LOCUS       sequence-472924-        6065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human CAPN15 ZF4 (337-369).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6065)
  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 6065)
  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 ZF4 (337-369)
FEATURES             Location/Qualifiers
     source          1..6065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(39..467)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(149..170)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     361..380
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(504..551)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     547..565
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(618..635)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(758..781)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(800..1453)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             complement(1454..1471)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1484..1506
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1521..1545
                     /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(1545..1564)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1546..1564)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1648..1667)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1873..1950
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1951..3033
                     /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(1970..1989)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3842..4033
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4027..4049)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4135..4274
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4190..4209
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4289..4306)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4460..5048)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4540..4559)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5159..5178)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5170..5985
                     /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             5170..5985
                     /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(5243..5262)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
       61 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
      121 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
      181 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
      241 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
      301 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
      361 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
      421 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccaat
      481 ccggatatag ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg
      541 ggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg
      601 ttagcagccg gatctcagtg gtggtggtgg tggtgtttaa atggtctaga aagctttagt
      661 gcagtttgga gcagccgcag gccgagcacc tgggggccac tgtggggttt ctgagcgtgc
      721 acttggcaca tgaccaggtg gtgaagtcgg ggctggacgg gccctgaaac agaacttcca
      781 gaccgctgct cagatccgat tttggaggat ggtcgccacc accaaacgtg gcttgccagc
      841 cctgcaaagg ccatgctata tacttgctgg atttcaagta cttatcaatt tgtgggatag
      901 cttcaatacg ttttttaaaa caaactaatt ttgggaacgc atccaggcac attgggtcca
      961 tgtataaaac aacatcaaga gcgtcataca acatgaagtc aggatgggtt acatgatcac
     1021 catttaaata tgttttatga cataaacgat cttcgaacat tttcagcatt tcaggtagct
     1081 tgctaagaaa atcaactttg agagtttcaa agtctttact atatgcaatt ctcgaaacac
     1141 cgtatctaat atccaaaacc gctccttcaa gcattgaaat ctctgcacgc tcttttggac
     1201 aaccacccaa catgttgtgc ttgtcagcta tataacgtat gatggccata gactgtgtta
     1261 atttaacatc accatcaata taataaggaa gattgggaaa ctccaaaccc aattcaaact
     1321 ttttgtttcg ccatttatca ccttcatcgc gctcatacaa atgctcttca tatttttctt
     1381 caagatattc caaaagaagt cgagtgggtt gcacaaggcc cttaattttc caataaccta
     1441 gtatagggga catgtgatgg tgatggtgat gtgccatggt atatctcctt cttaaagtta
     1501 aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt
     1561 attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca
     1621 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     1681 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     1741 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     1801 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     1861 gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     1921 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     1981 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     2041 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     2101 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     2161 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     2221 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     2281 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     2341 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     2401 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     2461 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     2521 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     2581 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     2641 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     2701 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     2761 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     2821 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     2881 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     2941 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     3001 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     3061 cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc
     3121 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     3181 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     3241 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     3301 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     3361 cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
     3421 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
     3481 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
     3541 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
     3601 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     3661 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     3721 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     3781 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     3841 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     3901 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     3961 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     4021 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     4081 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     4141 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     4201 cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa
     4261 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4321 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4381 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4441 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4501 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4561 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4621 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4681 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4741 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4801 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4861 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     4921 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4981 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5041 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5101 cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa cagtaataca
     5161 aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg attaaattcc
     5221 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     5281 gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     5341 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     5401 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     5461 accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt
     5521 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     5581 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     5641 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     5701 gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt
     5761 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     5821 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     5881 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     5941 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt aattcatgag
     6001 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6061 ccgaa
//
