LOCUS       sequence-177314-        6194 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses caspase 3 D3A mutant (D175A D28A D9A) in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6194)
  AUTHORS   Bose K, Pop C, Feeney B, Clark AC
  TITLE     An uncleavable procaspase-3 mutant has a lower catalytic efficiency 
            but an active site similar to that of mature caspase-3.
  JOURNAL   Biochemistry. 2003 Oct 28;42(42):12298-310.
  PUBMED    14567691
REFERENCE   2  (bases 1 to 6194)
  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: 
            "Biochemistry."; date: "2003-10-28"; volume: "42(42)"; pages: 
            "12298-310"
COMMENT     Sequence Label: pET21b-Caspase-3(D3A: D175A D28A D9A)
FEATURES             Location/Qualifiers
     source          1..6194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             926..943
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(996..1014)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1010..1057
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1094..1549
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1181..1200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1391..1412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1575..1679
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1680..1748,1749..2540)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1898..1917)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2711..3299
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3200..3219
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3453..3470
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3485..3624
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3550..3569)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3710..3732
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3726..3917)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4726..5808)
                     /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     5770..5789
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5809..5886)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6092..6111
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat ggagaacact gaaaactcag tggctagcaa
      121 atccattaaa aatttggaac ccaagatcat acatggaagc gaatcaatgg ccagtggaat
      181 atccctggac aacagttata aaatggatta tcctgagatg ggtttatgta taataattaa
      241 taataagaat tttcataaaa gcactggaat gacatctcgg tctggtacag atgtcgatgc
      301 agcaaacctc agggaaacat tcagaaactt gaaatatgaa gtcaggaata aaaatgatct
      361 tacacgtgaa gaaattgtgg aattgatgcg tgatgtttct aaagaagatc acagcaaaag
      421 gagcagtttt gtttgtgtgc ttctgagcca tggtgaagaa ggaataattt ttggaacaaa
      481 tggacctgtt gacctgaaaa aaataacaaa ctttttcaga ggggatcgtt gtagaagtct
      541 aactggaaaa cccaaacttt tcattattca ggcctgccgt ggtacagaac tggactgtgg
      601 cattgagaca gctagcggtg ttgatgatga catggcgtgt cataaaatac cagtggaggc
      661 cgacttcttg tatgcatact ccacagcacc tggttattat tcttggcgaa attcaaagga
      721 tggctcctgg ttcatccagt cgctttgtgc catgctgaaa cagtatgccg acaagcttga
      781 atttatgcac attcttaccc gggttaaccg aaaggtggca acagaatttg agtccttttc
      841 ctttgacgct acttttcatg caaagaaaca gattccatgt attgtttcca tgctcacaaa
      901 agaactctat ttttatcacc tcgagcacca ccaccaccac cactgagatc cggctgctaa
      961 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     1021 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     1081 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1141 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1201 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1261 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1321 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1381 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1441 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1501 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     1561 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1621 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1681 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1741 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1801 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1861 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1921 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1981 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     2041 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     2101 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2161 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2221 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2281 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2341 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     2401 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2461 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2521 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2581 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2641 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2701 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2761 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2821 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2881 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2941 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3001 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3061 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3121 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3181 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3241 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3301 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3361 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3421 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3481 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     3541 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3601 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3661 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3721 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3781 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3841 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3901 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3961 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     4021 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     4081 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     4141 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     4201 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     4261 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4321 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4381 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     4441 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     4501 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     4561 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     4621 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     4681 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     4741 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4801 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     4861 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     4921 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4981 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     5041 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5101 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5161 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5221 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5281 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5341 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5401 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5461 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5521 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     5581 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     5641 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     5701 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     5761 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     5821 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     5881 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     5941 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     6001 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     6061 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     6121 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     6181 cgatcccgcg aaat
//
