LOCUS       sequence-79802-e         986 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses human NKCC1 F728C 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 986)
  TITLE     Forbush Lab human NKCC1 NT clones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 986)
  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            "Unpublished"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 F728C (NT502)
FEATURES             Location/Qualifiers
     source          1..986
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     61..80
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        61..79
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      111..120
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             123..188
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(201..203,204..914)
                     /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(201..203,204..914)
                     /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(243..264)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(504..523)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     851..872
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
       61 taatacgact cactataggg agacccaagc tggctagccc gggcacggcc gccaccatgg
      121 gagactacaa agaccatgac ggtgattata aagatcacga catcgactac aaggatgacg
      181 atgacaaggg tgaccctacc gtcagcaagg gcgaggagct gttcaccggg gtggtgccca
      241 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      301 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      361 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cctgatgtgc ttcgcccgct
      421 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      481 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
      541 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
      601 gcaacatcct agggcacaag ctggagtaca actacaacag ccacaacgtt tatatcatgg
      661 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
      721 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
      781 tgctgcccga caaccactac ctgagctacc agtccaaact gagcaaagac cccaacgaga
      841 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
      901 acgagctcta caagggtgac ctggagccga ggcccacggc tcctagctcc ggcgcgccag
      961 gcctcgccgg agtcggcgaa acgccg
//
