LOCUS       sequence-378986-        7465 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression plasmid encoding full-length CAPN7 with L61D 
            mutation. Contains C-terminal OSF tag (One-Strep-FLAG).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7465)
  AUTHORS   Paine EL, Skalicky JJ, Whitby FG, Mackay DR, Ullman KS, Hill CP, 
            Sundquist WI
  TITLE     The Calpain-7 protease functions together with the ESCRT-III protein
            IST1 within the midbody to regulate the timing and completion of 
            abscission.
  JOURNAL   Elife. 2023 Sep 29;12:e84515. doi: 10.7554/eLife.84515.
  PUBMED    37772788
REFERENCE   2  (bases 1 to 7465)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2023-09-29"; volume: "12:e84515. doi"; pages: " 
            10.7554/eLife.84515"
COMMENT     Sequence Label: pCAG CAPN7 L61D-OSF
FEATURES             Location/Qualifiers
     source          1..7465
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..663
                     /label=chicken beta-actin promoter
     intron          664..1680
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1604..1626
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1688..1707
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1750..1759
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4225..4248
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4255..4278
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             4315..4338
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             4354..4383
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4399..4416
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4460..4477)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4487..4506)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4552..4607
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4606..4625)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4968..4984)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4968..4984)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4981..5003)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4992..5008
                     /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)."
     promoter        complement(join(5016..5022,5023..5040,5041..5046))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5061..5082
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5141..5336
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5187..5322
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5342..5476
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5379..5398)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5433..5452
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5544..5561)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5715..6303)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5795..5814)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6474..7265,7266..7334))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7097..7116
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7335..7439)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggcgc gcgccrggsg gggsggggsg gggsgrgggg
      541 sggggsgggg cgrggcggrg aggkgcggcg gcagccaatc agagcggcgc gctccgaaag
      601 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
      661 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      721 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      781 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
      841 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
      961 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     1021 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
     1081 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     1141 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     1201 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
     1261 gcggggccgc ctcgggccgg ggrgggctcg ggggrggggc gcggcggccc ccggagcgcc
     1321 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     1381 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     1441 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     1501 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     1561 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     1621 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     1681 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attatcgatc
     1741 cggaggtacg ccaccatgga cgccacagca ctggagcggg acgctgtgca gttcgcccgt
     1801 ctggcggttc agcgcgacca cgaaggccgc tactccgagg cggtgtttta ttacaaggaa
     1861 gctgcacaag ccttaattta tgctgagatg gcaggatcaa gcctagaaaa tattcaagaa
     1921 aaaataactg agtatgatga aagagttcaa gctctacatt cagcagttca gtcaaagagt
     1981 gctgatcctt tgaagtcaaa acatcagttg gacttagagc gtgctcattt ccttgttaca
     2041 caagcttttg atgaagatga aaaagagaat gttgaagatg ctatagaatt gtacacagaa
     2101 gctgtggatc tctgtctgaa aacatcttat gaaactgctg ataaagtcct gcaaaataaa
     2161 ctgaaacagt tggctcgaca ggcgctagac agagcagaag cgctgagtga gcctttgacc
     2221 aagccagttg gcaaaatcag ttcaacaagt gttaagccaa agccacctcc agtgagagca
     2281 cattttccac tgggcgctaa tcccttcctt gaaagacctc agtcatttat aagtcctcag
     2341 tcatgtgatg cacaaggaca gagatacaca gcagaagaaa tagaagtact caggacaaca
     2401 tcaaaaataa atggtataga atatgttcct ttcatgaatg ttgacctgag agaacgtttt
     2461 gcctatccaa tgcctttctg tgatagatgg ggcaagctac cattatcacc taaacaaaaa
     2521 actacatttt ccaagtgggt acgaccagaa gacctcacca acaatcctac aatgatatat
