LOCUS       sequence-348205-        7933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of eGFP along with SARS-CoV-2 
            sequence 20080-21171 (PS9) within the 3'UTR. Resulting transcript is
            packaged into SARS-CoV-2 virus-like particles..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7933)
  AUTHORS   Syed AM, Taha TY, Tabata T, Chen IP, Ciling A, Khalid MM, Sreekumar 
            B, Chen PY, Hayashi JM, Soczek KM, Ott M, Doudna JA
  TITLE     Rapid assessment of SARS-CoV-2 evolved variants using virus-like 
            particles.
  JOURNAL   Science. 2021 Nov 4:eabl6184. doi: 10.1126/science.abl6184.
  PUBMED    34735219
REFERENCE   2  (bases 1 to 7933)
  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: "Science. 
            2021 Nov 4:eabl6184. doi: 10.1126/science.abl6184."
COMMENT     Sequence Label: GFP-PS9
FEATURES             Location/Qualifiers
     source          1..7933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        185..564
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        565..768
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     719..739
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     813..832
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        813..831
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      892..901
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(898..900,901..903,904..1620)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKG"
     primer_bind     complement(943..964)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1204..1223)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1551..1572
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2817..3405
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2870..2890)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3288..3299)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3474..3491)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3480..3704
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3750..4178
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3837..3856)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4047..4068
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4187..4207)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4192..4521
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4372..4507
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4434..4453
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4588..5382
                     /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(4642..4661)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5252..5271
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5556..5677
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5593..5612)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5647..5666
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5726..5742)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5726..5742)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5739..5761)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5750..5766
                     /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(5774..5780,5781..5798,5799..5804))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5819..5840
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5957..5974)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6128..6716)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6208..6227)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6887..7678,7679..7747))
                     /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     7510..7529
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7748..7852)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(7927..7933,1..13))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
       61 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      121 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      181 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      241 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      301 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      361 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      421 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      481 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      541 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      601 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      661 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      721 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      781 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      841 gctggctagc ttgttagacg aagcttgggc tgcaggtccg atccaccggt cgccaccatg
      901 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      961 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1021 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1081 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1141 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1201 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1261 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1321 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1381 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1441 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1501 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1561 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggataa
     1621 aatcaagaat tcgcttatgc ggccgcctcc tttaagaagg agatatagat ctctgtaggt
     1681 cccaaacaag ctagtcttaa tggagtcaca ttaattggag aagccgtaaa aacacagttc
     1741 aattattata agaaagttga tggtgttgtc caacaattac ctgaaactta ctttactcag
     1801 agtagaaatt tacaagaatt taaacccagg agtcaaatgg aaattgattt cttagaatta
     1861 gctatggatg aattcattga acggtataaa ttagaaggct atgccttcga acatatcgtt
     1921 tatggagatt ttagtcatag tcagttaggt ggtttacatc tactgattgg actagctaaa
     1981 cgttttaagg aatcaccttt tgaattagaa gattttattc ctatggacag tacagttaaa
     2041 aactatttca taacagatgc gcaaacaggt tcatctaagt gtgtgtgttc tgttattgat
     2101 ttattacttg atgattttgt tgaaataata aaatcccaag atttatctgt agtttctaag
     2161 gttgtcaaag tgactattga ctatacagaa atttcattta tgctttggtg taaagatggc
     2221 catgtagaaa cattttaccc aaaattacaa tctagtcaag cgtggcaacc gggtgttgct
     2281 atgcctaatc tttacaaaat gcaaagaatg ctattagaaa agtgtgacct tcaaaattat
     2341 ggtgatagtg caacattacc taaaggcata atgatgaatg tcgcaaaata tactcaactg
     2401 tgtcaatatt taaacacatt aacattagct gtaccctata atatgagagt tatacatttt
     2461 ggtgctggtt ctgataaagg agttgcacca ggtacagctg ttttaagaca gtggttgcct
     2521 acgggtacgc tgcttgtcga ttcagatctt aatgactttg tctctgatgc agattcaact
     2581 ttgattggtg attgtgcaac tgtacataca gctaataaat gggatctcat tattagtgat
     2641 atgtacgacc ctaagactaa aaatgttaca aaagaaaatg actctaaaga gggttttttc
     2701 acttacattt gtgggtttat acaacaaaag ctagctcttg gaggttccgt ggctataaag
     2761 atagatccca actccataag tgtgtcgaca tataaggatc cactcgacga ctcgacaatc
     2821 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     2881 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     2941 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3001 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3061 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     3121 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3181 gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct
     3241 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3301 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg catcttcgcc
     3361 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgga attaattcga
     3421 gctcggtacc ttgattctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     3481 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3541 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3601 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3661 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3721 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3781 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3841 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3901 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3961 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4021 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4081 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4141 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     4201 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4261 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4321 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4381 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4441 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     4501 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     4561 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     4621 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4681 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4741 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4801 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4861 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4921 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4981 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     5041 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     5101 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     5161 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     5221 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     5281 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     5341 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     5401 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     5461 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5521 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5581 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5641 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccctccacct
     5701 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5761 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5821 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5881 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5941 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     6001 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6061 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6121 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6181 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6241 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6301 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6361 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6421 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6481 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6541 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6601 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6661 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6721 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6781 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6841 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6901 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6961 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     7021 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7081 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7141 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7201 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7261 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7321 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7381 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7441 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7501 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7561 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7621 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7681 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7741 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7801 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7861 cccgaaaagt gccacctgac ctccacggat cgggagatct cccgatcccc tatggtgcac
     7921 tctcagtaca atc
//
