LOCUS       sequence-324838-        8375 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 8375)
  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 8375)
  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..8375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          661..1677
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1601..1623
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             5485..5508
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5512..5535
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             5542..5565
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5605..5628
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(5730..5749)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5795..5850
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5849..5868)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6211..6227)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6211..6227)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6224..6246)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6235..6251
                     /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(6259..6265,6266..6283,6284..6289))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6304..6325
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6454..6471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6625..7213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6705..6724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7384..8175,8176..8244))
                     /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     8007..8026
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8245..8349)
                     /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 tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggggsgcg csrggsgggg sggggsgggg sgrggggsgg
      541 ggsggggsgr ggsggrgrgg kgsggsggca gccaatcaga gcggcgcgct ccgaaagttt
      601 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      661 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      721 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      781 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
      841 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc
      901 gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc
      961 tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg
     1021 ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac
     1141 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     1201 ggcgcggggc tcgccgtgcc gggcgggggg kggcggcagg kgggggkgcc gggcggggcg
     1261 gggccgcckc gggccggggr gggckcgggg grggggcgcg gcggcccccg grgcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     1441 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     1501 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
     1561 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1621 accggcggct ctagcgcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1681 ctgggcaacg tgctggttgt tgtgctgtct catcattttg gcaaagaatt gcggccgtct
     1741 caggccgagt tcggtaccgc caccatgttc gtgttcctgg tgctcctgcc tctggtgagc
     1801 agccagtgcg tgaacctgac cacccgaacc cagctcccac cagcctacac caacagcttt
     1861 acacggggcg tgtactaccc tgacaaggtg ttcagatcta gcgtcctgca cagcactcag
     1921 gacctcttcc tgccgttctt cagcaacgtg acatggttcc acgccatcca cgtgagcggc
     1981 acaaacggaa ccaagcggtt tgataacccc gtcctgccat tcaatgatgg agtttacttc
     2041 gccagtaccg agaagagtaa catcatccgg ggctggatct tcggcaccac cctggatagc
     2101 aaaacacaga gcctcctgat cgtgaacaat gccacgaacg tcgtgatcaa ggtgtgcgag
     2161 ttccagtttt gcaatgatcc tttcctgggt gtgtactacc acaagaacaa caagagctgg
     2221 atggaaagcg agttcagagt ctacagcagc gccaacaact gcacattcga gtacgtctct
     2281 cagccttttc tgatggacct tgaggggaaa caaggcaact tcaagaacct gagagaattc
     2341 gtgttcaaga acatcgacgg ctacttcaaa atctactcca agcacacacc catcaacctg
     2401 gtccgggacc tccctcaggg cttcagcgcc ctggaacccc tggtcgacct gcccataggc
     2461 atcaacataa cgcggttcca aaccctgctg gccctgcata gatcctacct gactcctggc
     2521 gacagcagca gcggatggac cgccggagct gcagcctact atgtgggcta cctgcaacct
     2581 agaaccttcc tgctgaagta caacgagaac ggcacaatca cagacgccgt cgactgcgcc
     2641 ctggaccctc tctctgagac aaagtgcacc ctgaagtcct tcaccgtgga aaagggcatc
     2701 taccagacca gcaacttccg ggtgcagcct acagagagca tcgtgcgatt tccaaacatt
     2761 accaacctct gccccttcgg cgaggtgttt aacgccacaa gatttgcctc cgtttacgcc
     2821 tggaatagaa agagaatcag caattgtgtg gccgactact ccgtgctgta taacagcgcc
     2881 tctttcagca ccttcaagtg ctacggcgtt tccccaacaa agctgaatga cctgtgcttc
     2941 accaacgtgt acgccgactc cttcgtaatt agaggcgatg aggtgcggca gatcgcacca
     3001 ggccagaccg gtaagatcgc tgactacaac tataagctgc ctgatgattt tacaggctgc
     3061 gtgatcgcct ggaactctaa caacctggat agcaaggtgg gcggcaacta caactacctg
     3121 taccggctgt ttcgcaagtc taacctgaaa cctttcgaga gagacatctc cacagagatc
     3181 taccaggccg gttctacacc ttgtaacggg gtggaaggct tcaactgtta cttccctctg
     3241 caaagctacg gcttccagcc taccaatgga gtcggctacc agccataccg ggtggtcgtg
     3301 ctgtccttcg agttactcca cgcccccgcc accgtctgcg gtcctaagaa gtccaccaat
     3361 ctggttaaga acaaatgcgt gaacttcaac ttcaacggcc tgaccgggac cggcgtgctg
     3421 accgaaagca acaaaaagtt cctccccttc cagcagttcg gccgtgatat cgctgacacc
     3481 acagatgccg tcagagatcc acagaccctg gaaatcctgg atattacacc ctgctccttc
     3541 ggaggagttt ctgtgatcac ccccgggacc aataccagca accaggtggc tgtgctgtac
     3601 caaggtgtta actgcaccga ggttcctgtg gccatccacg ccgatcagct gacacctact
