LOCUS       sequence-472918-        6089 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human CAPN15 NZF2 (34-72).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6089)
  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 6089)
  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 NZF2 (34-72)
FEATURES             Location/Qualifiers
     source          1..6089
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1763..1954
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1948..1970)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2056..2195
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2111..2130
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2210..2227)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2381..2969)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2461..2480)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3080..3099)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3091..3906
                     /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             3091..3906
                     /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(3164..3183)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3998..4453)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4135..4156)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4347..4366
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4490..4537)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4533..4551
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4604..4621)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4768..4791)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(4810..5463)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             complement(5464..5481)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5494..5516
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5531..5555
                     /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(5555..5574)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5556..5574)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5658..5677)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5883..5960
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5961..6089,1..954)
                     /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(5980..5999)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
       61 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
      121 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
      181 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
      241 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
      301 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
      361 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
      421 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
      481 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
      541 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
      601 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
      661 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
      721 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
      781 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
      841 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
      901 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
      961 cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag
     1021 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     1081 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat
     1141 tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt
     1201 cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg
     1261 cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct
     1321 gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc
     1381 gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac
     1441 atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg
     1501 caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac
     1561 atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat
     1621 ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt
     1681 aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa
     1741 tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg
     1801 ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga
     1861 acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct
     1921 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     1981 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     2041 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     2101 cttaactatg cggcatcaga gcagattgta ctgagagtgc accattgcgg tgtgaaatac
     2161 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
     2221 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2281 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2341 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2401 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2461 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2521 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2581 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2641 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2701 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2761 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2821 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     2881 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     2941 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3001 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgaacaat aaaactgtct
     3061 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc
     3121 tctaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     3181 gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca
     3241 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     3301 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
     3361 cctgatgatg catggttact caccactgcg atccccggga aaacagcatt ccaggtatta
     3421 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     3481 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
     3541 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     3601 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     3661 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     3721 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     3781 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     3841 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     3901 ttctaagaat taattcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3961 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     4021 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4081 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     4141 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     4201 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     4261 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     4321 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     4381 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     4441 ctacagggcg cgtcccattc gccaatccgg atatagttcc tcctttcagc aaaaaacccc
     4501 tcaagacccg tttagaggcc ccaaggggtt atgctagtta ttgctcagcg gtggcagcag
     4561 ccaactcagc ttcctttcgg gctttgttag cagccggatc tcagtggtgg tggtggtggt
     4621 gtttaaatgg tctagaaagc tttacgcagg ctccggggtg aagccgcaca cctcgcaggc
     4681 ctccttgccc aggaagttgc ggaaggtgca gcgggcgcag ggccatttct gctcctccac
     4741 gctgagccgc aggatgtggt tgaggtccgg gccctgaaac agaacttcca gaccgctgct
     4801 cagatccgat tttggaggat ggtcgccacc accaaacgtg gcttgccagc cctgcaaagg
     4861 ccatgctata tacttgctgg atttcaagta cttatcaatt tgtgggatag cttcaatacg
     4921 ttttttaaaa caaactaatt ttgggaacgc atccaggcac attgggtcca tgtataaaac
     4981 aacatcaaga gcgtcataca acatgaagtc aggatgggtt acatgatcac catttaaata
     5041 tgttttatga cataaacgat cttcgaacat tttcagcatt tcaggtagct tgctaagaaa
     5101 atcaactttg agagtttcaa agtctttact atatgcaatt ctcgaaacac cgtatctaat
     5161 atccaaaacc gctccttcaa gcattgaaat ctctgcacgc tcttttggac aaccacccaa
     5221 catgttgtgc ttgtcagcta tataacgtat gatggccata gactgtgtta atttaacatc
     5281 accatcaata taataaggaa gattgggaaa ctccaaaccc aattcaaact ttttgtttcg
     5341 ccatttatca ccttcatcgc gctcatacaa atgctcttca tatttttctt caagatattc
     5401 caaaagaagt cgagtgggtt gcacaaggcc cttaattttc caataaccta gtatagggga
     5461 catgtgatgg tgatggtgat gtgccatggt atatctcctt cttaaagtta aacaaaatta
     5521 tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg
     5581 cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac
     5641 aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca
     5701 cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg
     5761 actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct
     5821 caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc
     5881 cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
     5941 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
     6001 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
     6061 cgcgggaaaa agtggaagcg gcgatggcg
//
