LOCUS       sequence-450674-        7669 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET24b-p97-T262A-His6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7669)
  AUTHORS   Blythe EE, Gates SN, Deshaies RJ, Martin A
  TITLE     Multisystem Proteinopathy Mutations in VCP/p97 Increase NPLOC4.UFD1L
            Binding and Substrate Processing.
  JOURNAL   Structure. 2019 Dec 3;27(12):1820-1829.e4. doi: 
            10.1016/j.str.2019.09.011. Epub 2019 Oct 14.
  PUBMED    31623962
REFERENCE   2  (bases 1 to 7669)
  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.str.2019.09.011"; journalName: "Structure"; date: 
            "2019-12-3- 3"; volume: "27"; issue: "12"; pages: "1820-1829.e4"
COMMENT     Sequence Label: pAM224
FEATURES             Location/Qualifiers
     source          1..7669
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..108
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        89..107
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    108..132
                     /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)."
     RBS             147..169
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2622..2639
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2692..2710)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2706..2753
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2790..3245
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2877..2896)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3087..3108
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3337..4152)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3337..4152)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4060..4079
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4144..4163
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4274..4862
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4763..4782
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5016..5033
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5048..5187
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5113..5132)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5273..5295
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5289..5480)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6289..7371)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7333..7352
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7372..7449)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(7655..7669,1..5)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
       61 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      121 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgg
      181 cttctggagc cgattcaaaa ggtgatgacc tatcaacagc cattctcaaa cagaagaacc
      241 gtcccaatcg gttaattgtt gatgaagcca tcaatgagga caacagtgtg gtgtccttgt
      301 cccagcccaa gatggatgaa ttgcagttgt tccgaggtga cacagtgttg ctgaaaggaa
      361 agaagagacg agaagctgtt tgcatcgtcc tttctgatga tacttgttct gatgagaaga
      421 ttcggatgaa tagagttgtt cggaataacc ttcgtgtacg cctaggggat gtcatcagca
      481 tccagccatg ccctgatgtg aagtacggca aacgtatcca tgtgctgccc attgatgaca
      541 cagtggaagg cattactggt aatctcttcg aggtatacct taagccgtac ttcctggaag
      601 cgtatcgacc catccggaaa ggagacattt ttcttgtccg tggtgggatg cgtgctgtgg
      661 agttcaaagt ggtggaaaca gatcctagcc cttattgcat tgttgctcca gacacagtga
      721 tccactgcga aggggagcct atcaaacgag aggatgagga agagtccttg aatgaagtag
      781 ggtatgatga cattggtggc tgcaggaagc agctagctca gataaaggag atggtggaac
      841 tgcccctgag acatcctgcc ctctttaagg caattggtgt gaagcctcct agaggaatcc
      901 tgctttacgg acctcctgga acaggaaaga ccctgattgc tcgagctgta gcaaatgagg
      961 ctggagcctt cttcttcttg atcaatggtc ctgagatcat gagcaaattg gctggtgagt
     1021 ctgagagcaa ccttcgtaaa gcctttgagg aggctgagaa gaatgctcct gccatcatct
     1081 tcattgatga gctagatgcc atcgctccca aaagagagaa aactcatggc gaggtggagc
     1141 ggcgcattgt atcacagttg ttgaccctca tggatggcct aaagcagagg gcacatgtga
     1201 ttgttatggc agcaaccaac agacccaaca gcattgaccc agctctacgg cgatttggtc
     1261 gctttgacag ggaggtagat attggaattc ctgatgctac aggacgctta gagattcttc
     1321 agatccatac caagaacatg aagctggcag atgatgtgga cctggaacag gtagccaatg
     1381 agactcacgg gcatgtgggt gctgacttag cagccctgtg ctcagaggct gctctgcaag
     1441 ccatccgcaa gaagatggat ctcattgacc tagaggatga gaccattgat gccgaggtca
     1501 tgaactctct agcagttact atggatgact tccggtgggc cttgagccag agtaacccat
     1561 cagcactgcg ggaaaccgtg gtagaggtgc cacaggtaac ctgggaagac atcgggggcc
     1621 tagaggatgt caaacgtgag ctacaggagc tggtccagta tcctgtggag cacccagaca
     1681 aattcctgaa gtttggcatg acaccttcca agggagttct gttctatgga cctcctggct
     1741 gtgggaaaac tttgttggcc aaagccattg ctaatgaatg ccaggccaac ttcatctcca
     1801 tcaagggtcc tgagctgctc accatgtggt ttggggagtc tgaggccaat gtcagagaaa
     1861 tctttgacaa ggcccgccaa gctgccccct gtgtgctatt ctttgatgag ctggattcga
     1921 ttgccaaggc tcgtggaggt aacattggag atggtggtgg ggctgctgac cgagtcatca
     1981 accagatcct gacagaaatg gatggcatgt ccacaaaaaa aaatgtgttc atcattggcg
     2041 ctaccaaccg gcctgacatc attgatcctg ccatcctcag acctggccgt cttgatcagc
     2101 tcatctacat cccacttcct gatgagaagt cccgtgttgc catcctcaag gctaacctgc
     2161 gcaagtcccc agttgccaag gatgtggact tggagttcct ggctaaaatg actaatggct
     2221 tctctggagc tgacctgaca gagatttgcc agcgtgcttg caagctggcc atccgtgaat
     2281 ccatcgagag tgagattagg cgagaacgag agaggcagac aaacccatca gccatggagg
     2341 tagaagagga tgatccagtg cctgagatcc gtcgagatca ctttgaagaa gccatgcgct
     2401 ttgcgcgccg ttctgtcagt gacaatgaca ttcggaagta tgagatgttt gcccagaccc
     2461 ttcagcagag tcggggcttt ggcagcttca gattcccttc agggaaccag ggtggagctg
     2521 gccccagtca gggcagtgga ggcggcacag gtggcagtgt atacacagaa gacaatgatg
     2581 atgacctgta tggcgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact
     2641 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     2701 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     2761 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     2821 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2881 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2941 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3001 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3061 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3121 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3181 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     3241 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3301 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     3361 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     3421 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     3481 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     3541 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     3601 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     3661 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     3721 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     3781 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     3841 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     3901 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     3961 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     4021 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     4081 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     4141 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     4201 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4261 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4321 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4381 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4441 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4501 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4561 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4621 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4681 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4741 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4801 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4861 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4921 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4981 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     5041 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     5101 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     5161 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     5221 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5281 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     5341 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     5401 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     5461 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     5521 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     5581 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     5641 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     5701 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     5761 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     5821 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     5881 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     5941 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     6001 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     6061 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     6121 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     6181 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     6241 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     6301 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     6361 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     6421 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     6481 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     6541 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     6601 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     6661 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     6721 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     6781 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6841 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6901 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6961 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     7021 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     7081 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     7141 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     7201 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     7261 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     7321 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     7381 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     7441 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     7501 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     7561 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     7621 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgat
//
