LOCUS       sequence-459043-        9215 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of SARS-CoV spike protein with glycan deletion at 
            N1080, N1116, N1140, N1155, N1176 by implementing Asn-to-Gln 
            mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9215)
  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 9215)
  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 spike all5KO mutant
FEATURES             Location/Qualifiers
     source          1..9215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        149..528
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        529..732
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     683..703
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     777..796
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        777..795
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4722..4739)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4728..4952
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4998..5426
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5085..5104)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5295..5316
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5435..5455)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5440..5769
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5620..5755
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5890..5909)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6498..6517
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6802..6923
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6839..6858)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6893..6912
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6972..6988)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6972..6988)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6985..7007)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6996..7012
                     /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(7020..7026,7027..7044,7045..7050))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7065..7086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7203..7220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7374..7962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7454..7473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8133..8924,8925..8993))
                     /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     8756..8775
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8994..9098)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9173..9192)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
       61 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
      121 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      181 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      241 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      301 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      361 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      421 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      481 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      541 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      601 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      661 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      721 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
      781 acgactcact atagggagac ccaagctggc tagcgtttaa acgggccctc tagaaaccat
      841 gcccatgggg tctctgcaac cgctggccac cttgtacctg ctggggatgc tggtcgcttc
      901 cgtgctagcc agcggcagcg acctggaccg ctgcaccacg ttcgatgacg tgcaggcgcc
      961 caactacacc cagcacacct ccagcatgcg cggcgtgtac tatcccgacg agatcttccg
     1021 cagcgacacc ctgtacctca cccaggacct gttcctgccc ttctactcca acgtgaccgg
     1081 cttccacacc atcaaccaca ccttcggcaa ccccgtgatc ccgttcaagg acggcatcta
     1141 cttcgccgcg accgagaaga gcaacgtggt ccgcggctgg gtgttcggga gcaccatgaa
     1201 taacaagtcc cagagcgtga tcattatcaa taactccacc aacgtcgtga tccgcgcctg
     1261 caacttcgag ctgtgtgaca acccgttctt tgccgtgagc aagcccatgg gcacccagac
     1321 gcacaccatg atcttcgaca acgccttcaa ctgcaccttc gagtacatca gcgacgcctt
     1381 ctccctggac gtgagcgaga agtccggaaa cttcaagcac ctgcgcgagt tcgtgttcaa
     1441 gaacaaagac ggcttcctgt acgtgtacaa gggctaccag cccatcgacg tcgtgcgcga
     1501 cctgcccagc ggcttcaaca ccctgaagcc catcttcaag ctgcccctcg gcattaacat
     1561 caccaacttc cgcgccatcc tgaccgcctt cagccctgcc caggacatct ggggcaccag
     1621 cgccgcggcc tacttcgtcg gctacctgaa gcccacgacc ttcatgctga agtacgacga
     1681 gaacggcacg atcaccgatg ccgtggactg cagccagaac ccgctcgccg agctgaagtg
     1741 cagcgtgaag tccttcgaga tcgacaaggg catctaccag accagcaact tccgcgtggt
     1801 acccagcggc gacgtggtcc gcttcccgaa catcaccaac ctgtgtccct tcggcgaggt
     1861 gttcaacgcc accaagttcc cgagcgtgta cgcctgggag cgcaagaaga tcagcaactg
     1921 cgtggccgac tacagcgtgc tgtacaactc caccttcttt agcaccttca agtgctacgg
     1981 cgtgagcgcc accaagctca acgacctgtg cttcagcaac gtgtacgccg acagcttcgt
     2041 ggtcaagggc gacgatgtgc gccagatcgc gcccggccag accggcgtga tcgccgacta
     2101 caactacaag ctgcccgatg acttcatggg ctgcgtgctg gcctggaaca cccgcaacat
     2161 cgacgccacg agcaccggca actacaacta caagtatcgc tacctgcgcc acggcaagct
     2221 gcggcccttc gagcgcgaca tctccaacgt gcccttcagc ccggacggca agccctgcac
     2281 gccgcccgcc ctgaactgct actggccgct gaacgactat ggcttctaca ctacgaccgg
     2341 tatcggctac cagccctacc gcgtggtcgt gctgagcttc gagctcctga acgcgcccgc
     2401 caccgtgtgc ggcccgaagc tgagcaccga cctgatcaag aaccagtgcg tgaacttcaa
     2461 tttcaacggc ctgaccggca ccggcgtgct gacgcccagc tccaagcgct tccagccctt
     2521 ccagcagttc ggccgcgacg tcagcgactt caccgacagc gtgcgcgacc cgaagaccag
     2581 cgagatcctg gacatctctc cctgctcctt cggcggtgtc agcgtgatca cgcccggcac
     2641 caacgccagc tccgaggtgg ccgtcctgta ccaggacgtg aactgcaccg acgtcagcac
     2701 cgccatccac gcggaccagc tgacgcccgc ctggcgcatc tacagcaccg gcaacaacgt
     2761 gttccagacc caggccggct gcctgatcgg cgccgagcac gtcgacacca gctacgagtg
     2821 cgacatcccg attggcgccg ggatctgcgc cagctaccac accgtgagcc tgctccgctc
     2881 caccagccag aagtccatcg tggcctacac catgagcctg ggcgccgaca gcagcatcgc
     2941 ctacagcaac aacaccatcg ccatcccgac caacttctcc atcagcatca cgaccgaggt
     3001 gatgcccgtg agcatggcca agaccagcgt ggactgcaac atgtacatct gcggcgacag
     3061 caccgagtgc gccaacctgc tcctgcagta cggctccttc tgcacccagc tgaaccgcgc
     3121 cctgagcggc atcgccgccg agcaggaccg caacacccgc gaggtgttcg cccaggtgaa
     3181 gcagatgtac aagacgccca ccctgaagta cttcggcggc ttcaacttca gccagatcct
     3241 gcccgacccg ctgaagccca ccaagcgcag cttcatcgag gacctgctct tcaacaaggt
