LOCUS       sequence-76290-e        6875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a tandem green ddFP heterodimer in mammalian cells, 
            construct 1 for a green to red colour switch-based fluorescent 
            caspase-3 biosensor, used together with a second plasmid RANLS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6875)
  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 6875)
  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: GANES-DEVD-BNLS
FEATURES             Location/Qualifiers
     source          1..6875
                     /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             2376..2396
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2400..2420
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2424..2444
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2469..2486)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2475..2699
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2745..3173
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2832..2851)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3042..3063
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3182..3202)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3187..3516
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3367..3502
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3429..3448
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3583..4377
                     /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(3637..3656)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4247..4266
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4551..4672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4588..4607)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4642..4661
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4721..4737)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4721..4737)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4734..4756)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4745..4761
                     /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(4769..4775,4776..4793,4794..4799))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4814..4835
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4952..4969)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5123..5708)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5203..5222)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5879..6670,6671..6739))
                     /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     6502..6521
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6740..6844)
                     /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 agaatggtga gcaagagcga ggaggtcatc aaagagttca
      961 tgcgcttcaa ggtgcgcttg gagggctcca tgaacggcca cgagttcgag atcgagggcg
     1021 agggcgaggg ccgcccttac gagggcaccc agaccgccaa gctgaaggtg accaagggcg
     1081 gccccctgcc cttcgcctgg gacatcctgt cccccctgat catgtacggc tccaagatgt
     1141 acgtgaagca ccccgccgac gtccccgatt acatgaagct gtccttcccc gagggcttca
     1201 agtgggagcg cgtgatgcac ttcgaggacg gcggtctggt gaccgcgaca caggactcct
     1261 ccctgcagga cggcacgctg atctacaagg tgaagatgcg cggcaccaac ttcccccccg
     1321 acggccccgt aatgcagaag aagaccttgg gctgggatta tgccaccgag cgcctgtacc
     1381 ccgaagaagg cgtgctgaag ggcgagctcc tggggcgcct gaagctgaag gacggcggcc
     1441 tcaacctggt ggagtccaag accatctaca tggccaagaa gcccgtgcaa ctgcccggct
     1501 actacttcgt ggacaccaag ctggacatca cctcccacaa cgaggactac accatcgtgg
     1561 aacagtacga gcgctccgag ggccgccacc acctggggca tggcaccggc agcaccttag
     1621 ccttgaaatt agcaggtctt gatatcggga gcggtaccgc ctccggcgat gaggtggatg
     1681 gagccaccat caaagagttc atgcgcttca aggtgcgcat ggagggctcc atgaacggcc
     1741 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     1801 agctgaaggt gaccaagggc ggccccctgc cctttgcctg ggacatcctg tccccccaga
     1861 tcatgtacgg ctccaaagcg tacgtgaggc accccgccga catccccgat tacaagaagc
     1921 tgcccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaagac ggcggtctgg
     1981 tgaccgttac ccaggactcc tccctgcagg acggcacgct gatctgcaag gtgaagattc
     2041 gcggcaccaa cttccccccc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     2101 cctccaccga gatgctgtac cccaaggacg gcgtgctgaa gggccatagc tatcaggccc
     2161 tgaagctgaa ggacggcggc cactacctgg tggagttcga gaccatctac atggccaaga
     2221 agcccgtgca actgcccggc gattactgtg tggacaccaa gctggacatc acctcccaca
     2281 acgaggacta caccatcgtg gaacagtacg ggcgctccga gggccgccac cgtctgggca
     2341 tggacgagcg gtacaaggga acggcgggaa gcgatccaaa aaagaagaga aaggtagatc
     2401 caaaaaagaa gagaaaggta gatccaaaaa agaagagaaa ggtataagct tggtaaaccg
     2461 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2521 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2581 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2641 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2701 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2761 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2821 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2881 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2941 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3001 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3061 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3121 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3181 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3241 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3301 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3361 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3421 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3481 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3541 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3601 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3661 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3721 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     3781 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3841 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3901 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3961 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4021 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4081 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     4141 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4201 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4261 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4321 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     4381 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4441 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4501 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4561 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4621 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4681 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4741 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4801 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4861 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4921 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4981 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5041 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5101 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5161 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5221 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5281 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5341 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5401 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5461 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5521 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5581 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5641 aaaccaccgc tggtagcggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5701 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5761 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5821 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5881 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5941 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6001 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6061 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6121 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6181 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6241 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6301 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6361 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6421 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6481 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6541 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6601 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6661 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6721 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6781 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6841 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
