LOCUS       sequence-78662-e         917 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses human NKCC1 with truncation of N-terminus, mutations at 
            P676C I730C and C-terminus replaced with that of NBCe. Contains an 
            N-terminal 3xFLAG-YFP tag for expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 917)
  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 917)
  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 P676C I730C N-termina...
FEATURES             Location/Qualifiers
     source          1..917
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     54..73
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        54..72
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      104..113
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             116..181
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(194..196,197..907)
                     /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(194..196,197..907)
                     /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(236..257)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(497..516)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     844..865
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg
       61 actcactata gggagaccca agctggctag cccgggcacg gccgccacca tgggagacta
      121 caaagaccat gacggtgatt ataaagatca cgacatcgac tacaaggatg acgatgacaa
      181 gggtgaccct accgtcagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      241 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
      301 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
      361 ctggcccacc ctcgtgacca ccttcggcta cggcctgatg tgcttcgccc gctaccccga
      421 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
      481 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
      541 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
      601 cctagggcac aagctggagt acaactacaa cagccacaac gtttatatca tggccgacaa
      661 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
      721 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
      781 cgacaaccac tacctgagct accagtccaa actgagcaaa gaccccaacg agaagcgcga
      841 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
      901 ctacaagggt gacctgg
//
