LOCUS       sequence-450668-        7669 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET24b-p97-R155H-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: pAM219
FEATURES             Location/Qualifiers
     source          1..7669
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     65..84
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        65..83
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    84..108
                     /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             123..145
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2598..2615
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2668..2686)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2682..2729
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2766..3221
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2853..2872)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3063..3084
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3313..4128)
                     /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(3313..4128)
                     /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     4036..4055
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4120..4139
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4250..4838
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4739..4758
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4992..5009
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5024..5163
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5089..5108)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5249..5271
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5265..5456)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6265..7347)
                     /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     7309..7328
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7348..7425)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7631..7650
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
       61 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
      121 attttgttta actttaagaa ggagatatac atatggcttc tggagccgat tcaaaaggtg
      181 atgacctatc aacagccatt ctcaaacaga agaaccgtcc caatcggtta attgttgatg
      241 aagccatcaa tgaggacaac agtgtggtgt ccttgtccca gcccaagatg gatgaattgc
      301 agttgttccg aggtgacaca gtgttgctga aaggaaagaa gagacgagaa gctgtttgca
      361 tcgtcctttc tgatgatact tgttctgatg agaagattcg gatgaataga gttgttcgga
      421 ataaccttcg tgtacgccta ggggatgtca tcagcatcca gccatgccct gatgtgaagt
      481 acggcaaacg tatccatgtg ctgcccattg atgacacagt ggaaggcatt actggtaatc
      541 tcttcgaggt ataccttaag ccgtacttcc tggaagcgta tcgacccatc cggaaaggag
      601 acatttttct tgtccatggt gggatgcgtg ctgtggagtt caaagtggtg gaaacagatc
      661 ctagccctta ttgcattgtt gctccagaca cagtgatcca ctgcgaaggg gagcctatca
      721 aacgagagga tgaggaagag tccttgaatg aagtagggta tgatgacatt ggtggctgca
      781 ggaagcagct agctcagata aaggagatgg tggaactgcc cctgagacat cctgccctct
      841 ttaaggcaat tggtgtgaag cctcctagag gaatcctgct ttacggacct cctggaacag
      901 gaaagaccct gattgctcga gctgtagcaa atgagactgg agccttcttc ttcttgatca
      961 atggtcctga gatcatgagc aaattggctg gtgagtctga gagcaacctt cgtaaagcct
     1021 ttgaggaggc tgagaagaat gctcctgcca tcatcttcat tgatgagcta gatgccatcg
     1081 ctcccaaaag agagaaaact catggcgagg tggagcggcg cattgtatca cagttgttga
     1141 ccctcatgga tggcctaaag cagagggcac atgtgattgt tatggcagca accaacagac
     1201 ccaacagcat tgacccagct ctacggcgat ttggtcgctt tgacagggag gtagatattg
     1261 gaattcctga tgctacagga cgcttagaga ttcttcagat ccataccaag aacatgaagc
     1321 tggcagatga tgtggacctg gaacaggtag ccaatgagac tcacgggcat gtgggtgctg
     1381 acttagcagc cctgtgctca gaggctgctc tgcaagccat ccgcaagaag atggatctca
     1441 ttgacctaga ggatgagacc attgatgccg aggtcatgaa ctctctagca gttactatgg
     1501 atgacttccg gtgggccttg agccagagta acccatcagc actgcgggaa accgtggtag
     1561 aggtgccaca ggtaacctgg gaagacatcg ggggcctaga ggatgtcaaa cgtgagctac
     1621 aggagctggt ccagtatcct gtggagcacc cagacaaatt cctgaagttt ggcatgacac
     1681 cttccaaggg agttctgttc tatggacctc ctggctgtgg gaaaactttg ttggccaaag
     1741 ccattgctaa tgaatgccag gccaacttca tctccatcaa gggtcctgag ctgctcacca
     1801 tgtggtttgg ggagtctgag gccaatgtca gagaaatctt tgacaaggcc cgccaagctg
     1861 ccccctgtgt gctattcttt gatgagctgg attcgattgc caaggctcgt ggaggtaaca
     1921 ttggagatgg tggtggggct gctgaccgag tcatcaacca gatcctgaca gaaatggatg
     1981 gcatgtccac aaaaaaaaat gtgttcatca ttggcgctac caaccggcct gacatcattg
     2041 atcctgccat cctcagacct ggccgtcttg atcagctcat ctacatccca cttcctgatg
     2101 agaagtcccg tgttgccatc ctcaaggcta acctgcgcaa gtccccagtt gccaaggatg
     2161 tggacttgga gttcctggct aaaatgacta atggcttctc tggagctgac ctgacagaga
     2221 tttgccagcg tgcttgcaag ctggccatcc gtgaatccat cgagagtgag attaggcgag
     2281 aacgagagag gcagacaaac ccatcagcca tggaggtaga agaggatgat ccagtgcctg
     2341 agatccgtcg agatcacttt gaagaagcca tgcgctttgc gcgccgttct gtcagtgaca
     2401 atgacattcg gaagtatgag atgtttgccc agacccttca gcagagtcgg ggctttggca
     2461 gcttcagatt cccttcaggg aaccagggtg gagctggccc cagtcagggc agtggaggcg
     2521 gcacaggtgg cagtgtatac acagaagaca atgatgatga cctgtatggc gtcgacaagc
     2581 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     2641 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     2701 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     2761 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2821 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2881 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2941 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3001 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3061 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3121 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3181 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     3241 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3301 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     3361 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     3421 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     3481 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     3541 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     3601 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     3661 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     3721 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     3781 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     3841 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     3901 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     3961 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     4021 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     4081 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     4141 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     4201 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4261 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4321 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4381 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4441 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4501 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4561 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4621 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4681 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4741 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4801 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4861 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4921 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4981 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     5041 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     5101 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     5161 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5221 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5281 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     5341 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     5401 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     5461 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     5521 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     5581 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     5641 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     5701 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     5761 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     5821 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     5881 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     5941 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     6001 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     6061 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     6121 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     6181 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     6241 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6301 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     6361 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     6421 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     6481 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     6541 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     6601 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     6661 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     6721 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     6781 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     6841 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     6901 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     6961 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     7021 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     7081 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     7141 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     7201 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     7261 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     7321 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     7381 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     7441 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     7501 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     7561 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     7621 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgt
//
