LOCUS       sequence-472922-        6485 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human CAPN15 (N)ZF1-3 (1-173).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6485)
  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 6485)
  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 (N)ZF1-3 (1-173)
FEATURES             Location/Qualifiers
     source          1..6485
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..108
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     192..211
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        192..210
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    211..235
                     /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             250..272
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             285..302
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             303..956
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             975..998
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1541..1558
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1611..1629)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1625..1672
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1709..2164
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1796..1815)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2006..2027
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2256..3071)
                     /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(2256..3071)
                     /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     2979..2998
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3063..3082
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3193..3781
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3682..3701
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3935..3952
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3967..4106
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4032..4051)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4192..4214
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4208..4399)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5208..6290)
                     /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     6252..6271
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6291..6368)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
       61 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
      121 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
      181 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
      241 agaaataatt ttgtttaact ttaagaagga gatataccat ggcacatcac catcaccatc
      301 acatgtcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc actcgacttc
      361 ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat gaaggtgata
      421 aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct tattatattg
      481 atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct gacaagcaca
      541 acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa ggagcggttt
      601 tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa actctcaaag
      661 ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt ttatgtcata
      721 aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat gacgctcttg
      781 atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta gtttgtttta
      841 aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc aagtatatag
      901 catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct ccaaaatcgg
      961 atctgagcag cggtctggaa gttctgtttc agggcccgat ggccacggtc ggagagtggt
     1021 cctgtgtgcg ctgcaccttc ctgaacccgg ccggccagcg ccagtgctcc atctgcgagg
     1081 ctccccggca caagcccgac ctcaaccaca tcctgcggct cagcgtggag gagcagaaat
     1141 ggccctgcgc ccgctgcacc ttccgcaact tcctgggcaa ggaggcctgc gaggtgtgcg
     1201 gcttcacccc ggagcctgcg cctggggctg ccttcctgcc agtcctcaac ggggtcctcc
     1261 ccaagccacc cgccatcctg ggggagccca agggcagctg ccaggaggaa gcaggtccag
     1321 tgaggactgc ggggctggtg gccacggagc ccgccagggg gcagtgcgag gacaaggacg
     1381 aggaggagaa ggaggagcag gaggaggagg agggagcggc ggagcccaga gggggctggg
     1441 cgtgtccgcg ttgcacgctg cacaacacgc ccgtggccag ctcctgctcc gtctgcgggg
     1501 gcccacgcag gctctcgtaa agctttctag accatttaaa caccaccacc accaccactg
     1561 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     1621 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     1681 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     1741 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1801 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1861 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1921 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1981 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2041 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2101 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     2161 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2221 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     2281 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2341 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2401 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2461 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     2521 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     2581 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     2641 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     2701 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     2761 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     2821 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     2881 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     2941 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     3001 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     3061 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     3121 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3181 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3241 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3301 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3361 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3421 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3481 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3541 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3601 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3661 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3721 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3781 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3841 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3901 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3961 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     4021 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     4081 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4141 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4201 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     4261 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     4321 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     4381 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     4441 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     4501 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     4561 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     4621 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     4681 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     4741 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     4801 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     4861 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     4921 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     4981 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     5041 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     5101 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     5161 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     5221 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5281 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     5341 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     5401 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     5461 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     5521 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     5581 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     5641 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     5701 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     5761 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     5821 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     5881 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     5941 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     6001 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     6061 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     6121 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     6181 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     6241 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     6301 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     6361 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     6421 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     6481 gccca
//
