LOCUS       sequence-429078-        7528 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of codon optimized SARS-CoV-2 
            Membrane and Envelope proteins (original Wuhan Hu1 sequence). Used 
            for generating RNA packaging virus-like particles..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7528)
  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 7528)
  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: CoV2-M-IRES-E
FEATURES             Location/Qualifiers
     source          1..7528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(17..591,592..594,595..603)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(184..201)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     411..430
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     misc_feature    874..1462
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(927..947)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1345..1356)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1552..1569)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1558..1782
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1828..2256
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1915..1934)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2125..2146
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2265..2285)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2270..2599
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2450..2585
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2512..2531
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2666..3460
                     /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(2720..2739)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3330..3349
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3634..3755
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3671..3690)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3725..3744
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3804..3820)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3804..3820)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3817..3839)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3828..3844
                     /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(3852..3858,3859..3876,3877..3882))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3897..3918
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4035..4052)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4206..4794)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4286..4305)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4965..5756,5757..5825))
                     /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     5588..5607
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5826..5930)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6005..6024)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6196..6575
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6576..6779
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6730..6750
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6824..6843
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6824..6842
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6862..6871
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 tccagtaagg atcccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct
       61 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg
      121 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt
      181 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg
      241 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac
      301 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg
      361 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct
      421 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg
      481 atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag
      541 gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa gcttgccaca
      601 acccacaagg agaaattcgc cgccaccatg tacagcttcg tatcagaaga aaccgggaca
      661 ctgatcgtaa attctgtgct cttgtttctg gcattcgtcg tatttctcct cgtcacactg
      721 gcaattctga ctgcattgag gctttgcgcc tactgttgta acattgtcaa tgtatctctc
      781 gtgaaaccct cattctacgt ttacagcagg gtgaagaatc tcaattctag cagggtgccg
      841 gatctcctcg ttctttaact cgaccgactc gacaatcaac ctctggatta caaaatttgt
      901 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
      961 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     1021 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     1081 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     1141 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     1201 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     1261 tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc
     1321 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     1381 ctgctgccgg ctctgcggcc tcttccgcat cttcgccttc gccctcagac gagtcggatc
     1441 tccctttggg ccgcctcccc gcctggaatt aattcgagct cggtaccttt aagaccaatg
     1501 acttacaagg cagcctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     1561 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     1621 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     1681 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     1741 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     1801 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     1861 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1921 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1981 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2041 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2101 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2161 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2221 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     2281 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2341 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2401 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     2461 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     2521 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     2581 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     2641 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     2701 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     2761 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     2821 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     2881 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     2941 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     3001 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     3061 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     3121 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     3181 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     3241 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     3301 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     3361 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     3421 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     3481 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     3541 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     3601 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     3661 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     3721 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     3781 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3841 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3901 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3961 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4021 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4081 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4141 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4201 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4261 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4321 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4381 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4441 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4501 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4561 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4621 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4681 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4741 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4801 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4861 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4921 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4981 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5041 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5101 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5161 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5221 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5281 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5341 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5401 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5461 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5521 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5581 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5641 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5701 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5761 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5821 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5881 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5941 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     6001 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     6061 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     6121 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     6181 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     6241 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     6301 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     6361 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     6421 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     6481 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     6541 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     6601 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     6661 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     6721 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     6781 tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata
     6841 gggagaccca agctggctag cgccaccatg gccgactcaa atgggaccat tactgtagag
     6901 gagctgaaaa aactgttgga gcaatggaac cttgtgattg gatttctctt cttgacatgg
     6961 atttgtctcc tgcaattcgc atacgcgaat cgcaaccgat tcctctatat tataaaactt
     7021 atattccttt ggctcctgtg gcctgtgacc cttgcctgct tcgtactcgc ggcggtgtat
     7081 cgaataaact ggataactgg cggcatagca atagctatgg catgcctggt aggtttgatg
     7141 tggctctcct attttatagc gtcattcaga ctttttgcac gcacgcgaag catgtggtca
     7201 tttaaccctg aaacaaatat cttgcttaat gtccccctgc atggcacaat tttgacccga
     7261 ccgctccttg aatcagaatt ggtgattgga gccgtgatcc tgaggggcca cttgaggatc
     7321 gcaggacatc atcttggtcg ctgtgatatt aaggatttgc caaaagaaat aactgtagct
     7381 accagtcgaa ctttgagcta ttacaagctt ggagcgtccc aaagggtcgc tggcgatagt
     7441 ggatttgcag cctattcacg ctacaggata gggaactata aactgaatac agaccactca
     7501 tcatcttctg ataacattgc tttgctcg
//