     3301 gacgctggcc gacgccggct tcatgaagca gtacggcgag tgcctgggcg acatcaacgc
     3361 ccgcgacctg atctgcgccc agaagttcaa cggcctgacc gtgctcccgc cgctgctgac
     3421 cgacgacatg atcgccgcgt acaccgccgc gctggtgagc ggcaccgcca cggccggctg
     3481 gaccttcggc gccggcgcgg ccctgcagat cccgttcgcc atgcagatgg cctaccgctt
     3541 caacggcatc ggcgtgaccc agaacgtgct gtacgagaac cagaagcaga tcgccaacca
     3601 gttcaacaag gccatcagcc agatccagga gagcctgacc acgaccagca cggccctggg
     3661 caagctgcag gacgtggtca accagaacgc ccaggccctg aacaccctgg tgaagcagct
     3721 gtccagcaac ttcggcgcca tcagctccgt gctgaacgat atcctgagcc gcctggacaa
     3781 ggtggaggcc gaggtgcaga tcgatcgcct gatcaccggc cgcctgcaga gcctgcagac
     3841 ctacgtgacc cagcagctga tccgcgccgc ggagatccgc gccagcgcga acctggccgc
     3901 caccaagatg tccgagtgcg tgctgggcca gagcaagcgc gtggacttct gcggcaaggg
     3961 ctaccacctg atgagcttcc cgcaggccgc gccgcacggc gtggtcttcc tgcacgtgac
     4021 ctacgtgccc agccaggagc gcaacttcac cacggcgccc gccatctgcc acgagggcaa
     4081 ggcctacttc ccgcgcgagg gcgtgttcgt gttccagggc accagctggt tcatcaccca
     4141 gcgcaacttc ttcagcccgc aaatcatcac cacggacaac accttcgtga gcggcaactg
     4201 cgacgtcgtg atcggcatca tccagaacac cgtgtacgac ccgctgcagc ccgagctgga
     4261 ctccttcaag gaggagctgg acaagtactt caagcagcac accagcccgg acgtggacct
     4321 gggcgacatc agcggcatcc aggccagcgt cgtgaacatc cagaaggaga tcgaccgcct
     4381 gcaggaggtg gccaagaacc tgaacgagag cctgatcgac ctccaggagc tgggcaagta
     4441 cgagcagtac atcaagtggc cttggtatgt gtggctgggc ttcatcgccg ggctgatcgc
     4501 catcgtgatg gtgaccatcc tgctctgctg catgaccagc tgctgctcct gcctgaaggg
     4561 cgcctgcagc tgcggcagct gctgcaagtt cgacgaggac gacagcgagc ccgtgctgaa
     4621 gggcgtgaag ctgcactaca cgggcggcac cgagaccagc caggtggcgc ccgcctagga
     4681 tccgagctcg gtaccaagct taagtttaaa ccgctgatca gcctcgactg tgccttctag
     4741 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     4801 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     4861 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     4921 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     4981 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5041 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     5101 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5161 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     5221 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5281 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     5341 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     5401 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     5461 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     5521 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     5581 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     5641 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     5701 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     5761 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     5821 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     5881 gtggagaggc tattcggcta tgactgggca acagacaatc ggctgctctg atgccgccgt
     5941 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     6001 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     6061 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     6121 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     6181 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     6241 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     6301 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     6361 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     6421 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     6481 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     6541 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     6601 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     6661 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     6721 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     6781 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     6841 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     6901 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     6961 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     7021 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     7081 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     7141 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     7201 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     7261 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     7321 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     7381 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     7441 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     7501 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     7561 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     7621 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     7681 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     7741 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     7801 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     7861 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     7921 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     7981 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     8041 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     8101 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     8161 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     8221 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     8281 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     8341 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     8401 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     8461 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     8521 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     8581 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     8641 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     8701 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     8761 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     8821 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     8881 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     8941 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     9001 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     9061 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     9121 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     9181 gctctgatgc cgcatagtta agccagtatc tgctc
//
