LOCUS       sequence-78649-e         948 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses human NKCC1 C723S C724V V729C mutant with an N-terminal 
            3xFLAG-YFP tag in mammalian cells. cDNA is synthetic, containing 
            convenient restriction sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 948)
  AUTHORS   Monette MY, Somasekharan S, Forbush B
  TITLE     Molecular Motions Involved in Na-K-Cl Cotransporter-mediated Ion 
            Transport and Transporter Activation Revealed by Internal 
            Crosslinking between Transmembrane Domains 10 and 11/12.
  JOURNAL   J Biol Chem. 2014 Jan 22.
  PUBMED    24451383
REFERENCE   2  (bases 1 to 948)
  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: "J Biol 
            Chem. 2014 Jan 22."
COMMENT     Sequence Label: pcDNA3.1 Flag YFP hNKCC1 C723S C724V V729C (NT...
FEATURES             Location/Qualifiers
     source          1..948
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     50..69
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        50..68
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      100..109
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             112..177
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(190..192,193..903)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(190..192,193..903)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(232..253)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(493..512)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     840..861
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
       61 actataggga gacccaagct ggctagcccg ggcacggccg ccaccatggg agactacaaa
      121 gaccatgacg gtgattataa agatcacgac atcgactaca aggatgacga tgacaagggt
      181 gaccctaccg tcagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      241 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      301 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      361 cccaccctcg tgaccacctt cggctacggc ctgatgtgct tcgcccgcta ccccgaccac
      421 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      481 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
      541 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatccta
      601 gggcacaagc tggagtacaa ctacaacagc cacaacgttt atatcatggc cgacaagcag
      661 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
      721 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
      781 aaccactacc tgagctacca gtccaaactg agcaaagacc ccaacgagaa gcgcgatcac
      841 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctctac
      901 aagggtgacc tggagccgag gcccacggct cctagctccg gcgcgcca
//
