LOCUS       Exported                7040 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Zipped GFP1-10 with TEV cleavage sequence T2A mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    ZipGFP1-10_TEV
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7040)
  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 7040)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7040
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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      917..926
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1037..1675
                     /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             1676..1696
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1832..1885
                     /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             1892..2602
                     /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(2037..2055)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2305..2324
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2634..2651)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2640..2864
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2910..3338
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2997..3016)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3207..3228
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3347..3367)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3352..3681
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3532..3667
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3594..3613
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3748..4542
                     /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(3802..3821)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4412..4431
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4716..4837
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4753..4772)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4807..4826
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4886..4902)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4886..4902)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4899..4921)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4910..4926
                     /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(4934..4964)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4979..5000
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5117..5134)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5288..5873)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5368..5387)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6044..6904)
                     /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     6667..6686
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6905..7009)
                     /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 gtttaaactt aagcttgcca ccatggaagt gagcgcgctg gaaaaagagg tctccgctct
      961 tgagaaggaa gtttctgcct tggagaaaga ggtgagcgcc ctggagaagg aagtctctgc
     1021 gcttgaaaaa ggatcctcca aaggagaaga actgtttacc ggtgttgtgc caattttggt
     1081 tgaactcgat ggtgatgtca acggacataa gttctcagtg agaggcgaag gagaaggtga
     1141 cgccaccatt ggaaaattga ctcttaaatt catctgtact actggtaaac ttcctgtacc
     1201 atggccgact ctcgtaacaa cgcttacgta cggagttcag tgcttttcga gatacccaga
     1261 ccatatgaaa agacatgact tttttaagtc ggctatgcct gaaggttacg tgcaagaaag
     1321 aacaatttcg ttcaaagatg atggaaaata taaaactaga gcagttgtta aatttgaagg
     1381 agatactttg gttaaccgca ttgaactgaa aggaacagat tttaaagaag atggtaatat
     1441 tcttggacac aaactcgaat acaattttaa tagtcataac gtatacatca ctgctgataa
     1501 gcaaaagaac ggaattaaag cgaatttcac agtacgccat aatgtagaag atggcagtgt
     1561 tcaacttgcc gaccattacc aacaaaacac ccctattgga gacggtccgg tacttcttcc
     1621 tgataatcac tacctctcaa cacaaacagt cctgagcaaa gatccaaatg aaaaagaaaa
     1681 tctctacttc cagagtgaat tcggcggcag caaggtgtct gcacttaaag aaaaggtttc
     1741 cgcactcaag gagaaagttt cagctttgaa agagaaggtc agtgccctta aggaaaaagt
     1801 gagcgcgctg aaagaaggcg gccgctctag agagggcaga ggaagtctgc taacatgcgg
     1861 tgacgtcgag gagaatcctg gcccagaatt gatggtgagc aagggcgagg aggataacat
     1921 ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     1981 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     2041 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     2101 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     2161 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     2221 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     2281 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     2341 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     2401 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     2461 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa
     2521 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     2581 catggacgag ctgtacaagt aatctagagg gcccgtttaa acccgctgat cagcctcgac
     2641 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2701 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2761 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2821 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2881 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2941 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3001 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3061 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3121 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3181 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3241 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3301 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3361 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3421 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3481 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3541 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3601 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     3661 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     3721 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     3781 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     3841 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     3901 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     3961 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4021 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4081 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4141 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4201 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4261 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4321 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4381 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4441 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4501 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     4561 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     4621 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4681 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     4741 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4801 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     4861 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4921 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4981 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5041 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5101 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5161 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5221 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5281 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5341 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5401 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5461 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5521 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5581 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5641 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5701 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5761 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5821 gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5881 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5941 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6001 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6061 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6121 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6181 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6241 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6301 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6361 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6421 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6481 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6541 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6601 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6661 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6721 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6781 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6841 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6901 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6961 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7021 gaaaagtgcc acctgacgtc
//