     3661 tggagagtgt actccactgg ctccaatgtg ttccagacca gggccggatg tctgatcggc
     3721 gccgagcacg tgaataacag ttacgagtgc gacatcccta tcggcgccgg catctgtgcc
     3781 agctaccaga cccagacaaa cagccctagg agagctcgct ctgtagctag ccagagcatc
     3841 atcgcctaca ccatgagcct gggcgcagag aacagcgtgg cctattccaa caactctatc
     3901 gccattccca ccaactttac aattagcgtc acaacagaga tcctgcccgt gagcatgacc
     3961 aagaccagcg tggactgtac aatgtacatc tgtggcgaca gcactgaatg cagcaacctg
     4021 ctgctgcaat acggctcctt ttgcacccaa ctgaaccggg cgctgaccgg aatcgccgtg
     4081 gaacaggaca aaaataccca ggaggtgttc gcccaagtga agcagatcta caagacccca
     4141 cctatcaagg acttcggcgg ctttaacttt agccagattc tccctgatcc ttctaagcct
     4201 agcaagcgga gctttatcga ggatctgctg ttcaacaagg tcaccctggc cgatgccggc
     4261 tttatcaaac agtatggcga ttgcctgggc gacatagccg ccagagatct gatctgcgcc
     4321 cagaaattca acggcctgac agttctccca cctctgctga ccgacgagat gatcgctcag
     4381 tacacctctg ccctgctggc tggcaccatc acatctgggt ggacatttgg cgccggcgcc
     4441 gccctgcaga tcccctttgc catgcagatg gcctatagat tcaacggaat cggcgtgacc
     4501 cagaacgtgc tgtatgaaaa ccagaagctg atcgctaacc agttcaattc tgccatcggc
     4561 aagatccagg actccctctc ctctaccgcc agcgccctgg gcaaactgca ggacgtggtg
     4621 aatcagaacg cccaagccct gaacaccctg gtgaagcagc tcagcagcaa ttttggcgcc
     4681 atcagctctg tgctgaacga tatcctgtct agactggaca aggtggaagc cgaagtccag
     4741 atcgatagac tgatcacagg cagactgcag tccctgcaaa cctacgtgac ccaacagctg
     4801 atcagggccg ctgaaataag agccagcgcc aatctcgccg ctaccaagat gtccgagtgt
     4861 gtgctgggac agtctaaacg cgttgacttc tgcggcaaag gctatcacct gatgagcttc
     4921 ccccagagcg cgccgcacgg cgtggtgttc ctgcatgtga catacgtgcc tgcccaagag
     4981 aagaatttca caaccgcccc tgccatctgc cacgacggca aggcccactt cccaagagag
     5041 ggcgttttcg tttccaatgg cacacactgg ttcgtgacac aaagaaactt ctacgaaccc
     5101 cagattatca ccaccgacaa caccttcgtg agtggcaatt gtgacgtggt catcggaatc
     5161 gtgaacaaca cagtgtacga ccctctgcaa cctgagctgg actcttttaa ggaagagctg
     5221 gacaagtact ttaaaaacca caccagcccc gatgtggacc tgggcgacat cagtggcatt
     5281 aacgccagcg tggtgaacat ccaaaaggaa atcgacagac tgaacgaggt ggccaagaac
     5341 ctgaacgagt ccctgatcga cctgcaggag ctcggcaaat acgagcaggg atccggatac
     5401 atccccgagg cccccagaga tggccaggcc tacgtgcgga aggacggcga gtgggtactg
     5461 ctgagcacat tcctgggcag atccctggag gtgctgttcc agggcccagg ccatcaccac
     5521 catcaccacc atcatagcgc ctggtcccac ccccagttcg agaagggcgg cggtagtgga
     5581 gggggcggat ctggcggctc agcttggagc cacccccagt tcgaaaagtg ataatgactc
     5641 gagcgataat tcactcctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc
     5701 aatgccctgg ctcacaaata ccactgagat ctttttccct ctgccaaaaa ttatggggac
     5761 atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca
     5821 atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat
     5881 ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca tatgctggct
     5941 gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc
     6001 cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat attttgtttt
     6061 gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact agccagattt
     6121 ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg gagatccctc
     6181 gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     6241 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     6301 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6361 aaacctgtcg tgccagcgga tcgatccgct gcattaatga atcggccaac gcgcggggag
     6421 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     6481 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     6541 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6601 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6661 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6721 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6781 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6841 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6901 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6961 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     7021 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     7081 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     7141 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     7201 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     7261 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     7321 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     7381 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     7441 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7501 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7561 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7621 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7681 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7741 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7801 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7861 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7921 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7981 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     8041 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     8101 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     8161 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     8221 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     8281 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     8341 ggttccgcgc acatttcccc gaaaagtgcc acctg
//
