LOCUS       sequence-203167-        6520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of light-activated caspase-3 with N7C-7 linker 
            variation.
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
FEATURES             Location/Qualifiers
     source          1..6520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     110..129
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     213..232
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        213..231
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    232..256
                     /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             271..293
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             312..329
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             330..350
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1596..1613
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1666..1684)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1680..1727
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1764..2219
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1851..1870)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2061..2082
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2312..3127)
                     /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(2312..3127)
                     /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     3035..3054
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3119..3138
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3249..3837
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3738..3757
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3991..4008
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4023..4162
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4088..4107)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4248..4270
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4264..4455)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5264..6346)
                     /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
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6308..6327
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6347..6424)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
       61 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
      121 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
      181 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
      241 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
      301 tgggttcttc tcaccatcac catcaccatg aaaacctgta cttccaatcc aatgcatctg
      361 gaatatccct ggacaacagt tataaaatgg attatcctga gatgggttta tgtataataa
      421 ttaataataa gaattttcat aaaagcactg gaatgacatc tcggtctggt acagatgtcg
      481 atgcagcaaa cctcagggaa acattcagaa acttgaaata tgaagtcagg aataaaaatg
      541 atcttacacg tgaagaaatt gtggaattga tgcgtgatgt ttctaaagaa gatcacagca
      601 aaaggagcag ttttgtttgt gtgcttctga gccatggtga agaaggaata atttttggaa
      661 caaatggacc tgttgacctg aaaaaaataa caaacttttt cagaggggat cgttgtagaa
      721 gtctaactgg aaaacccaaa cttttcatta ttcaggcctg ccgtggtaca gaactggact
      781 gtggcattga gacaaacggc ggtagcggca gtggcggcac caccctggaa cgcatcgaga
      841 agaacttcgt catcaccgat ccccgcctgc ccgataatcc catcatcttc gccagcgata
      901 gcttcttgca actgacagag tacagccgcg aggagatcct gggccgcaac tgccgcttcc
      961 tccagggacc cgagaccgat cgcgccaccg tgcgcaagat ccgcgatgcc atcgataacc
     1021 agaccgaggt gaccgtgcag ctgattaact acaccaagag cggcaaaaag ttttggaacc
     1081 tgttccacct gcagcccatg cgcgaccaga agggcgatgt gcagtacttc atcggcgtac
     1141 aactggatgg caccgaacac gtgcgggatg ccgcggagcg cgaggccgtg atgctgatca
     1201 agaagaccgc cgaggaaatc gacgaggccg ccaaggagct ggcgtgtcat aaaataccag
     1261 tggaggccga cttcttgtat gcatactcca cagcacctgg ttattattct tggcgaaatt
     1321 caaaggatgg ctcctggttc atccagtcgc tttgtgccat gctgaaacag tatgccgaca
     1381 agcttgaatt tatgcacatt cttacccggg ttaaccgaaa ggtggcaaca gaatttgagt
     1441 ccttttcctt tgacgctact tttcatgcaa agaaacagat tccatgtatt gtttccatgc
     1501 tcacaaaaga actctatttt tatcactaat aacattggaa gtggataacg gatccgaatt
     1561 cgagcgccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc
     1621 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     1681 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1741 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1801 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1861 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1921 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1981 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2041 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2101 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2161 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa ctagtaacgt ttacaatttc
     2221 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2281 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     2341 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     2401 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     2461 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     2521 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     2581 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     2641 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     2701 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     2761 catcaacaat gttttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     2821 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     2881 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     2941 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3001 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3061 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3121 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3181 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3241 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3301 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3361 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3421 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3481 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3541 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3601 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3661 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3721 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3781 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3841 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3901 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3961 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4021 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4081 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4141 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4201 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4261 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4321 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4381 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4441 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4501 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4561 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4621 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4681 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4741 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4801 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4861 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4921 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     4981 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5041 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5101 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5161 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5221 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5281 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5341 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     5401 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     5461 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     5521 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccgaac tcggtaatgg
     5581 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatac
     5641 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     5701 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     5761 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     5821 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     5881 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     5941 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     6001 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     6061 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     6121 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     6181 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     6241 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     6301 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     6361 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     6421 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     6481 aggttgaggc cgttgagcac cgccgccgca aggaatggtg
//
