LOCUS       sequence-390140-        7645 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian cell expression of D304N mutation in NEMO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7645)
  AUTHORS   Harding O, Holzer E, Riley JF, Martens S, Holzbaur ELF
  TITLE     Damaged mitochondria recruit the effector NEMO to activate NF-kappaB
            signaling.
  JOURNAL   Mol Cell. 2023 Sep 7;83(17):3188-3204.e7. doi: 
            10.1016/j.molcel.2023.08.005.
  PUBMED    37683611
REFERENCE   2  (bases 1 to 7645)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2023.08"; journalName: "Mol Cell"; date: 
            "2023-09-7- 7"; volume: "83"; issue: "17"; pages: "3188-3204.e7"
COMMENT     Sequence Label: HA-EGFP-NEMO-D304N
FEATURES             Location/Qualifiers
     source          1..7645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    7..28
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(145..162)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(316..904)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(396..415)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1075..1866,1867..1935))
                     /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     1698..1717
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1936..2040)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2115..2134)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2306..2685
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2686..2889
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2840..2860
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2934..2953
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2934..2952
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3029..3055
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(3071..3073,3074..3076,3077..3787)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3071..3073,3074..3076,3077..3787)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3116..3137)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3377..3396)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3724..3745
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(5269..5287)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5270..5287)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5307..5324)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5313..5537
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5583..6011
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5670..5689)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5880..5901
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6020..6040)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6025..6354
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6205..6340
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6267..6286
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6421..7215
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6475..6494)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7085..7104
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7389..7510
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7426..7445)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7480..7499
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7559..7575)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7559..7575)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7572..7594)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7583..7599
                     /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(7607..7613,7614..7631,7632..7637))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
       61 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
      121 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
      181 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      241 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      301 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      361 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      421 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      481 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      541 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      601 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      661 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      721 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
      781 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      841 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      901 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
      961 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     1021 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     1081 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1141 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1201 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1261 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1321 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1381 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1441 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1501 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1561 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1621 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1681 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1741 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     1801 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     1861 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     1921 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     1981 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     2041 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     2101 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     2161 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     2221 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     2281 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
     2341 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     2401 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     2461 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     2521 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     2581 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     2641 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     2701 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     2761 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     2821 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     2881 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg
     2941 actcactata gggagaccca agcttgctta catttgcttc tgacacaact gtgttcacta
     3001 gcaacctcaa acagacacca tgagatctta tccatatgat gtgccagatt atgcagccat
     3061 ggccggatcc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     3121 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     3181 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     3241 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     3301 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     3361 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     3421 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     3481 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     3541 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     3601 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3661 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     3721 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3781 gtacaaggaa ttcctcgaca acaagcaccc ctggaagaac cagctgagtg agatggtgca
     3841 gcccagtggt ggcccagcag aggaccagga catgctgggt gaagaatctt ctctggggaa
     3901 gcctgcaatg ctacatctgc cttcagagca gggtactcct gagaccctcc agcgctgcct
     3961 ggaagagaat caagagctcc gagacgctat ccggcagagc aatcagatgc tgagggaacg
     4021 ctgtgaggag ctgctgcatt tccaggtcag ccagcgggag gagaaggagt tccttatgtg
     4081 caaattccag gaagcccgga agctggtgga gagactgagc ttggagaagc ttgatcttcg
     4141 gagtcagagg gaacaggcct taaaggagtt ggagcaactg aagaaatgcc aacagcagat
     4201 ggctgaggac aaggcctctg tgaaagttca ggtgacatca ttgctcggag aactccagga
     4261 gagccagagc cgtttggagg ctgccaccaa ggatcggcaa gctttagagg gaaggattcg
     4321 agcagttagt gagcaggtca gacagctgga gagtgagcgg gaggtgctac agcagcagca
     4381 cagcgtccag gtggaccagc tgcgtatgca gaaccagagc gtggaggctg ccttgcgaat
     4441 ggggcggcag gctgcttcag aggagaagcg gaagctggct cagttgcagg cagcctatca
     4501 ccagctcttc caagactacg acagccacat taagagcaac aagggcatgc agctggaaga
     4561 tctgaggcaa cagctccagc aagctgagga ggccctggta gccaaacagg aattgattga
     4621 taagctgaaa gaggaggctg agcagcacaa gattgtgatg gagactgtgc cagtcttgaa
     4681 ggcccaggcg gatatctaca aggctaactt ccaagctgag aggcatgccc gggagaagct
     4741 ggtggagaag aaggagtatt tgcaggagca gctggagcag ctgcagcgcg agttcaacaa
     4801 gctgaaagtt ggctgccatg agtcagccag gattgaggat atgaggaagc ggcatgtaga
     4861 gactccccag cctcctttac tccctgctcc agctcaccac tcctttcatt tggccttgtc
     4921 caaccagcgg aggagccctc ctgaagaacc tcctgacttc tgttgtccga agtgccagta
     4981 tcaggctcct gatatggaca ctctacagat acatgtcatg gagtgcatag agtaggggca
     5041 gcagatgcaa ggccacttgc agtactatgt cctgatctgt gtgacttgtg ctttcctgtt
     5101 ttacctgcat agtccacact taagggcttg ctttagccct ttggtccccc atttagggta
     5161 gacagcccca ttcagggctt tttttttttt ctgtgtgcct gatccagttt gcctctggtg
     5221 gcttcttccc tcttctccca tagtcctagg gagtctagag ggccctattc tatagtgtca
     5281 cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     5341 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     5401 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     5461 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     5521 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     5581 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     5641 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     5701 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     5761 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     5821 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5881 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     5941 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     6001 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     6061 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     6121 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     6181 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     6241 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     6301 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     6361 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     6421 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     6481 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     6541 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     6601 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     6661 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     6721 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     6781 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     6841 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     6901 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     6961 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     7021 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     7081 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     7141 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     7201 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     7261 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     7321 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     7381 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     7441 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     7501 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     7561 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     7621 gaagcataaa gtgtaaagcc tgggg
//
