LOCUS       sequence-450660-        7704 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ybbR-p97-T262A-His6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7704)
  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 7704)
  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: pAM231
FEATURES             Location/Qualifiers
     source          1..7704
                     /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             2633..2650
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2703..2721)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2717..2764
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2801..3256
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2888..2907)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3098..3119
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3348..4163)
                     /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(3348..4163)
                     /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     4071..4090
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4155..4174
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4285..4873
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4774..4793
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5027..5044
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5059..5198
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5124..5143)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5284..5306
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5300..5491)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6300..7382)
                     /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     7344..7363
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7383..7460)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7666..7685
                     /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 catggattct cttgagttca tcgctagtaa
      181 gcttgcgggt ggcgggtcga ctgcttctgg agccgattca aaaggtgatg acctatcaac
      241 agccattctc aaacagaaga accgtcccaa tcggttaatt gttgatgaag ccatcaatga
      301 ggacaacagt gtggtgtcct tgtcccagcc caagatggat gaattgcagt tgttccgagg
      361 tgacacagtg ttgctgaaag gaaagaagag acgagaagct gtttgcatcg tcctttctga
      421 tgatacttgt tctgatgaga agattcggat gaatagagtt gttcggaata accttcgtgt
      481 acgcctaggg gatgtcatca gcatccagcc atgccctgat gtgaagtacg gcaaacgtat
      541 ccatgtgctg cccattgatg acacagtgga aggcattact ggtaatctct tcgaggtata
      601 ccttaagccg tacttcctgg aagcgtatcg acccatccgg aaaggagaca tttttcttgt
      661 ccgtggtggg atgcgtgctg tggagttcaa agtggtggaa acagatccta gcccttattg
      721 cattgttgct ccagacacag tgatccactg cgaaggggag cctatcaaac gagaggatga
      781 ggaagagtcc ttgaatgaag tagggtatga tgacattggt ggctgcagga agcagctagc
      841 tcagataaag gagatggtgg aactgcccct gagacatcct gccctcttta aggcaattgg
      901 tgtgaagcct cctagaggaa tcctgcttta cggacctcct ggaacaggaa agaccctgat
      961 tgctcgagct gtagcaaatg aggctggagc cttcttcttc ttgatcaatg gtcctgagat
     1021 catgagcaaa ttggctggtg agtctgagag caaccttcgt aaagcctttg aggaggctga
     1081 gaagaatgct cctgccatca tcttcattga tgagctagat gccatcgctc ccaaaagaga
     1141 gaaaactcat ggcgaggtgg agcggcgcat tgtatcacag ttgttgaccc tcatggatgg
     1201 cctaaagcag agggcacatg tgattgttat ggcagcaacc aacagaccca acagcattga
     1261 cccagctcta cggcgatttg gtcgctttga cagggaggta gatattggaa ttcctgatgc
     1321 tacaggacgc ttagagattc ttcagatcca taccaagaac atgaagctgg cagatgatgt
     1381 ggacctggaa caggtagcca atgagactca cgggcatgtg ggtgctgact tagcagccct
     1441 gtgctcagag gctgctctgc aagccatccg caagaagatg gatctcattg acctagagga
     1501 tgagaccatt gatgccgagg tcatgaactc tctagcagtt actatggatg acttccggtg
     1561 ggccttgagc cagagtaacc catcagcact gcgggaaacc gtggtagagg tgccacaggt
     1621 aacctgggaa gacatcgggg gcctagagga tgtcaaacgt gagctacagg agctggtcca
     1681 gtatcctgtg gagcacccag acaaattcct gaagtttggc atgacacctt ccaagggagt
     1741 tctgttctat ggacctcctg gctgtgggaa aactttgttg gccaaagcca ttgctaatga
     1801 atgccaggcc aacttcatct ccatcaaggg tcctgagctg ctcaccatgt ggtttgggga
     1861 gtctgaggcc aatgtcagag aaatctttga caaggcccgc caagctgccc cctgtgtgct
     1921 attctttgat gagctggatt cgattgccaa ggctcgtgga ggtaacattg gagatggtgg
     1981 tggggctgct gaccgagtca tcaaccagat cctgacagaa atggatggca tgtccacaaa
     2041 aaaaaatgtg ttcatcattg gcgctaccaa ccggcctgac atcattgatc ctgccatcct
     2101 cagacctggc cgtcttgatc agctcatcta catcccactt cctgatgaga agtcccgtgt
     2161 tgccatcctc aaggctaacc tgcgcaagtc cccagttgcc aaggatgtgg acttggagtt
     2221 cctggctaaa atgactaatg gcttctctgg agctgacctg acagagattt gccagcgtgc
     2281 ttgcaagctg gccatccgtg aatccatcga gagtgagatt aggcgagaac gagagaggca
     2341 gacaaaccca tcagccatgg aggtagaaga ggatgatcca gtgcctgaga tccgtcgaga
     2401 tcactttgaa gaagccatgc gctttgcgcg ccgttctgtc agtgacaatg acattcggaa
     2461 gtatgagatg tttgcccaga cccttcagca gagtcggggc tttggcagct tcagattccc
     2521 ttcagggaac cagggtggag ctggccccag tcagggcagt ggaggcggca caggtggcag
     2581 tgtatacaca gaagacaatg atgatgacct gtatggcgcg gccgccctcg agcaccacca
     2641 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     2701 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     2761 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     2821 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2881 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2941 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3001 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3061 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3121 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     3181 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     3241 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3301 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     3361 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     3421 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     3481 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     3541 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     3601 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     3661 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     3721 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     3781 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     3841 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     3901 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     3961 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     4021 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     4081 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     4141 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     4201 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4261 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4321 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4381 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4441 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4501 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4561 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4621 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4681 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4741 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4801 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4861 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4921 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4981 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     5041 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5101 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     5161 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     5221 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5281 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     5341 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     5401 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     5461 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     5521 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     5581 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     5641 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     5701 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     5761 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     5821 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     5881 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     5941 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     6001 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     6061 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     6121 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     6181 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     6241 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     6301 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6361 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     6421 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     6481 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     6541 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     6601 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     6661 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     6721 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     6781 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     6841 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     6901 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     6961 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     7021 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     7081 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     7141 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     7201 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     7261 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     7321 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     7381 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     7441 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     7501 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     7561 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     7621 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     7681 ataggcgcca gcaaccgcac ctgt
//
