LOCUS       sequence-320052-        8370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SARS-CoV-2 Spike protein with D614G mutation and Furin site restored
            (S-RRAR-D614G variant).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8370)
  AUTHORS   Gobeil SM, Janowska K, McDowell S, Mansouri K, Parks R, Manne K, 
            Stalls V, Kopp MF, Henderson R, Edwards RJ, Haynes BF, Acharya P
  TITLE     D614G Mutation Alters SARS-CoV-2 Spike Conformation and Enhances 
            Protease Cleavage at the S1/S2 Junction.
  JOURNAL   Cell Rep. 2021 Jan 12;34(2):108630. doi: 
            10.1016/j.celrep.2020.108630. Epub 2020 Dec 26.
  PUBMED    33417835
REFERENCE   2  (bases 1 to 8370)
  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: "Cell Rep.";
            date: "2021-01-12"; volume: "34(2):108630. doi"; pages: " 
            10.1016/j.celrep.2020.108630. Epub 2020 Dec 26"
COMMENT     Sequence Label: p-alpha-H-S-RRAR/D614G
FEATURES             Location/Qualifiers
     source          1..8370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             5482..5505
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5509..5532
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             5539..5562
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5602..5625
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(5727..5746)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5792..5847
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5846..5865)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6206..6222)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6206..6222)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6219..6241)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6230..6246
                     /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(6254..6260,6261..6278,6279..6284))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6299..6320
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6449..6466)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6620..7208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6700..6719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7379..8170,8171..8239))
                     /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     8002..8021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8240..8344)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gcgcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttgttgt gctgtctcat cattttggca aagaattgcg gccgtctcag
     1741 gccgagttcg gtaccgccac catgttcgtg ttcctggtgc tcctgcctct ggtgagcagc
     1801 cagtgcgtga acctgaccac ccgaacccag ctcccaccag cctacaccaa cagctttaca
     1861 cggggcgtgt actaccctga caaggtgttc agatctagcg tcctgcacag cactcaggac
     1921 ctcttcctgc cgttcttcag caacgtgaca tggttccacg ccatccacgt gagcggcaca
     1981 aacggaacca agcggtttga taaccccgtc ctgccattca atgatggagt ttacttcgcc
     2041 agtaccgaga agagtaacat catccggggc tggatcttcg gcaccaccct ggatagcaaa
     2101 acacagagcc tcctgatcgt gaacaatgcc acgaacgtcg tgatcaaggt gtgcgagttc
     2161 cagttttgca atgatccttt cctgggtgtg tactaccaca agaacaacaa gagctggatg
     2221 gaaagcgagt tcagagtcta cagcagcgcc aacaactgca cattcgagta cgtctctcag
     2281 ccttttctga tggaccttga ggggaaacaa ggcaacttca agaacctgag agaattcgtg
     2341 ttcaagaaca tcgacggcta cttcaaaatc tactccaagc acacacccat caacctggtc
     2401 cgggacctcc ctcagggctt cagcgccctg gaacccctgg tcgacctgcc cataggcatc
     2461 aacataacgc ggttccaaac cctgctggcc ctgcatagat cctacctgac tcctggcgac
     2521 agcagcagcg gatggaccgc cggagctgca gcctactatg tgggctacct gcaacctaga
     2581 accttcctgc tgaagtacaa cgagaacggc acaatcacag acgccgtcga ctgcgccctg
     2641 gaccctctct ctgagacaaa gtgcaccctg aagtccttca ccgtggaaaa gggcatctac
     2701 cagaccagca acttccgggt gcagcctaca gagagcatcg tgcgatttcc aaacattacc
     2761 aacctctgcc ccttcggcga ggtgtttaac gccacaagat ttgcctccgt ttacgcctgg
     2821 aatagaaaga gaatcagcaa ttgtgtggcc gactactccg tgctgtataa cagcgcctct
     2881 ttcagcacct tcaagtgcta cggcgtttcc ccaacaaagc tgaatgacct gtgcttcacc
     2941 aacgtgtacg ccgactcctt cgtaattaga ggcgatgagg tgcggcagat cgcaccaggc
     3001 cagaccggta agatcgctga ctacaactat aagctgcctg atgattttac aggctgcgtg
     3061 atcgcctgga actctaacaa cctggatagc aaggtgggcg gcaactacaa ctacctgtac
     3121 cggctgtttc gcaagtctaa cctgaaacct ttcgagagag acatctccac agagatctac
     3181 caggccggtt ctacaccttg taacggggtg gaaggcttca actgttactt ccctctgcaa
     3241 agctacggct tccagcctac caatggagtc ggctaccagc cataccgggt ggtcgtgctg
     3301 tccttcgagt tactccacgc ccccgccacc gtctgcggtc ctaagaagtc caccaatctg
     3361 gttaagaaca aatgcgtgaa cttcaacttc aacggcctga ccgggaccgg cgtgctgacc
     3421 gaaagcaaca aaaagttcct ccccttccag cagttcggcc gtgatatcgc tgacaccaca
     3481 gatgccgtca gagatccaca gaccctggaa atcctggata ttacaccctg ctccttcgga
     3541 ggagtttctg tgatcacccc cgggaccaat accagcaacc aggtggctgt gctgtaccaa
     3601 ggtgttaact gcaccgaggt tcctgtggcc atccacgccg atcagctgac acctacttgg
     3661 agagtgtact ccactggctc caatgtgttc cagaccaggg ccggatgtct gatcggcgcc
     3721 gagcacgtga ataacagtta cgagtgcgac atccctatcg gcgccggcat ctgtgccagc
     3781 taccagaccc agacaaacag ccctaggaga gctcgctctg tagctagcca gagcatcatc
