LOCUS       sequence-450661-        6225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MSS-His6-UFD1L.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6225)
  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 6225)
  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: pAM227
FEATURES             Location/Qualifiers
     source          1..6225
                     /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             161..178
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             188..205
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1154..1171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1224..1242)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1238..1285
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1322..1777
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1409..1428)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1619..1640
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1869..2684)
                     /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(1869..2684)
                     /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     2592..2611
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2676..2695
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2806..3394
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3295..3314
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3548..3565
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3580..3719
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3645..3664)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3805..3827
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3821..4012)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4821..5903)
                     /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     5865..5884
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5904..5981)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6187..6206
                     /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 catgagcagc catcatcatc atcatcacag
      181 cagcggcctg gtgccgcgcg gcagccatat gttctctttc aacatgttcg accaccctat
      241 tcccagggtc ttccaaaacc gcttctccac acagtaccgc tgcttctctg tgtccatgct
      301 agcagggcct aatgacaggt cagatgtgga gaaaggaggg aagataatta tgccaccctc
      361 ggccctggac caactcagcc gacttaacat tacctatccc atgctgttca aactgaccaa
      421 taagaattcg gaccgcatga cgcattgtgg cgtgctggag tttgtggctg atgagggcat
      481 ctgctacctc ccacactgga tgatgcagaa cttactcttg gaagaaggcg gcctggtcca
      541 ggtggagagc gtcaaccttc aagtggccac ctactccaaa ttccaacctc agagccctga
      601 cttcctggac atcaccaacc ccaaagccgt attagaaaac gcacttagga actttgcctg
      661 tctgaccacc ggggatgtga ttgccatcaa ctataatgaa aagatctacg aactgcgtgt
      721 gatggagacc aaacccgaca aggcagtgtc catcattgag tgtgacatga acgtggactt
      781 tgatgctccc ctgggctaca aggaacccga aagacaagtc cagcatgagg agtcgacaga
      841 aggtgaagcc gaccacagtg gctatgctgg agagctgggc ttccgcgctt tctctggatc
      901 tggcaataga ctggatggaa agaagaaagg ggtagagccc agcccctccc caatcaagcc
      961 tggagatatt aaaagaggaa ttcccaatta tgaatttaaa cttggtaaga taactttcat
     1021 cagaaattca cgtccccttg tcaaaaaggt tgaagaggat gaagctggag gcagattcgt
     1081 cgctttctct ggagaaggac agtcattgcg taaaaaggga agaaagccct aaaagcttgc
     1141 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     1201 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1261 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     1321 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1381 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1441 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1501 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1561 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1621 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     1681 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     1741 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     1801 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1861 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     1921 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     1981 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     2041 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     2101 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     2161 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     2221 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     2281 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     2341 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     2401 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     2461 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     2521 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     2581 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     2641 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     2701 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     2761 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2821 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2881 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2941 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3001 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3061 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3121 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3181 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3241 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3301 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3361 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3421 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3481 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3541 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3601 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     3661 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3721 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3781 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3841 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     3901 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     3961 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4021 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4081 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4141 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4201 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4261 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4321 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4381 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4441 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4501 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4561 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4621 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4681 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     4741 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4801 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4861 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4921 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     4981 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5041 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5101 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5161 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5221 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5281 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5341 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5401 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5461 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5521 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5581 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5641 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5701 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5761 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5821 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5881 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     5941 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6001 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6061 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     6121 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     6181 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgt
//
