LOCUS       sequence-444748-        6341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP-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 6341)
  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 6341)
  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: GFP-VCF1 N167A
FEATURES             Location/Qualifiers
     source          1..6341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        168..547
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        548..751
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     702..722
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    753..771
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    774..792
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     802..826
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(917..919,920..922,923..1633)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(917..919,920..922,923..1633)
                     /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(962..983)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1223..1242)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1570..1591
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2226..2243)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2232..2456
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2589..2608)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2740..2787
                     /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             2795..3817
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2795..3817
                     /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    3947..4068
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3984..4003)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4038..4057
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4117..4133)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4117..4133)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4130..4152)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4141..4157
                     /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(4165..4171,4172..4189,4190..4195))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4210..4231
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4348..4365)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4519..5107)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4599..4618)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5278..6069,6070..6138))
                     /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     5901..5920
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6139..6243)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6318..6337)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
       61 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
      121 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
      181 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      241 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      301 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      361 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      421 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      481 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      541 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      601 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      661 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      721 tgtacggtgg gaggtctata taagcagagc tctccctatc agtgatagag atctccctat
      781 cagtgataga gatcgtcgac gagctcgttt agtgaaccgt cagatcgcct ggagacgcca
      841 tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc ggactctagc
      901 gtttaaactt aagcttatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      961 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1021 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1081 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     1141 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1201 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1261 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1321 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1381 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1441 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1501 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1561 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1621 cgagctgtac aagggtacca tgggggggcg cggagcagac gccggaagta gtggtggtac
     1681 gggtccgact gaggggtact cgccaccggc tgcgtccacc cgagcggctg cgagagccaa
     1741 ggcccgaggg ggtgggcgtg gcggccgccg aaacacaacc ccctctgttc cctctcttcg
     1801 cggagcggcg ccgcgtagct tccatccgcc agctgccatg agcgagcgcc tccgtcccag
     1861 gaaaaggaga aggaatggca acgaagaaga caaccatctt cccccccaga ccaaaagaag
     1921 tagcagaaac cctgtctttc aggattcctg ggacacagag tcttcaggca gtgacagtgg
     1981 tgggagcagc agcagcagca gcagcagcat caatagcccg gacagggcca gcgggccgga
     2041 aggcagcttg agccagacca tggccggatc cagccctaac acgcctcagc ccgtgcccga
     2101 gcagtccgcg ctgtgccaag gcctctactt ccacatcgcg cagaccctga gggaggccca
     2161 cttccacagc ctacagcacc gagggcggcc tctgacatga gggcccgttt aaacccgctg
     2221 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2281 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2341 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2401 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2461 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     2521 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2581 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2641 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2701 ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc
     2761 ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc
     2821 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     2881 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     2941 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     3001 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     3061 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     3121 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     3181 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     3241 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     3301 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     3361 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     3421 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     3481 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     3541 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     3601 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     3661 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     3721 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     3781 ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc
     3841 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     3901 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     3961 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4021 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4081 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4141 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4201 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4261 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4321 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4381 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4441 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4501 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4561 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4621 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4681 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4741 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4801 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4861 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4921 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4981 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5041 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5101 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5161 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5221 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5281 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5341 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5401 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5461 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5521 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5581 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5641 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5701 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5761 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5821 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5881 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5941 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6001 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6061 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6121 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6181 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6241 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     6301 ctatggtgca ctctcagtac aatctgctct gatgccgcat a
//
