LOCUS Exported 913 bp ds-DNA linear SYN 21-AUG-2021 DEFINITION Expresses human NKCC1 I721C C723S C724V mutant with an N-terminal 3xFLAG-YFP tag in mammalian cells. cDNA is synthetic, containing convenient restriction sites.. ACCESSION . VERSION . KEYWORDS pcDNA3.1 Flag YFP hNKCC1 I721C C723S C724V (NT535) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 913) TITLE Forbush Lab human NKCC1 NT clones JOURNAL Unpublished REFERENCE 2 (bases 1 to 913) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 21, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..913 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 115..180 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 196..906 /codon_start=1 /product="enhanced variant of YFP (Heikal et al., 2001)" /label=Citrine /note="mammalian codon-optimized" /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEF VTAAGITLGMDELYK" CDS 196..906 /codon_start=1 /product="enhanced YFP with monomerizing A206K mutation (Zacharias et al., 2002)" /label=mEYFP /note="mammalian codon-optimized" /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEF VTAAGITLGMDELYK" 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 acc //