LOCUS       Exported                7189 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses FlipCherry (TEV cleavage sequence) and T2A GFP in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3-FlipCherry-T2A-GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7189)
  AUTHORS   Zhang Q, Schepis A, Huang H, Yang J, Ma W, Torra J, Zhang SQ, Yang 
            L, Wu H, Nonell S, Dong Z, Kornberg TB, Coughlin SR, Shu X
  TITLE     Designing a green fluorogenic protease reporter by flipping a beta 
            strand of GFP for imaging apoptosis in animals.
  JOURNAL   J Am Chem Soc. 2019 Mar 1. doi: 10.1021/jacs.8b13042.
  PUBMED    30821975
REFERENCE   2  (bases 1 to 7189)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7189
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             946..966
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1084..1095
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1087..1140
                     /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"
     primer_bind     complement(1286..1304)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             1735..1788
                     /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             1795..2514
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1840..1861)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2101..2120)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2448..2469
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2546..2563)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2552..2776
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2822..3250
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2909..2928)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3119..3140
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3259..3279)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3264..3593
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3444..3579
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3506..3525
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3660..4454
                     /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(3714..3733)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4324..4343
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4628..4749
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4665..4684)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4719..4738
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4798..4814)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4798..4814)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4811..4833)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4822..4838
                     /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(4846..4876)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4891..4912
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5029..5046)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5200..5788)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5280..5299)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5959..6819)
                     /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     6582..6601
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6820..6924)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6999..7018)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgcca ccatgcagct tcccggcgcc tacaacgtgg acatcaagct ggacatcacc
      721 agccacaacg aggactccgg actcagatct ggcagcggtc tcgagatgga agttagcgct
      781 ctggaaaaag aagtgtctgc actcgagaaa gaagtaagtg cccttgagaa ggaggtgtcc
      841 gcactcgaga aggaggtcag cgccctggaa aagggcggca gcggcgacta caccatcgtg
      901 gagcagtacg agagagccga ggccagacgc agcaccgacg caagcgaaaa cctgtacttt
      961 cagtccggcg gcggcaaggt gtccgccctg aaggaaaaag taagcgcact gaaagaaaag
     1021 gtgagcgcgc tgaaggagaa agtgagcgcc ctgaaagaga aagtctctgc ccttaaggag
     1081 gatatcgagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca
     1141 aagcttgcca ccatgaagga ggacaacatg gccatcatca aggagttcat gagattcaag
     1201 gtgcacatgg atggctgcgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1261 cacccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc
     1321 ttcgcctggg acatcctgag cccccagttc atgtacggca gcaaggccta cgtgaagcac
     1381 cccgccgaca tccccgacta cctgaagctg agcttccccg agggcttcac ctgggagaga
     1441 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggacagcag cctgcaggac
     1501 ggcgagttca tctacaaggt gaagctgctg ggcatcaact tccccagcga cggccccgtg
     1561 atgcagaaga agaccatggg ctgggaggcc agcaccgaga gaatgtaccc cgaggacggc
     1621 gccctgaagg gcgagatcaa ccagagactg aagctgaagg acggcggcca ctacgacgcc
     1681 gaggtgaaga ccacctacaa ggccaagaag cccgtgcagg gcggccgctc tagagagggc
     1741 agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccaga attgatggtg
     1801 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1861 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1921 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1981 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     2041 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     2101 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     2161 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     2221 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     2281 aaggccaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     2341 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2401 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2461 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaatctaga
     2521 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2581 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2641 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2701 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2761 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     2821 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2881 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2941 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3001 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3061 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3121 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3181 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3241 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3301 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3361 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3421 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3481 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3541 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3601 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     3661 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3721 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3781 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     3841 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     3901 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3961 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4021 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4081 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4141 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     4201 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4261 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4321 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4381 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4441 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4501 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4561 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4621 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4681 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4741 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     4801 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4861 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4921 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4981 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5041 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5101 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5161 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5221 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5281 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5341 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5401 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5461 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5521 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5581 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5641 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5701 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5761 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5821 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5881 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5941 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6001 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6061 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6121 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6181 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6241 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6301 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6361 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6421 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6481 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6541 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6601 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6661 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6721 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     6781 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     6841 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     6901 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     6961 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
     7021 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
     7081 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
     7141 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgtt
//
