LOCUS       sequence-76292-e        6136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses one ddFP in mammalian cells, the second plasmid for a 
            green to red colour switch-based fluorescent caspase-3 biosensor, 
            used together with plasmid GANES-DEVD-BNLS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6136)
  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 6136)
  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: RANLS
FEATURES             Location/Qualifiers
     source          1..6136
                     /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"
     primer_bind     complement(1057..1075)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1325..1344
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1635..1655
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1659..1679
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1683..1703
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1730..1747)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1736..1960
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2006..2434
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2093..2112)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2303..2324
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2443..2463)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2448..2777
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2628..2763
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2690..2709
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2844..3638
                     /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(2898..2917)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3508..3527
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3812..3933
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3849..3868)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3903..3922
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3982..3998)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3982..3998)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3995..4017)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4006..4022
                     /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(4030..4036,4037..4054,4055..4060))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4075..4096
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4213..4230)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4384..4969)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4464..4483)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5140..5931,5932..6000))
                     /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     5763..5782
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6001..6105)
                     /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 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg
     1081 gccccctgcc cttcgcctgg gacatcctgt ccccccagat catgtacggc tccaaggcgt
     1141 acgtgaagca ccccgccgac gtccccgatt acatgaagct gtccttcccc gagggcttca
     1201 agtgggagcg cgtgatgcac ttcgaggacg gcggtctggt gaccgtgact caggacacct
     1261 ccctgcagga cggcacgctg atctacaagg tgaagatgcg cggcaccaac ttcccccccg
     1321 acggccccgt aatgcagaag aagaccttgg gctgggatta tgccaccgag cgcctgtacc
     1381 ccgaagacgg cgtgctgaag ggcgagcttc tggggcgcct gaagctgaag gacggcggcc
     1441 tcaacctggt ggagttcaag accatctaca tggccaagaa gcccgtgcaa ctgcccggct
     1501 actacttcgt ggacaccaag ctgggcatca cctcccacaa cgaggactac accatcgtgg
     1561 aacagtacga gcgctccgag ggccgccacc acctgggcat ggacgagctg tacaagggaa
     1621 cggcgggaag cgatccaaaa aagaagagaa aggtagatcc aaaaaagaag agaaaggtag
     1681 atccaaaaaa gaagagaaag gtatagaagc ttggtaaacc gctgatcagc ctcgactgtg
     1741 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1801 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1861 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1921 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     1981 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2041 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2101 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2161 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2221 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2281 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2341 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2401 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     2461 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2521 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2581 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2641 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2701 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2761 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     2821 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     2881 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     2941 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3001 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     3061 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3121 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3181 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3241 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3301 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3361 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3421 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3481 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3541 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3601 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     3661 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     3721 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3781 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     3841 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3901 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     3961 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4021 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4081 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4141 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4201 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4261 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4321 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4381 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4441 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4501 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4561 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4621 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4681 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4741 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4801 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4861 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4921 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4981 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5041 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5101 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5161 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5221 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5281 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5341 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5401 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     5461 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5521 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5581 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5641 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5701 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5761 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5821 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     5881 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5941 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6001 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6061 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6121 agtgccacct gacgtc
//
