LOCUS       sequence-450586-        7693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET24b-p97-FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7693)
  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 7693)
  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: pAM232
FEATURES             Location/Qualifiers
     source          1..7693
                     /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             2595..2618
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2646..2663
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2716..2734)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2730..2777
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2814..3269
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2901..2920)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3111..3132
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3361..4176)
                     /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(3361..4176)
                     /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     4084..4103
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4168..4187
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4298..4886
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4787..4806
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5040..5057
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5072..5211
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5137..5156)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5297..5319
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5313..5504)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6313..7395)
                     /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     7357..7376
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7396..7473)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(7679..7693,1..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 gcaaatgaga
      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 tggcgattat aaagatgatg atgataaata ggacaagctt gcggccgcac
     2641 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     2701 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     2761 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     2821 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2881 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2941 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3001 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3061 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3121 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3181 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3241 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     3301 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     3361 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     3421 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     3481 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     3541 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     3601 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     3661 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     3721 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     3781 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     3841 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     3901 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     3961 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     4021 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     4081 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     4141 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     4201 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     4261 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4321 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4381 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4441 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4501 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4561 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4621 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4681 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4741 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4801 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4861 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4921 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4981 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     5041 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     5101 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     5161 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     5221 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5281 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5341 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     5401 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     5461 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     5521 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     5581 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     5641 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     5701 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     5761 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     5821 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     5881 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5941 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     6001 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     6061 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     6121 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     6181 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     6241 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     6301 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6361 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     6421 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     6481 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     6541 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     6601 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     6661 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     6721 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     6781 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     6841 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     6901 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     6961 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     7021 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     7081 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     7141 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     7201 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     7261 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     7321 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     7381 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     7441 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     7501 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     7561 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     7621 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     7681 tgatgtcggc gat
//
