LOCUS       sequence-315898-        7290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of CAPN2_EF-hand.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7290)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
REFERENCE   2  (bases 1 to 7290)
  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: "Mol Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_CAPN2_EF-hand
FEATURES             Location/Qualifiers
     source          1..7290
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      1485..1532
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3125..3580)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3262..3283)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3474..3493
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3622..3644)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3730..3870
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3785..3804
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3885..3902)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4056..4643)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4814..5605,5606..5674))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5437..5456
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5675..5779)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5847..5865
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5888..5965
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             5966..7048
                     /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(5985..6004)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggccgatga cgaaattgaa gcaaacctgg aagaatttga tatttccgaa gatgacatcg
      961 atgacggctt tcgtcgcctg ttcgcgcagc tggccggtga agatgcggaa attagcgcct
     1021 ttgaactgca gaccatcctg cgtcgcgttc tggccaagcg tcaagatatt aaaagtgacg
     1081 gcttctccat tgaaacgtgc aaaatcatgg tcgatatgct ggatagcgac ggctctggca
     1141 agctgggtct gaaagaattc tatattctgt ggaccaagat ccagaagtac caaaagatct
     1201 accgtgaaat cgatgtggac cgctcaggca cgatgaattc gtacgaaatg cgtaaggcac
     1261 tggaagaagc tggttttaaa atgccgtgcc agctgcatca agtgattgtt gcacgcttcg
     1321 ctgatgacca gctgattatc gattttgaca acttcgtccg ttgtctggtg cgcctggaaa
     1381 ccctgtttaa gatcttcaaa caactggatc cggaaaatac cggcacgatt gaactggatc
     1441 tgatcagctg gctgtgtttt tctgttctgg cggccgcata ataaaccgat acaattaaag
     1501 gctccttttg gagccttttt tttggagatt ttcaacgtga aaaaattatt attcgcaatt
     1561 cctttagttg ttcctttcta ttctcactcc gctgaaactg ttgaaagttg tttagcaaaa
     1621 ccccatacag aaaattcatt tactaacgtc tggaaagacg acaaaacttt agatcgttac
     1681 gctaactatg agggctgtct gtggaatgct acaggcgttg tagtttgtac tggtgacgaa
     1741 actcagtgtc tagctagagt ggcggtggct ctggttccgg tgattttgat tatgaaaaga
     1801 tggcaaacgc taataagggg gctatgaccg aaaatgccga tgaaaacgcg ctacagtctg
     1861 acgctaaagg caaacttgat tctgtcgcta ctgattacgg tgctgctatc gatggtttca
     1921 ttggtgacgt ttccggcctt gctaatggta atggtgctac tggtgatttt gctggctcta
     1981 attcccaaat ggctcaagtc ggtgacggtg ataattcacc tttaatgaat aatttccgtc
     2041 aatatttacc ttccctccct caatcggttg aatgtcgccc ttttgtcttt ggcgctggta
     2101 aaccatatga attttctatt gattgtgaca aaataaactt attccgtggt gtctttgcgt
     2161 ttcttttata tgttgccacc tttatgtatg tattttctac gtttgctaac atactgcgta
     2221 ataaggagtc ttaatcatgc cagttctttt ggctagcgcc gccctatacc ttgtctgcct
     2281 ccccgcgttg cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg
     2341 cacctcgcta acggattcac cactccaaga attggagcca atcaattctt gcggagaact
     2401 gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg cgtccgccat ctccagcagc
     2461 cgcacgcggc gcatctcggg cagcgttggg tcctggccac gggtgcgcat gatcgtgctc
     2521 ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca
     2581 ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca
     2641 acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc
     2701 tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct
     2761 acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc
     2821 atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca
     2881 gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga
     2941 aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc
     3001 cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat
     3061 gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcaggatc
     3121 cgcgaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     3181 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     3241 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     3301 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     3361 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     3421 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     3481 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     3541 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cgcggatcct gcctcgcgcg
     3601 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     3661 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     3721 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
     3781 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     3841 agatgcgtaa gaagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     3901 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3961 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4021 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4081 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4141 taccagggtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4201 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4261 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4321 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4381 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4441 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     4501 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4561 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4621 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4681 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4741 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4801 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4861 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4921 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4981 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5041 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5101 atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg
     5161 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5221 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5281 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5341 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5401 ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa
     5461 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5521 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5581 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5641 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5701 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5761 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     5821 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag
     5881 aattcccgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga
     5941 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     6001 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     6061 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     6121 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     6181 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     6241 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     6301 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     6361 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     6421 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     6481 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     6541 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     6601 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     6661 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     6721 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     6781 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     6841 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     6901 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     6961 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     7021 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     7081 attaggcaca attctcatgt ttgacagctt atcatcgact gcacggtgca ccaatgcttc
     7141 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
     7201 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
     7261 cggttctggc aaatattctg aaatgagctg
//