     3841 gcctacacca tgagcctggg cgcagagaac agcgtggcct attccaacaa ctctatcgcc
     3901 attcccacca actttacaat tagcgtcaca acagagatcc tgcccgtgag catgaccaag
     3961 accagcgtgg actgtacaat gtacatctgt ggcgacagca ctgaatgcag caacctgctg
     4021 ctgcaatacg gctccttttg cacccaactg aaccgggcgc tgaccggaat cgccgtggaa
     4081 caggacaaaa atacccagga ggtgttcgcc caagtgaagc agatctacaa gaccccacct
     4141 atcaaggact tcggcggctt taactttagc cagattctcc ctgatccttc taagcctagc
     4201 aagcggagct ttatcgagga tctgctgttc aacaaggtca ccctggccga tgccggcttt
     4261 atcaaacagt atggcgattg cctgggcgac atagccgcca gagatctgat ctgcgcccag
     4321 aaattcaacg gcctgacagt tctcccacct ctgctgaccg acgagatgat cgctcagtac
     4381 acctctgccc tgctggctgg caccatcaca tctgggtgga catttggcgc cggcgccgcc
     4441 ctgcagatcc cctttgccat gcagatggcc tatagattca acggaatcgg cgtgacccag
     4501 aacgtgctgt atgaaaacca gaagctgatc gctaaccagt tcaattctgc catcggcaag
     4561 atccaggact ccctctcctc taccgccagc gccctgggca aactgcagga cgtggtgaat
     4621 cagaacgccc aagccctgaa caccctggtg aagcagctca gcagcaattt tggcgccatc
     4681 agctctgtgc tgaacgatat cctgtctaga ctggacaagg tggaagccga agtccagatc
     4741 gatagactga tcacaggcag actgcagtcc ctgcaaacct acgtgaccca acagctgatc
     4801 agggccgctg aaataagagc cagcgccaat ctcgccgcta ccaagatgtc cgagtgtgtg
     4861 ctgggacagt ctaaacgcgt tgacttctgc ggcaaaggct atcacctgat gagcttcccc
     4921 cagagcgcgc cgcacggcgt ggtgttcctg catgtgacat acgtgcctgc ccaagagaag
     4981 aatttcacaa ccgcccctgc catctgccac gacggcaagg cccacttccc aagagagggc
     5041 gttttcgttt ccaatggcac acactggttc gtgacacaaa gaaacttcta cgaaccccag
     5101 attatcacca ccgacaacac cttcgtgagt ggcaattgtg acgtggtcat cggaatcgtg
     5161 aacaacacag tgtacgaccc tctgcaacct gagctggact cttttaagga agagctggac
     5221 aagtacttta aaaaccacac cagccccgat gtggacctgg gcgacatcag tggcattaac
     5281 gccagcgtgg tgaacatcca aaaggaaatc gacagactga acgaggtggc caagaacctg
     5341 aacgagtccc tgatcgacct gcaggagctc ggcaaatacg agcagggatc cggatacatc
     5401 cccgaggccc ccagagatgg ccaggcctac gtgcggaagg acggcgagtg ggtactgctg
     5461 agcacattcc tgggcagatc cctggaggtg ctgttccagg gcccaggcca tcaccaccat
     5521 caccaccatc atagcgcctg gtcccacccc cagttcgaga agggcggcgg tagtggaggg
     5581 ggcggatctg gcggctcagc ttggagccac ccccagttcg aaaagtgata atgactcgag
     5641 cgataattca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat
     5701 gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta tggggacatc
     5761 atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata
     5821 gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta
     5881 aaacatcaga atgagtattt ggtttagagt ttggcaacat atgccatatg ctggctgcca
     5941 tgaacaaagg tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc
     6001 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt
     6061 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct
     6121 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatgaagat ccctcgacct
     6181 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     6241 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     6301 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     6361 tgtcgtgcca gcggatcgat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6421 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     6481 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6541 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6601 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6661 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6721 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6781 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
     6841 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6901 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6961 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     7021 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     7081 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     7141 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     7201 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     7261 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     7321 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     7381 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     7441 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7501 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7561 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7621 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7681 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7741 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7801 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7861 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7921 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7981 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     8041 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     8101 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     8161 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     8221 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     8281 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     8341 cgcgcacatt tccccgaaaa gtgccacctg
//
