LOCUS       sequence-473101-        6071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human NPL4 NZF (574-608).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6071)
  AUTHORS   Michel MA, Scutts S, Komander D
  TITLE     Secondary interactions in ubiquitin-binding domains achieve linkage 
            or substrate specificity.
  JOURNAL   Cell Rep. 2024 Aug 27;43(8):114545. doi: 
            10.1016/j.celrep.2024.114545. Epub 2024 Jul 23.
  PUBMED    39052481
REFERENCE   2  (bases 1 to 6071)
  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: "Cell Rep.";
            date: "2024-08-27"; volume: "43(8):114545. doi"; pages: " 
            10.1016/j.celrep.2024.114545. Epub 2024 Jul 23"
COMMENT     Sequence Label: pOPINK-NPL4 NZF (574-608)
FEATURES             Location/Qualifiers
     source          1..6071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1754..1945
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1939..1961)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2047..2186
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2102..2121
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2201..2218)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2372..2960)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2452..2471)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3071..3090)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3082..3897
                     /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             3082..3897
                     /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     complement(3155..3174)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3989..4444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4126..4147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4338..4357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4481..4528)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4524..4542
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4595..4612)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4741..4764)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(4783..5436)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             complement(5437..5454)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5467..5489
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5504..5528
                     /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)."
     primer_bind     complement(5528..5547)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5529..5547)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5631..5650)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5856..5933
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5934..6071,1..945)
                     /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     complement(5953..5972)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
       61 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
      121 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
      181 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
      241 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
      301 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
      361 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
      421 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
      481 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
      541 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
      601 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
      661 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
      721 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
      781 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
      841 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
      901 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
      961 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa
     1021 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     1081 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     1141 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     1201 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     1261 ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     1321 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     1381 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     1441 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     1501 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     1561 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     1621 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     1681 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     1741 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     1801 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     1861 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     1921 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     1981 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     2041 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     2101 gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata ccgcacagat
     2161 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     2221 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2281 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2341 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2401 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2461 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2521 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2581 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2641 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2701 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2761 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     2821 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2881 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2941 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3001 ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa taaaactgtc tgcttacata
     3061 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg ctctaggccg
     3121 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     3181 gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt
     3241 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     3301 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     3361 gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat
     3421 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     3481 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     3541 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     3601 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     3661 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     3721 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     3781 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     3841 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagaa
     3901 ttaattcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     3961 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     4021 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     4081 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     4141 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     4201 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     4261 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     4321 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     4381 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     4441 gcgtcccatt cgccaatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc
     4501 gtttagaggc cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag
     4561 cttcctttcg ggctttgtta gcagccggat ctcagtggtg gtggtggtgg tgtttaaatg
     4621 gtctagaaag ctttaggtcc ggggaaggct gcacatctcg cagtggcctg tgccaggctg
     4681 gttcatgaag gtgcagtgct gacaagccca catggcagag gtagcagcgc gtgcagagcc
     4741 cgggccctga aacagaactt ccagaccgct gctcagatcc gattttggag gatggtcgcc
     4801 accaccaaac gtggcttgcc agccctgcaa aggccatgct atatacttgc tggatttcaa
     4861 gtacttatca atttgtggga tagcttcaat acgtttttta aaacaaacta attttgggaa
     4921 cgcatccagg cacattgggt ccatgtataa aacaacatca agagcgtcat acaacatgaa
     4981 gtcaggatgg gttacatgat caccatttaa atatgtttta tgacataaac gatcttcgaa
     5041 cattttcagc atttcaggta gcttgctaag aaaatcaact ttgagagttt caaagtcttt
     5101 actatatgca attctcgaaa caccgtatct aatatccaaa accgctcctt caagcattga
     5161 aatctctgca cgctcttttg gacaaccacc caacatgttg tgcttgtcag ctatataacg
     5221 tatgatggcc atagactgtg ttaatttaac atcaccatca atataataag gaagattggg
     5281 aaactccaaa cccaattcaa actttttgtt tcgccattta tcaccttcat cgcgctcata
     5341 caaatgctct tcatattttt cttcaagata ttccaaaaga agtcgagtgg gttgcacaag
     5401 gcccttaatt ttccaataac ctagtatagg ggacatgtga tggtgatggt gatgtgccat
     5461 ggtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc
     5521 acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc
     5581 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
     5641 cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg
     5701 cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc
     5761 accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
     5821 gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct
     5881 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     5941 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     6001 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     6061 cggagctgaa t
//
