LOCUS       sequence-472916-        6092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human CAPN15 NZF1 (1-42).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6092)
  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 6092)
  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-CAPN15 NZF1 (1-42)
FEATURES             Location/Qualifiers
     source          1..6092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(32..51)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     242..263
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(492..1307)
                     /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(492..1307)
                     /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     1215..1234
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1299..1318
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1429..2017
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1918..1937
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2171..2188
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2203..2342
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2268..2287)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2428..2450
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2444..2635)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3444..4526)
                     /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     4488..4507
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4527..4604)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4810..4829
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4913..4932
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4913..4931
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4932..4956
                     /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             4971..4993
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5006..5023
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5024..5677
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             5696..5719
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5869..5886
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5939..5957)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5953..6000
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(6037..6092,1..400)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
       61 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
      121 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
      181 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
      241 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
      301 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
      361 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
      421 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
      481 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
      541 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
      601 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
      661 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
      721 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
      781 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
      841 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
      901 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
      961 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     1021 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     1081 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     1141 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     1201 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     1261 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     1321 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     1381 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     1441 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     1501 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     1561 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     1621 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     1681 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     1741 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     1801 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     1861 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     1921 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     1981 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     2041 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     2101 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     2161 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     2221 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     2281 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     2341 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     2401 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     2461 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     2521 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     2581 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     2641 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     2701 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     2761 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     2821 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     2881 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     2941 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     3001 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     3061 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     3121 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     3181 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     3241 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     3301 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     3361 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     3421 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3481 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     3541 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     3601 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     3661 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     3721 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     3781 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     3841 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     3901 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     3961 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     4021 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     4081 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     4141 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     4201 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     4261 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     4321 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     4381 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     4441 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     4501 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     4561 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     4621 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     4681 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     4741 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     4801 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     4861 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
     4921 ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac
     4981 tttaagaagg agatatacca tggcacatca ccatcaccat cacatgtccc ctatactagg
     5041 ttattggaaa attaagggcc ttgtgcaacc cactcgactt cttttggaat atcttgaaga
     5101 aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt
     5161 tgaattgggt ttggagtttc ccaatcttcc ttattatatt gatggtgatg ttaaattaac
     5221 acagtctatg gccatcatac gttatatagc tgacaagcac aacatgttgg gtggttgtcc
     5281 aaaagagcgt gcagagattt caatgcttga aggagcggtt ttggatatta gatacggtgt
     5341 ttcgagaatt gcatatagta aagactttga aactctcaaa gttgattttc ttagcaagct
     5401 acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt taaatggtga
     5461 tcatgtaacc catcctgact tcatgttgta tgacgctctt gatgttgttt tatacatgga
     5521 cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat
     5581 cccacaaatt gataagtact tgaaatccag caagtatata gcatggcctt tgcagggctg
     5641 gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg gatctgagca gcggtctgga
     5701 agttctgttt cagggcccga tggccacggt cggagagtgg tcctgtgtgc gctgcacctt
     5761 cctgaacccg gccggccagc gccagtgctc catctgcgag gctccccggc acaagcccga
     5821 cctcaaccac atcctgcggc tcagctaaag ctttctagac catttaaaca ccaccaccac
     5881 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     5941 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     6001 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     6061 gcgcggcggg tgtggtggtt acgcgcagcg tg
//
