LOCUS       sequence-473026-        6053 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human Sharpin NZF (347-376).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6053)
  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 6053)
  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-Sharpin NZF (347-376)
FEATURES             Location/Qualifiers
     source          1..6053
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(114..161)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     157..175
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(228..245)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(356..379)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(398..1051)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             complement(1052..1069)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1082..1104
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1119..1143
                     /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(1143..1162)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1144..1162)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1246..1265)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1471..1548
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1549..2631
                     /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(1568..1587)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3440..3631
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3625..3647)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3733..3872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3788..3807
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3887..3904)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4058..4646)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4138..4157)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4757..4776)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4768..5583
                     /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             4768..5583
                     /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(4841..4860)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5675..6053,1..77))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5812..5833)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6024..6043
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
       61 gccgctacag ggcgcgtccc attcgccaat ccggatatag ttcctccttt cagcaaaaaa
      121 cccctcaaga cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca
      181 gcagccaact cagcttcctt tcgggctttg ttagcagccg gatctcagtg gtggtggtgg
      241 tggtgtttaa atggtctaga aagctttagg gcctctgggt gctacacatc tcacagccag
      301 ggcggtctgg ggcattgatg aaggtgcagg aaggacagga ccagctgggc tggagtgggc
      361 cctgaaacag aacttccaga ccgctgctca gatccgattt tggaggatgg tcgccaccac
      421 caaacgtggc ttgccagccc tgcaaaggcc atgctatata cttgctggat ttcaagtact
      481 tatcaatttg tgggatagct tcaatacgtt ttttaaaaca aactaatttt gggaacgcat
      541 ccaggcacat tgggtccatg tataaaacaa catcaagagc gtcatacaac atgaagtcag
      601 gatgggttac atgatcacca tttaaatatg ttttatgaca taaacgatct tcgaacattt
      661 tcagcatttc aggtagcttg ctaagaaaat caactttgag agtttcaaag tctttactat
      721 atgcaattct cgaaacaccg tatctaatat ccaaaaccgc tccttcaagc attgaaatct
      781 ctgcacgctc ttttggacaa ccacccaaca tgttgtgctt gtcagctata taacgtatga
      841 tggccataga ctgtgttaat ttaacatcac catcaatata ataaggaaga ttgggaaact
      901 ccaaacccaa ttcaaacttt ttgtttcgcc atttatcacc ttcatcgcgc tcatacaaat
      961 gctcttcata tttttcttca agatattcca aaagaagtcg agtgggttgc acaaggccct
     1021 taattttcca ataacctagt ataggggaca tgtgatggtg atggtgatgt gccatggtat
     1081 atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat
     1141 tcccctatag tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac
     1201 gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
     1261 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
     1321 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     1381 tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg
     1441 agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc
     1501 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     1561 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     1621 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     1681 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     1741 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     1801 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     1861 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     1921 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     1981 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     2041 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     2101 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     2161 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     2221 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     2281 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     2341 ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
     2401 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     2461 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
     2521 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     2581 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     2641 aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc
     2701 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat
     2761 gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt
     2821 cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg
     2881 aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga
     2941 gaagcaggcc attatcgccg gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc
     3001 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     3061 acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg
     3121 aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac
     3181 cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc
     3241 tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca
     3301 taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac
     3361 ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc
     3421 cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt
     3481 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca
     3541 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc
     3601 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     3661 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     3721 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     3781 aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc
     3841 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     3901 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3961 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4021 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4081 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4141 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4201 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4261 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4321 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4381 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4441 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     4501 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttgttagct
     4561 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4621 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4681 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct
     4741 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct
     4801 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     4861 aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag
     4921 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     4981 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     5041 gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa
     5101 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     5161 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     5221 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     5281 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     5341 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     5401 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     5461 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     5521 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     5581 taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5641 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     5701 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     5761 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     5821 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     5881 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     5941 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     6001 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tag
//
