tcacatatag tggaagcaag tattagagct aaactgataa
      601 aaagtaagca aacatccgag ggcgagttca tcccattgaa tcagtctgac attaacgtgg
      661 gatactatac tggcgacgac cgtctttttt tagttagtcc acttatcatt tcccacgaaa
      721 ttaatcagca gtcccctttt tgggagattt ccaaggctca attgcctaag gaagaactag
      781 agattgtcgt gattcttgaa ggcatcgtcg aagccactgg catgacgtgc caagcacgtt
      841 cctcttacat cacatccgag atactatggg gatatagatt cacgcccgtc ttaaccatgg
      901 aagatggatt ctacgaagta gattataaca gtttccatga gacctatgag acgagtactc
      961 cttccctatc tgcaaaggaa ttggctgaac tggctaatag agccgagtcg aattctgaaa
     1021 acctgtattt tcagggttcc ggtagcactg ctgccgccgc tgtgagcaag ggcgaggagc
     1081 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1141 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1201 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1261 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1321 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1381 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1441 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     1501 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     1561 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1621 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc
     1681 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1741 ccgggatcac tctcggcatg gacgagctgt acaagtccgg aggtggtgca agctggagcc
     1801 acccgcagtt cgaaaagggt agccaccacc accatcatca ctaatagtct agaacaaaaa
     1861 ctcatctcag aagaggatct gaatagcgcc gtcgaccatc atcatcatca tcattgagtt
     1921 tgtagcctta gacatgactg ttcctcagtt caagttgggc acttacgaga agaccggtct
     1981 tgctagattc taatcaagag gatgtcagaa tgccatttgc ctgagagatg caggcttcat
     2041 ttttgatact tttttatttg taacctatat agtataggat tttttttgtc attttgtttc
     2101 ttctcgtacg agcttgctcc tgatcagcct atctcgcagc tgatgaatat cttgtggtag
     2161 gggtttggga aaatcattcg agtttgatgt ttttcttggt atttcccact cctcttcaga
     2221 gtacagaaga ttaagtgaga ccttcgtttg tgcggatctc ccacacacca tagcttcaaa
     2281 atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat cgccgtacca
     2341 cttcaaaaca cccaagcaca gcatactaaa tttcccctct ttcttcctct agggtgtcgt
     2401 taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc
     2461 gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaaatt ttttttttga
     2521 tttttttctc tttcgatgac ctcccattga tatttaagtt aataaacggt cttcaatttc
     2581 tcaagtttca gtttcatttt tcttgttcta ttacaacttt ttttacttct tgctcattag
     2641 aaagaaagca tagcaatcta atctaagggc ggtgttgaca attaatcatc ggcatagtat
     2701 atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt gaccagtgcc
     2761 gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc
     2821 gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc
     2881 ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg
     2941 gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg
     3001 gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc
     3061 ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtc
     3121 cgacgcggcc cgacgggtcc gaggcctcgg agatccgtcc cccttttcct ttgtcgatat
     3181 catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga
     3241 aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt
     3301 agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta
     3361 cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt
     3421 aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3481 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3541 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3601 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3661 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     3721 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3781 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     3841 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     3901 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     3961 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4021 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4081 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4141 ctcacgttaa gggattttgg tcatgagatc agatctaaca tccaaagacg aaaggttgaa
     4201 tgaaaccttt ttgccatccg acatccacag gtccattctc acacataagt gccaaacgca
     4261 acaggagggg atacactagc agcagaccgt tgcaaacgca ggacctccac tcctcttctc
     4321 ctcaacaccc acttttgcca tcgaaaaacc agcccagtta ttgggcttga ttggagctcg
     4381 ctcattccaa ttccttctat taggctacta acaccatgac tttattagcc tgtctatcct
     4441 ggcccccctg gcgaggttca tgtttgttta tttccgaatg caacaagctc cgcattacac
     4501 ccgaacatca ctccagatga gggctttctg agtgtggggt caaatagttt catgttcccc
     4561 aaatggccca aaactgacag tttaaacgct gtcttggaac ctaatatgac aaaagcgtga
     4621 tctcatccaa gatgaactaa gtttggttcg ttgaaatgct aacggccagt tggtcaaaaa
     4681 gaaacttcca aaagtcggca taccgtttgt cttgtttggt attgattgac gaatgctcaa
     4741 aaataatctc attaatgctt agcgcagtct ctctatcgct tctgaacccc ggtgcacctg
     4801 tgccgaaacg caaatgggga aacacccgct ttttggatga ttatgcattg tctccacatt
     4861 gtatgcttcc aagattctgg tgggaatact gctgatagcc taacgttcat gatcaaaatt
     4921 taactgttct aacccctact tgacagcaat atataaacag aaggaagctg ccctgtctta
     4981 aacctttttt tttatcatca ttattagctt actttcataa ttgcgactgg ttccaattga
     5041 caagcttttg attttaacga cttttaacga caacttgaga agatcaaaaa acaactaatt
     5101 attcgaagct
//
