LOCUS       sequence-224851-        6901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of recombinant Npl4 in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6901)
  AUTHORS   Weith M, Seiler J, van den Boom J, Kracht M, Hulsmann J, Primorac I,
            Del Pino Garcia J, Kaschani F, Kaiser M, Musacchio A, Bollen M, 
            Meyer H
  TITLE     Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives
            PP1 Holoenzyme Formation.
  JOURNAL   Mol Cell. 2018 Oct 11. pii: S1097-2765(18)30791-3. doi: 
            10.1016/j.molcel.2018.09.020.
  PUBMED    30344098
REFERENCE   2  (bases 1 to 6901)
  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"; journalName: "Mol Cell. 
            2018 Oct 11. pii: S1097-2765(18)30791-3. doi: 
            10.1016/j.molcel.2018.09.020."
COMMENT     Sequence Label: pET41b+_Npl4
FEATURES             Location/Qualifiers
     source          1..6901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     86..105
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     193..212
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        193..211
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    212..236
                     /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             251..273
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2175..2198
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2261..2279)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2275..2322
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2359..2814
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2446..2465)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2656..2677
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2906..3721)
                     /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(2906..3721)
                     /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     3629..3648
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3713..3732
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3843..4431
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4332..4351
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4585..4602
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4617..4756
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4682..4701)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4842..4864
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4858..5049)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5621..6703)
                     /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     6665..6684
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6704..6781)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
       61 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      121 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg
      181 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      241 tagaaataat tttgtttaac tttaagaagg agatatacat atggccgaga gcatcataat
      301 tcgtgtccag tccccggatg gagtgaagcg gatcacagca acaaagagag aaacagcagc
      361 aacatttttg aaaaaggttg caaaggagtt tggcttccaa aataatggct tctcggttta
      421 catcaataga aacaagaccg gagagataac agcctcctcc aacaaatccc tcaacttgct
      481 aaaaatcaag catggcgatt tgttgttcct gtttccctcg agccttgctg ggccctcatc
      541 tgaaatggag acttcagttc caccgggctt caaagtcttt ggcgctccca acgtggtgga
      601 ggatgagatt gatcagtacc tcagcaaaca ggacgggaag atttacagaa gccgagaccc
      661 acagctatgc cgccacggcc ctttggggaa atgcgtgcac tgcgtccctc tagagccatt
      721 cgatgaggac tatctaaacc atctcgagcc tcccgtgaag cacatgtcct tccacgccta
      781 catccggaag ctgactggag gggctgacaa ggggaagttt gttgccctgg agaacatcag
      841 ctgcaagatt aagtcagggt gcgaggggca cctcccgtgg ccgaatggca tctgtactaa
      901 gtgccagccg agcgccatca cgctgaacag acagaagtac aggcatgtgg acaatatcat
      961 gtttgagaat cacaccgtcg ctgaccgctt tcttgacttc tggagaaaga cagggaacca
     1021 gcattttggg tacttatacg gacggtacac ggagcacaaa gacattcccc ttggcatcag
     1081 ggctgaagtg gctgcgattt atgagccacc tcagattggt acacagaaca gcttggagct
     1141 tcttgaggat ccaaaagctg aagtggtcga tgaaattgct gccaaacttg gcctgcggaa
     1201 ggttggctgg atatttacag acctcgtctc agaagatacc cgaaagggta ccgtccgcta
     1261 cagtcgaaat aaggacacct atttcctaag ttcagaagag tgcatcactg caggagactt
     1321 ccagaacaag catcccaaca tgtgccggct ctctccagac ggacattttg gatccaagtt
     1381 tgttactgca gtggctacag gtggtcctga caaccaagtc cactttgaag ggtaccaggt
     1441 gtccaatcag tgtatggcac tggtccgtga tgagtgtttg ctgccatgca aggacgcccc
     1501 ggagcttggc tacgccaagg agtctagcag tgagcagtac gtgcctgatg tgttttataa
     1561 ggacgtagac aagtttggca acgagatcac ccagctggcc cgtcccctgc ctgtggagta
     1621 tctcatcata gacatcacaa caactttccc caaggatcca gtttacactt tttctatttc
     1681 gcaaaatcca tttcctattg aaaaccggga tgtattgggt gagacacagg acttccatag
     1741 cttggccacc tatttgtctc agaatacctc atctgtgttc ttggatacca tctcagattt
     1801 ccacctcttg ctgttcctgg tcaccaatga agttatgcct ctgcaggaca gcatcagctt
     1861 gctgctggag gccgtgcgga ccagaaatga ggagctcgcc cagacatgga agaggtctga
     1921 gcagtgggcc accatcgagc agctgtgcag cacagttggc gggcagctcc caggtctcca
     1981 tgagtacggg gccgtcgggg gctccacaca cacggccact gcagccatgt gggcctgtca
     2041 gcactgcacg ttcatgaacc agccaggcac aggccactgc gagatgtgca gcctccccag
     2101 gacctaagaa ttctgtacag gccttggcgc gcctgcaggc gagctccgtc gacaagcttg
     2161 cggccgcact cgagcaccac caccaccacc accaccacta attgattaat acctaggctg
     2221 ctaaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     2281 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     2341 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2401 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2461 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2521 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2581 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2641 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2701 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2761 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     2821 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2881 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     2941 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     3001 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     3061 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     3121 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     3181 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     3241 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     3301 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     3361 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     3421 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     3481 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     3541 cgctaccttt gccatgtttc agaaacaact ctggcgcatc aggcttccca tacaatcgat
     3601 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     3661 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     3721 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     3781 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3841 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3901 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3961 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     4021 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4081 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4141 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4201 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4261 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4321 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4381 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4441 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4501 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4561 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     4621 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     4681 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4741 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4801 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4861 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     4921 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     4981 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     5041 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     5101 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     5161 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     5221 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     5281 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     5341 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     5401 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     5461 accccgccag cctagccggg tcctcaacga caggagcacg atcatgctag tcatgccccg
     5521 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     5581 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5641 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5701 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     5761 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     5821 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     5881 cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc
     5941 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     6001 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     6061 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     6121 gcgccgagac agaacttaat ggtcccgcta acagcgcgat ttgctggtga cccaatgcga
     6181 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     6241 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     6301 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     6361 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     6421 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     6481 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     6541 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     6601 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     6661 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     6721 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     6781 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     6841 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     6901 a
//
