LOCUS       sequence-349872-        6066 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  encodes human NKD2 132DFD134>AFA mutant (two D mutated to A)cDNA 
            with a C-terminal mCherry fusion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6066)
  AUTHORS   Wu B, Woo JS, Vila P, Jew M, Leung J, Sun Z, Srikanth S, Gwack Y
  TITLE     NKD2 mediates stimulation-dependent ORAI1 trafficking to augment 
            Ca(2+) entry in T cells.
  JOURNAL   Cell Rep. 2021 Aug 24;36(8):109603. doi: 
            10.1016/j.celrep.2021.109603.
  PUBMED    34433025
REFERENCE   2  (bases 1 to 6066)
  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: "2021-08-24"; volume: "36(8):109603. doi"; pages: " 
            10.1016/j.celrep.2021.109603"
COMMENT     Sequence Label: pN1mcherry-NKD2EFMUT
FEATURES             Location/Qualifiers
     source          1..6066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        101..404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        405..608
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     559..579
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1973..1999
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     regulatory      2057..2066
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2063..2773
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2208..2226)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2476..2495
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2894..3015
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2931..2950)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2985..3004
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3022..3477)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3159..3180)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3371..3390
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3504..3608
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3610..3967
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3610..3801
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3633..3653)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3818..3953
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4002..4796
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4002..4796
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4056..4075)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4666..4685
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4984..5003)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5028..5075
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5404..5992
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5893..5912
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atcccctgat tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa
       61 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      361 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      421 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      481 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      541 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      601 gcagagctgg tttagtgaac cgtcagatcc gctagcgcta ccggactcag atctcgagat
      661 ggggaaactg cagtcgaagc acgccgccgc cgcccgcaag cggagagaga gcccggaagg
      721 ggacagcttc gtggcgtccg cgtacgcgag cggccgcaaa ggcgcggagg aagcggagcg
      781 gcgcgcgcgg gacaagcagg agctgcccaa tggggacccc aaggaggggc ctttccggga
      841 ggaccagtgt cccctacagg tggcactccc cgctgagaaa gctgagggcc gcgagcaccc
      901 gggacaactc ctcagcgcag atgacggaga gagggcagca aaccgcgagg gcccgcgagg
      961 accgggcggg cagcgcctca acattgacgc actccagtgc gatgtctcgg tggaggagga
     1021 cgaccgccag gagtggacgt tcacgctcta tgcctttgcc aactgcggga aggtcaccag
     1081 ggaggacatg tccagcctca tgcacaccat ctatgaggtc gtggatgcct cggtcaacca
     1141 ctcctcgggc agcagcaaga ccctccgtgt gaagctaacc gtcagccctg agccctccag
     1201 caagaggaag gagggtcctc ctgctggcca ggaccgggag cccacccgtt gcaggatgga
     1261 gggtgaactg gcagaggagc caagggtggc tgacaggagg ttgtctgcac acgtcaggag
     1321 gcccagtact gacccccagc cctgctcgga gcgggggccc tactgcgtgg acgagaacac
     1381 ggagcgcaga aaccactacc tggacctcgc cgggattgag aactacacgt ccagattcgg
     1441 ccctgggtcc cctcctgtgc aagcaaagca ggagccccag ggcagggcct cgcacctcca
     1501 ggcccggtcc cgctcccagg agccagatac acatgccgta caccaccgca ggtcacaggt
     1561 gctggtggaa cacgtcgtgc cagcctcgga gcctgctgcc cgggccctgg acacgcagcc
     1621 ccggccgaag gggccggaga agcagttcct caagtccccc aagggctccg ggaagccgcc
     1681 tggggtgcca gccagcagca agtccgggaa agccttcagc tactacctgc cggccgtcct
     1741 gccgccccag gcccctcagg acggccacca cctcccgcag cccccaccgc caccctacgg
     1801 ccacaagcgg taccgccaaa agggcaggga gggccactcg ccactcaagg ccccacacgc
     1861 tcagcctgcc acagtggagc acgaggtggt gcgggacctg ccgcccacgc cagcaggaga
     1921 gggctacgcg gtgccagtga tccagcggca cgagcaccac caccaccacg agcaccacca
     1981 ccaccaccac caccaccact tccacccgtc ccgaattctg cagtcgacgg taccgcgggc
     2041 ccgggatcca ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat
     2101 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     2161 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     2221 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     2281 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     2341 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     2401 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     2461 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga
     2521 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     2581 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     2641 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
     2701 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     2761 gctgtacaag tagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt
     2821 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     2881 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     2941 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3001 catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     3061 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     3121 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     3181 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     3241 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     3301 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     3361 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     3421 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag
     3481 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3541 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3601 ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     3661 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3721 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3781 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3841 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3901 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3961 ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     4021 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     4081 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4141 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc
     4201 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4261 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4321 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4381 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4441 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4501 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga
     4561 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4621 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4681 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4741 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4801 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     4861 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4921 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
     4981 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
     5041 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     5101 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     5161 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     5221 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact
     5281 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5341 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5401 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5461 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5521 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5581 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5641 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5701 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5761 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     5821 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5881 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5941 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6001 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     6061 tgcgtt
//
