LOCUS       sequence-76280-e        6140 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a tandem ddFP heterodimer in mammalian cells, construct 1 
            for a translocation-based green fluorescent caspase-3 biosensor, 
            used together with plasmid 1 (GANLS-DEVD-BNES or RANLS-DEVD-BNES)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6140)
  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 6140)
  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: BNLS
FEATURES             Location/Qualifiers
     source          1..6140
                     /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(1063..1081)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1331..1350
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1525..1548
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1641..1661
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1665..1685
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1689..1709
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1734..1751)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1740..1964
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2010..2438
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2097..2116)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2307..2328
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2447..2467)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2452..2781
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2632..2767
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2694..2713
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2848..3642
                     /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(2902..2921)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3512..3531
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3816..3937
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3853..3872)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3907..3926
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3986..4002)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3986..4002)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3999..4021)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4010..4026
                     /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(4034..4040,4041..4058,4059..4064))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4079..4100
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4217..4234)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4388..4973)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4468..4487)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5144..5935,5936..6004))
                     /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     5767..5786
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6005..6109)
                     /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 agagcctcct ccgaggacaa caacatggcc accatcaaag
      961 agttcatgcg cttcaaggtg cgcatggagg gctccatgaa cggccacgag ttcgagatcg
     1021 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     1081 agggcggccc cctgcccttt gcctgggaca tcctgtcccc ccagatcatg tacggctcca
     1141 aagcgtacgt gaggcacccc gccgacatcc ccgattacaa gaagctgccc ttccccgagg
     1201 gcttcaagtg ggagcgcgtg atgaacttcg aagacggcgg tctggtgacc gttacccagg
     1261 actcctccct gcaggacggc acgctgatct gcaaggtgaa gattcgcggc accaacttcc
     1321 cccccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc accgagatgc
     1381 tgtaccccaa ggacggcgtg ctgaagggcc atagctatca ggccctgaag ctgaaggacg
     1441 gcggccacta cctggtggag ttcgagacca tctacatggc caagaagccc gtgcaactgc
     1501 ccggcgatta ctgtgtggac accaagctgg acatcacctc ccacaacgag gactacacca
     1561 tcgtggaaca gtacgggcgc tccgagggcc gccaccgtct gggcatggac gagcggtaca
     1621 agggaacggc gggaagcgat ccaaaaaaga agagaaaggt agatccaaaa aagaagagaa
     1681 aggtagatcc aaaaaagaag agaaaggtat aagcttggta aaccgctgat cagcctcgac
     1741 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1801 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1861 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1921 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     1981 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2041 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2101 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2161 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2221 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2281 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2341 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2401 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     2461 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2521 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2581 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2641 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2701 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     2761 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     2821 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     2881 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     2941 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     3001 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     3061 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     3121 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     3181 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3241 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3301 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     3361 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     3421 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     3481 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     3541 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     3601 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     3661 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     3721 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3781 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     3841 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3901 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     3961 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4021 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4081 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4141 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4201 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4261 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4321 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4381 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4441 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4501 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4561 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4621 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4681 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4741 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4801 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4861 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4921 gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4981 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5041 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5101 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5161 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5221 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5281 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5341 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5401 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5461 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5521 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5581 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5641 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5701 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5761 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5821 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5881 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5941 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6001 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6061 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6121 gaaaagtgcc acctgacgtc
//
