LOCUS       sequence-190008-        6704 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  caspase-8 BRET Sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6704)
  AUTHORS   den Hamer A, Dierickx P, Arts R, de Vries JSPM, Brunsveld L, Merkx M
  TITLE     Bright Bioluminescent BRET Sensor Proteins for Measuring 
            Intracellular Caspase Activity.
  JOURNAL   ACS Sens. 2017 Jun 23;2(6):729-734. doi: 10.1021/acssensors.7b00239.
            Epub 2017 May 31.
  PUBMED    28670623
REFERENCE   2  (bases 1 to 6704)
  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: 
            "10.1021/acssensors.7b00239"; journalName: "ACS Sens"; date: 
            "2017-06-23- 23"; volume: "2"; issue: "6"; pages: "729-734"
COMMENT     Sequence Label: mNeonGreen-IETD-NanoLuc
FEATURES             Location/Qualifiers
     source          1..6704
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      683..692
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             701..718
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             728..745
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      1004..1013
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2061..2078)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2067..2291
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2337..2765
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2424..2443)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2634..2655
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2774..2794)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2779..3108
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2959..3094
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3021..3040
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3175..3969
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3229..3248)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3839..3858
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4143..4264
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4180..4199)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4234..4253
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4313..4329)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4313..4329)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4326..4348)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4337..4353
                     /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(4361..4367,4368..4385,4386..4391))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4406..4427
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4544..4561)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4715..5303)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4795..4814)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5474..6265,6266..6334))
                     /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     6097..6116
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6335..6439)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6514..6533)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaagc ttgccaccat gggaagttca catcatcatc atcatcactc
      721 atcaggactg gtgccacggg ggtctcacat ggtctccaag ggggaagagg acaacatggc
      781 ctctctgcct gccacacacg agctgcacat cttcggcagc atcaatggcg tggactttga
      841 tatggtgggc cagggcacag gcaacccaaa tgacggctac gaggagctga acctgaagag
      901 caccaagggc gatctgcagt tctccccttg gatcctggtg ccacacatcg gctatggctt
      961 tcaccagtat ctgccctacc ctgacggcat gtcccccttc caggccgcca tggtggatgg
     1021 ctctggctac caggtgcaca ggaccatgca gtttgaggac ggcgcctccc tgacagtgaa
     1081 ttaccggtat acctacgagg gctctcacat caagggcgag gcccaggtga agggcacagg
     1141 cttcccagcc gatggccccg tgatgacaaa ctctctgacc gccgccgact ggtgccggag
     1201 caagaagacc taccctaatg ataagacaat catctccacc tttaagtggt cttataccac
     1261 aggcaacggc aagcggtaca gaagcacagc cagaaccaca tatacctttg ccaagccaat
     1321 ggccgccaac tatctgaaga atcagcccat gtacgtgttc aggaagacag agctgaagca
     1381 ctccaagacc gagctgaatt tcaaggagtg gcagaaggcc tttacagacg tgatgggcat
     1441 ggatgagctg tacaaggagt ttggcaccga gaacctgtat atagagaccg acggctctgg
     1501 cggcagcatg gtgttcacac tggaggactt tgtgggcgat tggcggcaga ccgccggcta
     1561 taatctggat caggtgctgg agcagggcgg cgtgagctcc ctgttccaga acctgggcgt
     1621 gagcgtgacc cctatccagc ggatcgtgct gtccggcgag aacggcctga agatcgacat
     1681 ccacgtgatc atcccatacg agggcctgtc tggcgatcag atgggccaga tcgagaagat
     1741 cttcaaggtg gtgtaccccg tggacgatca ccacttcaaa gtgatcctgc actatggcac
     1801 cctggtcatc gacggcgtga cccccaatat gatcgattat ttcggccggc cttacgaggg
     1861 catcgccgtg tttgacggca agaagatcac cgtgacaggc accctgtgga acggcaataa
     1921 gatcatcgac gagagactga tcaaccccga tggctccctg ctgttcaggg tgactattaa
     1981 cggagtgact ggctggcggc tgtgcgaaag gattctggct tgactcgagt ctagagggcc
     2041 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     2101 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     2161 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     2221 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     2281 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     2341 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2401 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2461 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2521 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2581 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2641 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2701 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2761 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2821 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2881 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2941 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3001 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     3061 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     3121 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     3181 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     3241 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     3301 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     3361 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     3421 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     3481 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     3541 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3601 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3661 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     3721 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     3781 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     3841 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     3901 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     3961 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     4021 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4081 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4141 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4201 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4261 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4321 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4381 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4441 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4501 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4561 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4621 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4681 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4741 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4801 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4861 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4921 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4981 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5041 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5101 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5161 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5221 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5281 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5341 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5401 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5461 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5521 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5581 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5641 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5701 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5761 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5821 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5881 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5941 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6001 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6061 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6121 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6181 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6241 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6301 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6361 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6421 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     6481 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     6541 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     6601 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     6661 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgtt
//
