LOCUS       sequence-213470-        6520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of light-activated caspase-3 with N7C-7 linker 
            variation on C450A mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6520)
  AUTHORS   Smart AD, Pache RA, Thomsen ND, Kortemme T, Davis GW, Wells JA
  TITLE     Engineering a light-activated caspase-3 for precise ablation of 
            neurons in vivo.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Sep 26;114(39):E8174-E8183. doi: 
            10.1073/pnas.1705064114. Epub 2017 Sep 11.
  PUBMED    28893998
REFERENCE   2  (bases 1 to 6520)
  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"; doi: 
            "10.1073/pnas.1705064114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-09-26- 26"; volume: "114"; issue: "39"; pages: 
            "E8174-E8183"
COMMENT     Sequence Label: LIC1B_Caspase-LOVN7C-7 C450A
FEATURES             Location/Qualifiers
     source          1..6520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             326..346
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1592..1609
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1662..1680)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1676..1723
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1760..2215
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1847..1866)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2057..2078
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2308..3123)
                     /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(2308..3123)
                     /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     3031..3050
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3115..3134
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3245..3833
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3734..3753
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3987..4004
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4019..4158
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4084..4103)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4244..4266
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4260..4451)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5260..6342)
                     /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
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITEFGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARPVSRLESGQ"
     primer_bind     6304..6323
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6343..6420)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 ttcttctcac catcaccatc accatgaaaa cctgtacttc caatccaatg catctggaat
      361 atccctggac aacagttata aaatggatta tcctgagatg ggtttatgta taataattaa
      421 taataagaat tttcataaaa gcactggaat gacatctcgg tctggtacag atgtcgatgc
      481 agcaaacctc agggaaacat tcagaaactt gaaatatgaa gtcaggaata aaaatgatct
      541 tacacgtgaa gaaattgtgg aattgatgcg tgatgtttct aaagaagatc acagcaaaag
      601 gagcagtttt gtttgtgtgc ttctgagcca tggtgaagaa ggaataattt ttggaacaaa
      661 tggacctgtt gacctgaaaa aaataacaaa ctttttcaga ggggatcgtt gtagaagtct
      721 aactggaaaa cccaaacttt tcattattca ggcctgccgt ggtacagaac tggactgtgg
      781 cattgagaca aacggcggta gcggcagtgg cggcaccacc ctggaacgca tcgagaagaa
      841 cttcgtcatc accgatcccc gcctgcccga taatcccatc atcttcgcca gcgatagctt
      901 cttgcaactg acagagtaca gccgcgagga gatcctgggc cgcaacgccc gcttcctcca
      961 gggacccgag accgatcgcg ccaccgtgcg caagatccgc gatgccatcg ataaccagac
     1021 cgaggtgacc gtgcagctga ttaactacac caagagcggc aaaaagtttt ggaacctgtt
     1081 ccacctgcag cccatgcgcg accagaaggg cgatgtgcag tacttcatcg gcgtacaact
     1141 ggatggcacc gaacacgtgc gggatgccgc ggagcgcgag gccgtgatgc tgatcaagaa
     1201 gaccgccgag gaaatcgacg aggccgccaa ggagctggcg tgtcataaaa taccagtgga
     1261 ggccgacttc ttgtatgcat actccacagc acctggttat tattcttggc gaaattcaaa
     1321 ggatggctcc tggttcatcc agtcgctttg tgccatgctg aaacagtatg ccgacaagct
     1381 tgaatttatg cacattctta cccgggttaa ccgaaaggtg gcaacagaat ttgagtcctt
     1441 ttcctttgac gctacttttc atgcaaagaa acagattcca tgtattgttt ccatgctcac
     1501 aaaagaactc tatttttatc actaataaca ttggaagtgg ataacggatc cgaattcgag
     1561 cgccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc
     1621 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1681 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1741 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     1801 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1861 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     1921 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     1981 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2041 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2101 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     2161 ctgatttaac aaaaatttaa cgcgaatttt aacaaactag taacgtttac aatttcaggt
     2221 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2281 aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg
     2341 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     2401 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     2461 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     2521 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
     2581 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     2641 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     2701 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     2761 aacaatgttt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     2821 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     2881 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     2941 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     3001 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     3061 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct
     3121 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa
     3181 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3241 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3301 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3361 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3421 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3481 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3541 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3601 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3661 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3721 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3781 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3841 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3901 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3961 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4021 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     4081 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     4141 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4201 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4261 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4321 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4381 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4441 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4501 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4561 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4621 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4681 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4741 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4801 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4861 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     4921 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     4981 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     5041 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     5101 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     5161 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     5221 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5281 ggaaaccggt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5341 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     5401 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     5461 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     5521 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccgaactcgg taatggcgcg
     5581 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgataccctc
     5641 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     5701 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     5761 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     5821 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     5881 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     5941 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     6001 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     6061 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     6121 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     6181 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     6241 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     6301 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     6361 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     6421 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     6481 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
//
