LOCUS       sequence-76294-e        6913 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a tandem red ddFP heterodimer in mammalian cells, 
            construct 1 for a red to green colour switch-based fluorescent 
            caspase-3 biosensor, used together with a second plasmid GANLS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6913)
  AUTHORS   Ding Y, Li J, Enterina JR, Shen Y, Zhang I, Tewson PH, Mo GC, Zhang 
            J, Quinn AM, Hughes TE, Maysinger D, Alford SC, Zhang Y, Campbell RE
  TITLE     Ratiometric biosensors based on dimerization-dependent fluorescent 
            protein exchange.
  JOURNAL   Nat Methods. 2015 Jan 26. doi: 10.1038/nmeth.3261.
  PUBMED    25622108
REFERENCE   2  (bases 1 to 6913)
  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: "Nat 
            Methods. 2015 Jan 26. doi: 10.1038/nmeth.3261."
COMMENT     Sequence Label: NESRA-DEVD-BNLS
FEATURES             Location/Qualifiers
     source          1..6913
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      906..915
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2412..2432
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2436..2456
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2460..2480
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2507..2524)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2513..2737
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2783..3211
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2870..2889)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3080..3101
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3220..3240)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3225..3554
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3405..3540
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3467..3486
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3621..4415
                     /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(3675..3694)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4285..4304
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4589..4710
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4626..4645)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4680..4699
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4759..4775)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4759..4775)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4772..4794)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4783..4799
                     /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(4807..4813,4814..4831,4832..4837))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4852..4873
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4990..5007)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5161..5746)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5241..5260)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5917..6708,6709..6777))
                     /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     6540..6559
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6778..6882)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtgccgccac catggcctcg agaatgaacc tggtggacct ccaaaagaag ctggaggagc
      961 tggagctgga cgagcagcag atggtgagca agagcgagga ggtcatcaaa gagttcatgc
     1021 gcttcaaggt gcgcttggag ggctccatga acggccacga gttcgagatc gagggcgagg
     1081 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc
     1141 ccctgccctt cgcctgggac atcctgtccc cccagatcat gtacggctcc aaggcgtacg
     1201 tgaagcaccc cgccgacgtc cccgattaca tgaagctgtc cttccccgag ggcttcaagt
     1261 gggagcgcgt gatgcacttc gaggacggcg gtctggtgac cgtgactcag gacacctccc
     1321 tgcaggacgg cacgctgatc tacaaggtga agatgcgcgg caccaacttc ccccccgacg
     1381 gccccgtaat gcagaagaag accttgggct gggattatgc caccgagcgc ctgtaccccg
     1441 aagacggcgt gctgaagggc gagcttctgg ggcgcctgaa gctgaaggac ggcggcctca
     1501 acctggtgga gttcaagacc atctacatgg ccaagaagcc cgtgcaactg cccggctact
     1561 acttcgtgga caccaagctg ggcatcacct cccacaacga ggactacacc atcgtggaac
     1621 agtacgagcg ctccgagggc cgccaccacc tgggcatgga cgagctgtac aagggaacgg
     1681 cgggaagcgg taccgcctcc ggcgatgagg tggatggagc caccatcaaa gagttcatgc
     1741 gcttcaaggt gcgcatggag ggctccatga acggccacga gttcgagatc gagggcgagg
     1801 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc
     1861 ccctgccctt tgcctgggac atcctgtccc cccagatcat gtacggctcc aaagcgtacg
     1921 tgaggcaccc cgccgacatc cccgattaca agaagctgcc cttccccgag ggcttcaagt
     1981 gggagcgcgt gatgaacttc gaagacggcg gtctggtgac cgttacccag gactcctccc
     2041 tgcaggacgg cacgctgatc tgcaaggtga agattcgcgg caccaacttc ccccccgacg
     2101 gccccgtaat gcagaagaag accatgggct gggaggcctc caccgagatg ctgtacccca
     2161 aggacggcgt gctgaagggc catagctatc aggccctgaa gctgaaggac ggcggccact
     2221 acctggtgga gttcgagacc atctacatgg ccaagaagcc cgtgcaactg cccggcgatt
     2281 actgtgtgga caccaagctg gacatcacct cccacaacga ggactacacc atcgtggaac
     2341 agtacgggcg ctccgagggc cgccaccgtc tgggcatgga cgagcggtac aagggaacgg
     2401 cgggaagcga tccaaaaaag aagagaaagg tagatccaaa aaagaagaga aaggtagatc
     2461 caaaaaagaa gagaaaggta taaaagcttg gtaaaccgct gatcagcctc gactgtgcct
     2521 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2581 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2641 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     2701 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     2761 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2821 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2881 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2941 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3001 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3061 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3121 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3181 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     3241 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3301 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3361 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3421 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3481 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3541 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     3601 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3661 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3721 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3781 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     3841 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3901 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3961 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     4021 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4081 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4141 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4201 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4261 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4321 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4381 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     4441 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     4501 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4561 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     4621 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4681 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     4741 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4801 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4861 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4921 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4981 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5041 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5101 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5161 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5221 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5281 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5341 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5401 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5461 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5521 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5581 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     5641 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggttt
     5701 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5761 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5821 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5881 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5941 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6001 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6061 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6121 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6181 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6241 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6301 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6361 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6421 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6481 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6541 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6601 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6661 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6721 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6781 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6841 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6901 gccacctgac gtc
//
