LOCUS       sequence-448287-        5335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  endogenously tag WNK3 with N-terminal mNeon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5335)
  AUTHORS   Xiao YX, Lee SY, Aguilera-Uribe M, Samson R, Au A, Khanna Y, Liu Z, 
            Cheng R, Aulakh K, Wei J, Farias AG, Reilly T, Birkadze S, Habsid A,
            Brown KR, Chan K, Mero P, Huang JQ, Billmann M, Rahman M, Myers C, 
            Andrews BJ, Youn JY, Yip CM, Rotin D, Derry WB, Forman-Kay JD, Moses
            AM, Pritisanac I, Gingras AC, Moffat J
  TITLE     The TSC22D, WNK, and NRBP gene families exhibit functional buffering
            and evolved with Metazoa for cell volume regulation.
  JOURNAL   Cell Rep. 2024 Jul 23;43(7):114417. doi: 
            10.1016/j.celrep.2024.114417. Epub 2024 Jul 8.
  PUBMED    38980795
REFERENCE   2  (bases 1 to 5335)
  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: "Cell Rep.";
            date: "2024-07-23"; volume: "43(7):114417. doi"; pages: " 
            10.1016/j.celrep.2024.114417. Epub 2024 Jul 8"
COMMENT     Sequence Label: pYX-N-Puro-mNeon-WNK3
FEATURES             Location/Qualifiers
     source          1..5335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1023..1619
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1023..1042)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1519..1539
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1629..1685
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1692..1718
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1692..1718
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1722..1748
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1758..2465
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      2013..2022
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3114..3130)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3114..3130)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3127..3149)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3138..3154
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3162..3168,3169..3186,3187..3192))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3207..3228
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3345..3362)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3516..4104)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3596..3615)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4275..5066,5067..5135))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4898..4917
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5136..5240)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5308..5326
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cccactgaat tgtataatta
      421 aaaatggtta aaatggtaaa tttgacgata tgtatatttt agtacagtaa aagaaactac
      481 tattgatgaa ttccagaatt ttagacaaaa accacctaga aaccatgtgg ttgttttact
      541 tactgtgttt gtcacattgt ggctaagtga actcagggtt tgggtactca ttttgaattt
      601 gatggattga cagatggagc tgagaactag gtttctaacc aatattcatg caatatgaag
      661 acagattgag ctatatacta agtagtctgc tagacactga ggactaatgg taaacaagat
      721 aagacacagt ccctagagtc tgctggttcc taagattgga cagaaaaaag aataaacttc
      781 ttattaagga aaaagggatt aaaaaatcag gtgatttgtt ttctccttaa tgatttcctt
      841 tgtagggtga ttttaaaata tctttttccc ccccttcagg ttgctatgga aatacatgac
      901 cacgcaaaag gaagtccatt ctgataattc tgatacctga gatgtaactg gactgaagag
      961 tagaaacagg aaaaatttta gtgccaactt taattacaat gggcaagagc aagaccggta
     1021 gcatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     1081 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     1141 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     1201 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     1261 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     1321 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     1381 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     1441 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     1501 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     1561 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg
     1621 gatccggagc tactaacttc agcctgctga agcaggccgg agacgtggag gagaaccctg
     1681 gaccttctag atacccctac gacgtgcccg actacgccgg ctatccgtat gatgtcccgg
     1741 actatgcagg aagcggaatg gtgagcaagg gcgaggagga taacatggcc tctctcccag
     1801 cgacacatga gttacacatc tttggctcca tcaacggtgt ggactttgac atggtgggtc
     1861 agggcaccgg caatccaaat gatggttatg aggagttaaa cctgaagtcc accaagggtg
     1921 acctccagtt ctccccctgg attctggtcc ctcatatcgg gtatggcttc catcagtacc
     1981 tgccctaccc tgacgggatg tcgcctttcc aggccgccat ggtagatggc tccggatacc
     2041 aagtccatcg cacaatgcag tttgaagatg gtgcctccct tactgttaac taccgctaca
     2101 cctacgaggg aagccacatc aaaggagagg cccaggtgaa ggggactggt ttccctgctg
     2161 acggtcctgt gatgaccaac tcgctgaccg ctgcggactg gtgcaggtcg aagaagactt
     2221 accccaacga caaaaccatc atcagtacct ttaagtggag ttacaccact ggaaatggca
     2281 agcgctaccg gagcactgcg cggaccacct acacctttgc caagccaatg gcggctaact
     2341 atctgaagaa ccagccgatg tacgtgttcc gtaagacgga gctcaagcac tccaagaccg
     2401 agctcaactt caaggagtgg caaaaggcct ttaccgatgt gatgggcatg gacgagctgt
     2461 acaagggagg aagcggagga tctggcggta ccgccactga ttcaggggat ccagccagca
     2521 cagaagattc tgagaaacct gatggaattt catttgaaaa cagagttccc caggtcgctg
     2581 caactttgac agtagaagct agactaaagg agaaaaacag taccttctct gcttctgggg
     2641 aaactgtaga aaggaagaga tttttccgaa agagtgttga aatgacggaa gatgacaaag
     2701 ttgccgaatc atcccccaaa gatgagagaa ttaaggctgc aatgaatatt ccaagagtag
     2761 ataagcttcc ttcaaatgtg ttgagaggtg gacaagaagt taaatatgaa cagtgttcaa
     2821 agtcaacctc agaaatctca aaagattgtt tcaaggagaa aaatgaaaag gaaatggaag
     2881 aagaagcaga aatgaaggct gtagctactt ctcctagtgg cagattcctg aaatttgaca
     2941 tagaactagg aagaggagca tttaaaacag tatataaagg actggacact gaaacatggg
     3001 ttgaggttgc ttggtgtgag ctgcaggtag gtatataatc ttcttggtta taataaattc
     3061 aagttttagc tggcctggcc ttctgtgtga ttcataagct tggcgtaatc atggtcatag
     3121 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     3181 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     3241 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     3301 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3361 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3421 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3481 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3541 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3601 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3661 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3721 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3781 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3841 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3901 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     3961 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4021 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4081 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4141 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4201 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4261 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4321 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4381 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4441 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4501 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4561 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4621 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4681 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4741 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4801 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4861 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4921 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4981 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5041 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5101 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5161 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5221 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     5281 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
