LOCUS       sequence-390059-        7778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian cell expression of Halo-tagged NEMO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7778)
  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 7778)
  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"; 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: Halo-NEMO
FEATURES             Location/Qualifiers
     source          1..7778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      902..911
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             908..1798
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1811..1831
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     promoter        complement(3331..3349)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3332..3349)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3369..3386)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3375..3599
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3645..4073
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3732..3751)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3942..3963
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4082..4102)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4087..4416
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4267..4402
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4329..4348
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4483..5277
                     /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(4537..4556)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5147..5166
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5451..5572
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5488..5507)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5542..5561
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5621..5637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5621..5637)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5634..5656)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5645..5661
                     /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(5669..5675,5676..5693,5694..5699))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5714..5735
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5852..5869)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6023..6611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6103..6122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6782..7573,7574..7642))
                     /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     7405..7424
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7643..7747)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctctagcaa
      901 agccaccatg gcagaaatcg gtactggctt tccattcgac ccccattatg tggaagtcct
      961 gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat ggcacccctg tgctgttcct
     1021 gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg ttgcaccgac
     1081 ccatcgctgc attgctccag acctgatcgg tatgggcaaa tccgacaaac cagacctggg
     1141 ttatttcttc gacgaccacg tccgcttcat ggatgccttc atcgaagccc tgggtctgga
     1201 agaggtcgtc ctggtcattc acgactgggg ctccgctctg ggtttccact gggccaagcg
     1261 caatccagag cgcgtcaaag gtattgcatt tatggagttc atccgcccta tcccgacctg
     1321 ggacgaatgg ccagaatttg cccgcgagac cttccaggcc ttccgcacca ccgacgtcgg
     1381 ccgcaagctg atcatcgatc agaacgtttt tatcgagggt acgctgccga tgggtgtcgt
     1441 ccgcccgctg actgaagtcg agatggacca ttaccgcgag ccgttcctga atcctgttga
     1501 ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc gccggtgagc cagcgaacat
     1561 cgtcgcgctg gtcgaagaat acatggactg gctgcaccag tcccctgtcc cgaagctgct
     1621 gttctggggc accccaggcg ttctgatccc accggccgaa gccgctcgcc tggccaaaag
     1681 cctgcctaac tgcaaggctg tggacatcgg cccgggtctg aatctgctgc aagaagacaa
     1741 cccggacctg atcggcagcg agatcgcgcg ctggctgtcg acgctcgaga tttccggcga
     1801 gccaaccact gaggatctgt actttcagag cgataacgcg atcgcttccg aattcctcga
     1861 caacaagcac ccctggaaga accagctgag tgagatggtg cagcccagtg gtggcccagc
     1921 agaggaccag gacatgctgg gtgaagaatc ttctctgggg aagcctgcaa tgctacatct
     1981 gccttcagag cagggtactc ctgagaccct ccagcgctgc ctggaagaga atcaagagct
     2041 ccgagacgct atccggcaga gcaatcagat gctgagggaa cgctgtgagg agctgctgca
     2101 tttccaggtc agccagcggg aggagaagga gttccttatg tgcaaattcc aggaagcccg
     2161 gaagctggtg gagagactga gcttggagaa gcttgatctt cggagtcaga gggaacaggc
     2221 cttaaaggag ttggagcaac tgaagaaatg ccaacagcag atggctgagg acaaggcctc
     2281 tgtgaaagtt caggtgacat cattgctcgg agaactccag gagagccaga gccgtttgga
     2341 ggctgccacc aaggatcggc aagctttaga gggaaggatt cgagcagtta gtgagcaggt
     2401 cagacagctg gagagtgagc gggaggtgct acagcagcag cacagcgtcc aggtggacca
     2461 gctgcgtatg cagaaccaga gcgtggaggc tgccttgcga atggggcggc aggctgcttc
     2521 agaggagaag cggaagctgg ctcagttgca ggcagcctat caccagctct tccaagacta
     2581 cgacagccac attaagagca acaagggcat gcagctggaa gatctgaggc aacagctcca
     2641 gcaagctgag gaggccctgg tagccaaaca ggaattgatt gataagctga aagaggaggc
     2701 tgagcagcac aagattgtga tggagactgt gccagtcttg aaggcccagg cggatatcta
     2761 caaggctgac ttccaagctg agaggcatgc ccgggagaag ctggtggaga agaaggagta
     2821 tttgcaggag cagctggagc agctgcagcg cgagttcaac aagctgaaag ttggctgcca
     2881 tgagtcagcc aggattgagg atatgaggaa gcggcatgta gagactcccc agcctccttt
     2941 actccctgct ccagctcacc actcctttca tttggccttg tccaaccagc ggaggagccc
     3001 tcctgaagaa cctcctgact tctgttgtcc gaagtgccag tatcaggctc ctgatatgga
     3061 cactctacag atacatgtca tggagtgcat agagtagggg cagcagatgc aaggccactt
     3121 gcagtactat gtcctgatct gtgtgacttg tgctttcctg ttttacctgc atagtccaca
     3181 cttaagggct tgctttagcc ctttggtccc ccatttaggg tagacagccc cattcagggc
     3241 tttttttttt ttctgtgtgc ctgatccagt ttgcctctgg tggcttcttc cctcttctcc
     3301 catagtccta gggagtctag agggccctat tctatagtgt cacctaaatg ctagagctcg
     3361 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3421 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3481 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3541 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3601 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3661 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3721 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3781 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3841 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3901 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3961 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4021 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     4081 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4141 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     4201 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     4261 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4321 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     4381 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     4441 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     4501 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     4561 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     4621 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     4681 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     4741 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     4801 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     4861 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4921 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4981 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     5041 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     5101 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     5161 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     5221 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     5281 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     5341 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     5401 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     5461 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     5521 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     5581 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     5641 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     5701 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     5761 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5821 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5881 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5941 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     6001 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     6061 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6121 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6181 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6241 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6301 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6361 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6421 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     6481 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6541 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6601 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6661 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6721 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6781 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6841 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6901 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6961 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     7021 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     7081 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     7141 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     7201 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     7261 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     7321 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     7381 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7441 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7501 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7561 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7621 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7681 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7741 ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
