LOCUS       sequence-472925-        6068 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human CAPN15 ZF5 (411-444).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6068)
  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 6068)
  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 ZF5 (411-444)
FEATURES             Location/Qualifiers
     source          1..6068
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     102..123
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(352..1167)
                     /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(352..1167)
                     /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     1075..1094
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1159..1178
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1289..1877
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1778..1797
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2031..2048
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2063..2202
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2128..2147)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2288..2310
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2304..2495)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3304..4386)
                     /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     4348..4367
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4387..4464)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4670..4689
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4773..4792
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4773..4791
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4792..4816
                     /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             4831..4853
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4866..4883
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4884..5537
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             5556..5579
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5705..5722
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5775..5793)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5789..5836
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5873..6068,1..260)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5960..5979)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
       61 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
      121 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
      181 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
      241 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
      301 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
      361 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
      421 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
      481 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
      541 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
      601 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
      661 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
      721 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
      781 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
      841 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
      901 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
      961 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     1021 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     1081 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     1141 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     1201 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     1261 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     1321 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     1381 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     1441 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     1501 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     1561 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     1621 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     1681 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     1741 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     1801 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     1861 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     1921 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     1981 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     2041 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     2101 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     2161 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     2221 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     2281 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     2341 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     2401 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     2461 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     2521 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     2581 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     2641 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     2701 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     2761 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     2821 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     2881 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     2941 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     3001 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     3061 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     3121 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     3181 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     3241 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     3301 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3361 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     3421 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     3481 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     3541 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     3601 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     3661 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     3721 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     3781 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     3841 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     3901 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     3961 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     4021 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     4081 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     4141 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     4201 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     4261 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     4321 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     4381 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     4441 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     4501 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     4561 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     4621 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     4681 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     4741 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     4801 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
     4861 tggcacatca ccatcaccat cacatgtccc ctatactagg ttattggaaa attaagggcc
     4921 ttgtgcaacc cactcgactt cttttggaat atcttgaaga aaaatatgaa gagcatttgt
     4981 atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc
     5041 ccaatcttcc ttattatatt gatggtgatg ttaaattaac acagtctatg gccatcatac
     5101 gttatatagc tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt gcagagattt
     5161 caatgcttga aggagcggtt ttggatatta gatacggtgt ttcgagaatt gcatatagta
     5221 aagactttga aactctcaaa gttgattttc ttagcaagct acctgaaatg ctgaaaatgt
     5281 tcgaagatcg tttatgtcat aaaacatatt taaatggtga tcatgtaacc catcctgact
     5341 tcatgttgta tgacgctctt gatgttgttt tatacatgga cccaatgtgc ctggatgcgt
     5401 tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat cccacaaatt gataagtact
     5461 tgaaatccag caagtatata gcatggcctt tgcagggctg gcaagccacg tttggtggtg
     5521 gcgaccatcc tccaaaatcg gatctgagca gcggtctgga agttctgttt cagggcccgg
     5581 agcgcccggg ccagtgggcc tgccctgcct gtaccctgct caacgcactg cgggccaagc
     5641 actgcgccgc ctgccacacg cctcagctcc tggtggccca gtaaagcttt ctagaccatt
     5701 taaacaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     5761 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     5821 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     5881 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5941 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     6001 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     6061 cggcacct
//
