LOCUS       sequence-458823-        8601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of SARS-CoV-2 spike protein with glycan deletion at 
            N1098, N1134, N1158, N1173, N1194 by implementing Asn-to-Gln 
            mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8601)
  AUTHORS   Yang Q, Kelkar A, Manicassamy B, Neelamegham S
  TITLE     Conserved role of spike S2 domain N-glycosylation across 
            betacoronaviruses.
  JOURNAL   Npj Viruses. 2025 Jan 25;3(1):4. doi: 10.1038/s44298-024-00085-7.
  PUBMED    40295734
REFERENCE   2  (bases 1 to 8601)
  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: "Npj 
            Viruses."; date: "2025-01-25"; volume: "3(1):4. doi"; pages: " 
            10.1038/s44298-024-00085-7"
COMMENT     Sequence Label: SARS-CoV-2 spike G12345 mutant
FEATURES             Location/Qualifiers
     source          1..8601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          662..1682
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1690..1709
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             5571..5594
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(5622..5641)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5687..5742
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5741..5760)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6103..6119)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6103..6119)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6116..6138)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6127..6143
                     /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(6151..6157,6158..6175,6176..6181))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6196..6217
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6276..6471
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6322..6457
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6477..6611
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6514..6533)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6568..6587
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6679..6696)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6850..7438)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6930..6949)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7609..8400,8401..8469))
                     /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     8232..8251
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8470..8574)
                     /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 gggggggggc gccagggggg gggggggggg gggggggggg
      541 gggggggggg gggggggggg gggggggggg ggccaagggg gggggggggc gcgcgaagtt
      601 ttcttttttg tggggggggg gggggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagctaga gcctctgcta accatgttca tgccttcttc tttttcctac
     1681 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattcgccg
     1741 ccaccatgtt tgtgttcctg gtcctgctcc cactggtttc cagccaatgt gttaacctca
     1801 caacccgcac ccagctgcct cccgcctata ccaacagttt tactcgggga gtgtattacc
     1861 ctgacaaagt tttcaggagc tcagttctgc actcaaccca ggatctcttt ctgccttttt
     1921 tttccaacgt gacttggttc catgcaattc acgtttccgg gaccaacggc accaaaaggt
     1981 tcgataaccc cgtgctgccc tttaacgacg gcgtctattt cgcaagcact gagaaaagca
     2041 atatcatcag gggctggatt tttgggacaa ccttggatag caagacccag tcactgctga
     2101 tcgttaataa tgcaaccaat gtggtgatca aagtgtgcga atttcagttt tgcaatgacc
     2161 cctttctggg cgtctactat cataaaaaca ataaatcttg gatggagtct gagtttaggg
     2221 tgtattctag cgcgaataat tgtacctttg aatatgtatc acaaccattt ctcatggatc
     2281 tcgaaggcaa acaaggcaat ttcaaaaact tgagagagtt cgtcttcaaa aacatcgatg
     2341 gatattttaa aatatacagc aaacataccc caataaacct ggtccgcgac ctccctcagg
     2401 gtttcagcgc cctcgagcct ctcgtcgatc ttcctatcgg gatcaacatc acaagatttc
     2461 aaaccttgct ggctcttcat cgatcctacc tgacccctgg cgattcctct tccgggtgga
     2521 ccgccggcgc cgctgcctat tatgtggggt atctgcaacc tcgcacgttt ctcctcaagt
     2581 ataatgagaa tggaactatc accgatgcgg tagactgtgc gttggaccca ctctctgaga
     2641 ccaagtgcac tttgaagtcc tttaccgttg aaaaggggat ctatcagaca tccaatttta
     2701 gagtccaacc cacggaatcc atcgttcggt tccccaatat cacaaatctc tgccctttcg
     2761 gagaggtatt caacgcaact cgcttcgctt ctgtgtatgc gtggaacaga aaacgcatct
     2821 ccaactgcgt cgctgattac tctgttctgt ataactccgc ctcctttagc accttcaagt
     2881 gttacggcgt cagccccaca aagctcaacg acttgtgttt cactaacgtc tacgccgact
     2941 ccttcgtgat ccggggggac gaagttcggc agattgcgcc tggacagact ggtaaaatcg
     3001 ctgactataa ctataagctc ccagatgact ttaccggctg tgtgattgct tggaactcta
     3061 ataatctcga cagcaaagtt ggaggcaact ataattatct ctaccggttg tttcggaaga
     3121 gcaatctcaa gccattcgag agagacataa gcacggagat ctaccaagcc ggctccactc
     3181 catgcaatgg ggttgaaggc tttaattgtt attttcccct ccaatcctat ggttttcaac
     3241 cgactaatgg agtcgggtat caaccctaca gggttgtcgt cttgagcttc gagctcttgc
     3301 acgctcctgc tacggtgtgt ggtccgaaaa aatcaacaaa cctcgtcaaa aacaaatgtg
     3361 tgaattttaa ctttaacgga ctgactggca ccggggtgct gactgagagc aataagaagt
     3421 ttcttccatt tcaacaattc ggtcgggata tcgcggacac cactgatgct gtgcgcgatc
     3481 cgcaaacctt ggagatcttg gacataactc cgtgcagctt cggaggcgtc tctgttatca
     3541 cccctggaac gaatacatct aaccaagttg cggtccttta tcagggcgtt aactgcacag
     3601 aagtacccgt cgcgatacat gcggatcagc tgacccctac ttggcgggtc tactcaaccg
     3661 gctctaatgt gtttcaaact cgggcgggat gtttgatcgg agctgagcac gtgaataact
     3721 cttatgaatg cgatatacca atcggcgcag ggatttgcgc atcttaccaa acccaaacca
