LOCUS       sequence-78648-e         916 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses human NKCC1 C723S C724V W732C 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 916)
  TITLE     Forbush Lab human NKCC1 NT clones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 916)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pcDNA3.1 Flag YFP hNKCC1 C723S C724V W732C (NT...
FEATURES             Location/Qualifiers
     source          1..916
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     53..72
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        53..71
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      103..112
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             115..180
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(193..195,196..906)
                     /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(193..195,196..906)
                     /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(235..256)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(496..515)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     843..864
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
       61 ctcactatag ggagacccaa gctggctagc ccgggcacgg ccgccaccat gggagactac
      121 aaagaccatg acggtgatta taaagatcac gacatcgact acaaggatga cgatgacaag
      181 ggtgacccta ccgtcagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
      241 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
      301 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
      361 tggcccaccc tcgtgaccac cttcggctac ggcctgatgt gcttcgcccg ctaccccgac
      421 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
      481 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
      541 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
      601 ctagggcaca agctggagta caactacaac agccacaacg tttatatcat ggccgacaag
      661 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
      721 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
      781 gacaaccact acctgagcta ccagtccaaa ctgagcaaag accccaacga gaagcgcgat
      841 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctc
      901 tacaagggtg acctgg
//
