LOCUS       Exported                7394 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  A ZipGFP-based Caspase-3 protease reporter T2A mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    ZipGFP-Casp3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7394)
  AUTHORS   To TL, Schepis A, Ruiz-Gonzalez R, Zhang Q, Yu D, Dong Z, Coughlin 
            SR, Shu X
  TITLE     Rational Design of a GFP-Based Fluorogenic Caspase Reporter for 
            Imaging Apoptosis In Vivo.
  JOURNAL   Cell Chem Biol. 2016 Jul 21;23(7):875-82. doi: 
            10.1016/j.chembiol.2016.06.007.
  PUBMED    27447051
REFERENCE   2  (bases 1 to 7394)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7394
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     865..884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        865..883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      919..928
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1042..1089
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             1213..1266
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      1273..1282
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1393..2031
                     /codon_start=1
                     /product="fragment of superfolder GFP consisting of the 
                     first 10 beta-strands"
                     /label=GFP(1-10)
                     /note="associates with GFP11, the 11th beta-strand, to 
                     reconstitute fluorescent GFP (Cabantous et al., 2005; 
                     Kamiyama et al., 2016)"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGK
                     YKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK"
     CDS             2185..2238
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2245..2955
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2390..2408)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2658..2677
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2987..3004)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2993..3217
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3263..3691
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3350..3369)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3560..3581
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3700..3720)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3705..4034
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3885..4020
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3947..3966
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4101..4895
                     /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(4155..4174)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4765..4784
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5069..5190
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5106..5125)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5160..5179
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5239..5255)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5239..5255)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5252..5274)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5263..5279
                     /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(5287..5317)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5332..5353
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5470..5487)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5641..6229)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5721..5740)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6400..7260)
                     /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     7023..7042
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7261..7365)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta
      901 gcgtttaaac ttaagcttgc caccatgggc ggcagcaagg tgtctgcact taaagaaaag
      961 gtttccgcac tcaaggagaa agtttcagct ttgaaagaga aggtcagtgc ccttaaggaa
     1021 aaagtgagcg cgctgaaaga acgggaccac atggtgctgc acgagtacgt gaacgccgcc
     1081 ggcatcacag gtgatgaagt tgatggtgaa gtgagcgcgc tggaaaaaga ggtctccgct
     1141 cttgagaagg aagtttctgc cttggagaaa gaggtgagcg ccctggagaa ggaagtctct
     1201 gcgcttgaaa aagagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct
     1261 ggcccaaagc ttgccaccat ggaagtgagc gcgctggaaa aagaggtctc cgctcttgag
     1321 aaggaagttt ctgccttgga gaaagaggtg agcgccctgg agaaggaagt ctctgcgctt
     1381 gaaaaaggat cctccaaagg agaagaactg tttaccggtg ttgtgccaat tttggttgaa
     1441 ctcgatggtg atgtcaacgg acataagttc tcagtgagag gcgaaggaga aggtgacgcc
     1501 accattggaa aattgactct taaattcatc tgtactactg gtaaacttcc tgtaccatgg
     1561 ccgactctcg taacaacgct tacgtacgga gttcagtgct tttcgagata cccagaccat
     1621 atgaaaagac atgacttttt taagtcggct atgcctgaag gttacgtgca agaaagaaca
     1681 atttcgttca aagatgatgg aaaatataaa actagagcag ttgttaaatt tgaaggagat
     1741 actttggtta accgcattga actgaaagga acagatttta aagaagatgg taatattctt
     1801 ggacacaaac tcgaatacaa ttttaatagt cataacgtat acatcactgc tgataagcaa
     1861 aagaacggaa ttaaagcgaa tttcacagta cgccataatg tagaagatgg cagtgttcaa
     1921 cttgccgacc attaccaaca aaacacccct attggagacg gtccggtact tcttcctgat
     1981 aatcactacc tctcaacaca aacagtcctg agcaaagatc caaatgaaaa aggtgatgaa
     2041 gttgatggtg aattcggcgg cagcaaggtg tctgcactta aagaaaaggt ttccgcactc
     2101 aaggagaaag tttcagcttt gaaagagaag gtcagtgccc ttaaggaaaa agtgagcgcg
     2161 ctgaaagaag gcggccgctc tagagagggc agaggaagtc tgctaacatg cggtgacgtc
     2221 gaggagaatc ctggcccaga attgatggtg agcaagggcg aggaggataa catggccatc
     2281 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
     2341 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     2401 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
     2461 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     2521 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     2581 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     2641 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc
     2701 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
     2761 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
     2821 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
     2881 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
     2941 gagctgtaca agtaatctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     3001 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3061 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3121 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3181 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     3241 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3301 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3361 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3421 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3481 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3541 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3601 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3661 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     3721 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3781 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3841 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3901 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3961 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4021 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     4081 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     4141 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     4201 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     4261 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     4321 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     4381 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     4441 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     4501 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4561 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4621 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4681 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4741 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4801 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4861 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     4921 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     4981 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     5041 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     5101 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5161 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     5221 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5281 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5341 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5401 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5461 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5521 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5581 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5641 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5701 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5761 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5821 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5881 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5941 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6001 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6061 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6121 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6181 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6241 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6301 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6361 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6421 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6481 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6541 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6601 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6661 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6721 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6781 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6841 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6901 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6961 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7021 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7081 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7141 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7201 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     7261 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     7321 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     7381 agtgccacct gacg
//