     3781 actctcctcg gcgggctcgc tccgtcgcat cccaatccat tatcgcctat accatgagtc
     3841 tcggagctga gaactctgtc gcttacagca acaattctat agccatccca accaacttca
     3901 cgattagcgt tacaacagag atactccccg tcagcatgac gaaaacttcc gttgattgta
     3961 cgatgtatat ctgtggggat tctacagagt gtagcaatct gctgttgcag tatggctcct
     4021 tctgtaccca attgaatcgg gcactcaccg gcatcgctgt ggaacaagac aaaaatacgc
     4081 aagaagtttt cgcacaggtg aagcaaatct acaagacgcc tccaatcaaa gactttggcg
     4141 gattcaattt ttctcagatt ctgcctgatc cctcaaagcc cagcaaaaga agcttcatcg
     4201 aggacctgct cttcaacaag gtcactctcg cagatgccgg gttcatcaaa caatatggag
     4261 attgcttggg cgacattgct gcgcgggatc ttatttgtgc tcaaaagttc aatgggctga
     4321 ccgtcctgcc gcccctcttg accgatgaaa tgattgctca atacacctcc gcgttgctcg
     4381 cgggaactat cacttccggc tggaccttcg gcgccggcgc tgccctccag ataccgtttg
     4441 ccatgcaaat ggcctacaga ttcaacggca tcggagtcac acagaatgtc ctgtacgaaa
     4501 atcaaaaatt gatagccaat cagtttaatt ctgcaatcgg caaaatacaa gactccctgt
     4561 cctctactgc ctccgcactt gggaaattgc aagacgtcgt gaaccaaaac gcacaggccc
     4621 ttaacactct ggtgaaacag ttgagctcca acttcggcgc tataagctcc gtcttgaacg
     4681 atatactctc tcgcctggac aaagtcgagg ctgaagtcca gatcgacaga ttgataacag
     4741 gcaggcttca atccctgcaa acctacgtca cgcagcagct gatcagagcc gcagagatca
     4801 gggcttccgc aaatcttgct gcaacaaaaa tgtccgaatg cgtcttgggc caatcaaagc
     4861 gcgtcgattt ctgtggaaag ggctaccatc tcatgagctt tccccaaagc gccccgcacg
     4921 gcgtggtttt tctccacgtc acctacgtcc ccgctcaaga gaagaacttt acaaccgctc
     4981 ctgcaatttg ccatgatggg aaagcgcact ttcctcgcga aggcgtcttt gtctcccagg
     5041 gcacacattg gtttgttact caacggaact tttatgaacc acaaatcatt accacggaca
     5101 acacgttcgt gtccggcaac tgtgacgttg tcatcggaat tgttcagaac acggtttacg
     5161 atcctctgca acccgagctt gactccttta aggaagaact ggacaagtac ttcaaacagc
     5221 acacctcacc ggatgtggac ttgggcgata tctctggcat acaggcgtca gtcgtgaaca
     5281 ttcaaaagga aattgatcga ctcaacgagg tcgcaaagaa tcttcaggaa tccctgatag
     5341 atctgcagga gttggggaag tacgagcagt atataaagtg gccttggtat atatggcttg
     5401 gctttatcgc cggcctcatt gcaatcgtga tggtcaccat catgctgtgc tgcatgacct
     5461 catgttgctc ctgtcttaag gggtgctgct cctgcggttc ctgttgcaaa tttgacgagg
     5521 acgacagcga accggtgctt aagggtgtga agctgcatta caccggaggc gattacaagg
     5581 atgacgatga caagtgacgg ccgcctgcag gttaattaag atctttttcc ctctgccaaa
     5641 aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt
     5701 attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg
     5761 agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc
     5821 catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag
     5881 ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta gatttttttt
     5941 atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta
     6001 ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta
     6061 tggagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt
     6121 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     6181 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     6241 tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata
     6301 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     6361 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     6421 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact
     6481 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     6541 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     6601 atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     6661 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     6721 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     6781 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6841 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     6901 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6961 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     7021 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     7081 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     7141 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     7201 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     7261 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     7321 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     7381 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     7441 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     7501 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     7561 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     7621 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     7681 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     7741 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     7801 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7861 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     7921 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7981 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     8041 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     8101 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     8161 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     8221 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     8281 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     8341 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     8401 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     8461 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     8521 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     8581 tccccgaaaa gtgccacctg g
//
