LOCUS       sequence-320823-        7183 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses FlipGFP-based activity reporter of SARS-CoV-2 main 
            protease Mpro and also mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7183)
  AUTHORS   Li X, Lidsky P, Xiao Y, Wu CT, Garcia-Knight M, Yang J, Nakayama T, 
            Nayak JV, Jackson PK, Andino R, Shu X
  TITLE     Ethacridine inhibits SARS-CoV-2 by inactivating viral particles.
  JOURNAL   PLoS Pathog. 2021 Sep 3;17(9):e1009898. doi: 
            10.1371/journal.ppat.1009898.
  PUBMED    34478458
REFERENCE   2  (bases 1 to 7183)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS 
            Pathog."; date: "2021-09-3"; volume: "17(9):e1009898. doi"; pages: "
            10.1371/journal.ppat.1009898"
COMMENT     Sequence Label: pcDNA3 FlipGFP(Mpro) T2A mCherry
FEATURES             Location/Qualifiers
     source          1..7183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1066..1077
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1069..1122
                     /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             1738..1791
                     /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             1798..2508
                     /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(1943..1961)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2211..2230
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2540..2557)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2546..2770
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2816..3244
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2903..2922)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3113..3134
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3253..3273)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3258..3587
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3438..3573
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3500..3519
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3654..4448
                     /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(3708..3727)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4318..4337
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4622..4743
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4659..4678)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4713..4732
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4792..4808)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4792..4808)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4805..4827)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4816..4832
                     /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(4840..4846,4847..4864,4865..4870))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4885..4906
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5023..5040)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5194..5782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5274..5293)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5953..6744,6745..6813))
                     /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     6576..6595
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6814..6918)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6993..7012)
                     /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 ccatggacct gcctgacgac cactacctgt ccacccagac catcctgtcc
      721 aaggacctga actccggact cagatctggc agcggtctcg agatggaagt tagcgctctg
      781 gaaaaagaag tgtctgcact agagaaagaa gtaagtgccc ttgagaagga ggtgtccgca
      841 ctagagaagg aggtcagcgc cctggaaaag gaaaagcgag accatatggt tttgcttgag
      901 tatgttacag cggctggcat taccgatgca tcagccgtgc tgcagagcgg cttcaggaag
      961 gtgtccgccc tgaaggaaaa agtaagcgca ctgaaagaaa aggtgagcgc gctgaaggag
     1021 aaagtgagcg ccctgaaaga gaaagtctct gcccttaagg aggatatcga gggcagagga
     1081 agtctgctaa catgcggtga cgtcgaggag aatcctggcc caaagcttgc caccatgcgc
     1141 aaaggcgaag aactgtttac cggcattgtg ccgattctgg tggaactgga tggcgatgtg
     1201 aacggccata aattttttgt gcgcggcgaa ggcgaaggcg atgcgaccat tggcaaactg
     1261 agcctgaaat ttatttgcac caccggcaaa ctgccggtgc cgtggccgac cctggtgacc
     1321 accctgacct atggcgtgca gtgctttagc cgctatccgg atcatatgaa acgccatgat
     1381 ttttttaaaa gcgcgatgcc ggaaggctat gtgcaggaac gcaccattta ttttaaagat
     1441 gatggcacct ataaaacccg cgcggaagtg aaatttgaag gcgataccct ggtgaaccgc
     1501 attgaactga aaggcattga ttttaaagaa gatggcaaca ttctgggcca taaactggaa
     1561 tataacttta acagccataa agtgtatatt accgcggata aacagaacaa cggcattaaa
     1621 gcgaacttta ccattcgcca taacgtggaa gatggcagcg tgcagctggc ggatcattat
     1681 cagcagaaca ccccgattgg cgatggcccg gttcttcttc ctggcggccg ctctagagag
     1741 ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc agaattgatg
     1801 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     1861 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1921 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     1981 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     2041 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     2101 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     2161 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     2221 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     2281 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     2341 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     2401 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     2461 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaatc tagagggccc
     2521 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2581 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2641 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2701 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2761 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2821 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2881 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2941 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3001 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3061 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3121 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3181 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3241 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3301 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3361 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3421 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3481 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3541 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3601 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3661 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3721 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3781 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     3841 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3901 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3961 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4021 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4081 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4141 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4201 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4261 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4321 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4381 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4441 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4501 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4561 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4621 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4681 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4741 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     4801 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4861 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4921 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4981 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5041 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5101 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5161 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5221 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5281 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5341 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5401 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5461 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5521 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5581 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5641 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5701 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5761 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5821 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5881 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5941 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6001 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6061 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6121 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6181 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6241 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6301 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6361 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6421 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6481 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6541 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6601 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6661 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6721 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6781 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6841 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6901 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     6961 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     7021 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     7081 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     7141 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//
