LOCUS       sequence-444739-        5780 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Strep-HA-tagged VCF1 (p97 binding-deficient N167A 
            mutant) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5780)
  AUTHORS   Mirsanaye AS, Hoffmann S, Weisser M, Mund A, Lopez Mendez B, Typas 
            D, van den Boom J, Benedict B, Hendriks IA, Nielsen ML, Meyer H, 
            Duxin JP, Montoya G, Mailand N
  TITLE     VCF1 is a p97/VCP cofactor promoting recognition of ubiquitylated 
            p97-UFD1-NPL4 substrates.
  JOURNAL   Nat Commun. 2024 Mar 19;15(1):2459. doi: 10.1038/s41467-024-46760-4.
  PUBMED    38503733
REFERENCE   2  (bases 1 to 5780)
  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: "Nat 
            Commun."; date: "2024-03-19"; volume: "15(1):2459. doi"; pages: " 
            10.1038/s41467-024-46760-4"
COMMENT     Sequence Label: Strep-HA-VCF1 N167A
FEATURES             Location/Qualifiers
     source          1..5780
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        152..531
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        532..735
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     686..706
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    737..755
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    758..776
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     786..810
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             910..933
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             970..993
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1021..1047
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1649..1666)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1655..1879
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2012..2031)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2163..2210
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2218..3240
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2218..3240
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3370..3491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3407..3426)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3461..3480
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3540..3556)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3540..3556)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3553..3575)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3564..3580
                     /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(3588..3594,3595..3612,3613..3618))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3633..3654
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3771..3788)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3942..4530)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4022..4041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4701..5492,5493..5561))
                     /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     5324..5343
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5562..5666)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5741..5760)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
       61 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
      121 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
      181 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      241 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      301 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      361 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      421 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      481 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      541 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      601 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      661 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      721 tatataagca gagctctccc tatcagtgat agagatctcc ctatcagtga tagagatcgt
      781 cgacgagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
      841 ctccatagaa gacaccggga ccgatccagc ctccggactc tagcgtttaa acttaagctt
      901 atggctagct ggagccaccc gcagttcgag aaaggtggag gttccggagg tggatcggga
      961 ggtggatcgt ggagccaccc gcagttcgaa aaaggcgccg agaccgcggt cccgaattct
     1021 tacccttatg acgtccccga ttacgccgct gcagccggta ccatgggggg gcgcggagca
     1081 gacgccggaa gtagtggtgg tacgggtccg actgaggggt actcgccacc ggctgcgtcc
     1141 acccgagcgg ctgcgagagc caaggcccga gggggtgggc gtggcggccg ccgaaacaca
     1201 accccctctg ttccctctct tcgcggagcg gcgccgcgta gcttccatcc gccagctgcc
     1261 atgagcgagc gcctccgtcc caggaaaagg agaaggaatg gcaacgaaga agacaaccat
     1321 cttccccccc agaccaaaag aagtagcaga aaccctgtct ttcaggattc ctgggacaca
     1381 gagtcttcag gcagtgacag tggtgggagc agcagcagca gcagcagcag catcaatagc
     1441 ccggacaggg ccagcgggcc ggaaggcagc ttgagccaga ccatggccgg atccagccct
     1501 aacacgcctc agcccgtgcc cgagcagtcc gcgctgtgcc aaggcctcta cttccacatc
     1561 gcgcagaccc tgagggaggc ccacttccac agcctacagc accgagggcg gcctctgaca
     1621 tgagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1681 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1741 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1801 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1861 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     1921 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1981 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2041 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2101 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc
     2161 tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc cttggccaaa
     2221 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     2281 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     2341 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     2401 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     2461 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     2521 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     2581 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     2641 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     2701 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     2761 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     2821 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     2881 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     2941 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     3001 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     3061 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     3121 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     3181 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     3241 cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     3301 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     3361 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3421 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3481 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     3541 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     3601 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     3661 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3721 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3781 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3841 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3901 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3961 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4021 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4081 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4141 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4201 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4261 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4321 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4381 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4441 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4501 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4561 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4621 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4681 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4741 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4801 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4861 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4921 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4981 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5041 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5101 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5161 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5221 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5281 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5341 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5401 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5461 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5521 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5581 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5641 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     5701 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     5761 catagttaag ccagtatctg
//