     2581 actgtgtcca gttttagcat aaagcagaca atagtatcgg attgctcctt tgtggcatca
     2641 ctggccatca gtgcagctta tgaaagacgt tttaataaga agttaattac cggcataatt
     2701 taccctcaaa acaaggatgg tgaaccagaa tacaatccat gtgggaagta tatggtaaaa
     2761 cttcacctca atggtgtccc aagaaaggtg ataattgatg accagttacc tgttgatcac
     2821 aagggagaat tgctctgttc ttattccaac aacaaaagtg aattatgggt ttctctcata
     2881 gaaaaagcat acatgaaagt catgggagga tatgattttc caggatccaa ctccaatatt
     2941 gatcttcatg cactgactgg ctggatacca gaaagaattg ctatgcattc agatagccaa
     3001 actttcagta aggataattc tttcagaatg ctttatcaaa gatttcacaa aggagatgtc
     3061 ctcatcactg cgtcaactgg aatgatgaca gaagctgaag gagagaagtg gggtctggtt
     3121 cccacacacg catatgctgt tttggatatt agagagttca aggggctgcg atttatccag
     3181 ttgaaaaatc cttggagtca tttacgttgg aaaggaagat acagtgaaaa tgatgtaaaa
     3241 aactggactc cagagttgca aaagtattta aactttgatc cccgaacagc tcagaaaata
     3301 gacaacggaa tattttggat ttcctgggat gatctctgcc agtattatga tgtgatttat
     3361 ttgagttgga atccaggtct ttttaaagaa tcaacatgta ttcacagtac ttgggatgct
     3421 aagcaaggac ctgtgaaaga tgcctatagc ctggccaaca acccccagta caaactggag
     3481 gtgcagtgtc cacagggggg tgctgcagtt tgggttttgc ttagtagaca cataacagac
     3541 aaggatgatt ttgcgaataa tcgagaattt atcacaatgg ttgtatacaa gactgatggg
     3601 aaaaaagttt attacccagc tgacccacct ccatacattg atggaattcg aattaacagc
     3661 cctcattatt tgactaagat aaagctgacc acacctggca cccatacctt tacattagtg
     3721 gtttctcaat atgaaaaaca gaacacaatc cattacacgg ttcgggtata ttcagcatgc
     3781 agctttactt tttcaaagat tccttcacca tacaccttat caaaacggat taatggaaag
     3841 tggagtggtc agagtgctgg aggatgtgga aatttccaag agactcacaa aaataacccc
     3901 atctaccaat tccatataga aaagactggg ccgttactga ttgagctacg aggaccaagg
     3961 caatatagcg ttggatttga ggttgtaaca gtttctactc taggagatcc tggtccccat
     4021 ggctttctga ggaaatctag tggtgactat aggtgtgggt tttgctacct ggaattagaa
     4081 aatatacctt ctgggatctt caatatcatt cctagtacct ttttgcctaa acaagaagga
     4141 ccttttttct tggactttaa tagtattatc cccatcaaga tcacacaact tcagctcgag
     4201 aagcttgcta gcagatctgg attcgattac aaggatgacg atgacaagag cgcttggagc
     4261 cacccgcagt tcgagaaagg tggaggttcc ggaggtggat cgggaggtgg atcgtggagc
     4321 cacccgcagt tcgaaaaata aaagcttggg cccgaacaaa aactcatctc agaagaggat
     4381 ctgaatagcg ccgtcgacca tcatcatcat catcattgag tttaaacggt ctccagctta
     4441 agtttaaacc gctgatcagc ctcgactgtg ccttctagtt gccggatctt tttccctctg
     4501 ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga
     4561 aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat
     4621 atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat
     4681 atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga
     4741 aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt
     4801 tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg
     4861 ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc
     4921 tcttatggag atccctcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt
     4981 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5041 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5101 cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa
     5161 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     5221 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     5281 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     5341 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5401 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5461 ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5521 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5581 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5641 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5701 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5761 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5821 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5881 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5941 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6001 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6061 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6121 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6181 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6241 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6301 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6361 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6421 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6481 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6541 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6601 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6661 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6721 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6781 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6841 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6901 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6961 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7021 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7081 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7141 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7201 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7261 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7321 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7381 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7441 acatttcccc gaaaagtgcc acctg
//
