LOCUS       sequence-386852-        7547 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Mammalian cell expression of SARS-CoV-2 Spike protein with mutation 
            N317P with furin side replaced by GSAS and with 2 Proline 
            substitution at 986 and 987.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7547)
  AUTHORS   Manrique PD, Chakraborty S, Henderson R, Edwards RJ, Mansbach R, 
            Nguyen K, Stalls V, Saunders C, Mansouri K, Acharya P, Korber B, 
            Gnanakaran S
  TITLE     Network analysis uncovers the communication structure of SARS-CoV-2 
            spike protein identifying sites for immunogen design.
  JOURNAL   iScience. 2023 Jan 20;26(1):105855. doi: 10.1016/j.isci.2022.105855.
            Epub 2022 Dec 26.
  PUBMED    36590900
REFERENCE   2  (bases 1 to 7547)
  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: "iScience.";
            date: "2023-01-20"; volume: "26(1):105855. doi"; pages: " 
            10.1016/j.isci.2022.105855. Epub 2022 Dec 26"
COMMENT     Sequence Label: p-alpha-H-S-GSAS-2P-N317P
FEATURES             Location/Qualifiers
     source          1..7547
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        54..433
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        464..568
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(569..637,638..1429)
                     /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     complement(787..806)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1600..2188
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2089..2108
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2342..2359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2488..2509
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2524..2529,2530..2547,2548..2554)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2562..2578
                     /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)."
     primer_bind     2567..2589
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2586..2602
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2586..2602
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2945..2964
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    2963..3018
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     3064..3083
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(3185..3208)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(3248..3271)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(3278..3301)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             complement(3305..3328)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(7190..7212)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 ggggaggggg gggagatggg gagagtgaag cagaacgtgg ggctcacctc gaccatggta
       61 atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac
      121 tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc
      181 atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga
      241 cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat
      301 gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg
      361 aactccatat atgggctatg aactaatgac cccgtaattg attactatta ataactagtc
      421 aataatcaat gtcgacccag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      481 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      541 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      601 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      661 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      721 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      781 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
      841 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      901 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      961 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1021 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1081 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1141 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1201 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1261 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1321 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1381 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1441 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1501 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1561 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1621 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1681 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1741 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1801 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1861 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1921 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1981 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2041 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2101 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2161 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2221 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2281 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2341 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     2401 gcgttggccg attcattaat gcagcggatc gatccgctgg cacgacaggt ttcccgactg
     2461 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     2521 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     2581 tcacacagga aacagctatg accatgatta cgccaagctt gggctgcagg tcgagggatc
     2641 tccataagag aagagggaca gctatgactg ggagtagtca ggagaggagg aaaaatctgg
     2701 ctagtaaaac atgtaaggaa aattttaggg atgttaaaga aaaaaataac acaaaacaaa
     2761 atataaaaaa aatctaacct caagtcaagg cttttctatg gaataaggaa tggacagcag
     2821 ggggctgttt catatactga tgacctcttt atagccaacc tttgttcatg gcagccagca
     2881 tatgggcata tgttgccaaa ctctaaacca aatactcatt ctgatgtttt aaatgatttg
     2941 ccctcccata tgtccttccg agtgagagac acaaaaaatt ccaacacact attgcaatga
     3001 aaataaattt cctttattag ccagaagtca gatgctcaag gggcttcatg atgtccccat
     3061 aatttttggc agagggaaaa agatctcagt ggtatttgtg agccagggca ttggccacac
     3121 cagccaccac cttctgatag gcagcctgca cctgaggagt gaattatcgc tcgagtcatt
     3181 atcacttttc gaactggggg tggctccaag ctgagccgcc agatccgccc cctccactac
     3241 cgccgccctt ctcgaactgg gggtgggacc aggcgctatg atggtggtga tggtggtgat
     3301 ggcctgggcc ctggaacagc acctccaggg atctgcccag gaatgtgctc agcagtaccc
     3361 actcgccgtc cttccgcacg taggcctggc catctctggg ggcctcgggg atgtatccgg
     3421 atccctgctc gtatttgccg agctcctgca ggtcgatcag ggactcgttc aggttcttgg
     3481 ccacctcgtt cagtctgtcg atttcctttt ggatgttcac cacgctggcg ttaatgccac
     3541 tgatgtcgcc caggtccaca tcggggctgg tgtggttttt aaagtacttg tccagctctt
     3601 ccttaaaaga gtccagctca ggttgcagag ggtcgtacac tgtgttgttc acgattccga
     3661 tgaccacgtc acaattgcca ctcacgaagg tgttgtcggt ggtgataatc tggggttcgt
     3721 agaagtttct ttgtgtcacg aaccagtgtg tgccattgga aacgaaaacg ccctctcttg
     3781 ggaagtgggc cttgccgtcg tggcagatgg caggggcggt tgtgaaattc ttctcttggg
     3841 caggcacgta tgtcacatgc aggaacacca cgccgtgcgg cgcgctctgg gggaagctca
     3901 tcaggtgata gcctttgccg cagaagtcaa cgcgtttaga ctgtcccagc acacactcgg
     3961 acatcttggt agcggcgaga ttggcgctgg ctcttatttc agcggccctg atcagctgtt
     4021 gggtcacgta ggtttgcagg gactgcagtc tgcctgtgat cagtctatcg atctggactt
     4081 cggcttctgg agggtccagt ctagacagga tatcgttcag cacagagctg atggcgccaa
     4141 aattgctgct gagctgcttc accagggtgt tcagggcttg ggcgttctga ttcaccacgt
     4201 cctgcagttt gcccagggcg ctggcggtag aggagaggga gtcctggatc ttgccgatgg
     4261 cagaattgaa ctggttagcg atcagcttct ggttttcata cagcacgttc tgggtcacgc
     4321 cgattccgtt gaatctatag gccatctgca tggcaaaggg gatctgcagg gcggcgccgg
     4381 cgccaaatgt ccacccagat gtgatggtgc cagccagcag ggcagaggtg tactgagcga
     4441 tcatctcgtc ggtcagcaga ggtgggagaa ctgtcaggcc gttgaatttc tgggcgcaga
     4501 tcagatctct ggcggctatg tcgcccaggc aatcgccata ctgtttgata aagccggcat
     4561 cggccagggt gaccttgttg aacagcagat cctcgataaa gctccgcttg ctaggcttag
     4621 aaggatcagg gagaatctgg ctaaagttaa agccgccgaa gtccttgata ggtggggtct
     4681 tgtagatctg cttcacttgg gcgaacacct cctgggtatt tttgtcctgt tccacggcga
     4741 ttccggtcag cgcccggttc agttgggtgc aaaaggagcc gtattgcagc agcaggttgc
     4801 tgcattcagt gctgtcgcca cagatgtaca ttgtacagtc cacgctggtc ttggtcatgc
     4861 tcacgggcag gatctctgtt gtgacgctaa ttgtaaagtt ggtgggaatg gcgatagagt
     4921 tgttggaata ggccacgctg ttctctgcgc ccaggctcat ggtgtaggcg atgatgctct
     4981 ggctagctac agaggaagca gacccagggc tgtttgtctg ggtctggtag ctggcacaga
     5041 tgccggcgcc gatagggatg tcgcactcgt aactgttatt cacgtgctcg gcgccgatca
     5101 gacatccggc cctggtctgg aacacattgg agccagtgga gtacactctc caagtaggtg
     5161 tcagctgatc ggcgtggatg gccacaggaa cctcggtgca gttaacatct tggtacagca
     5221 cagccacctg gttgctggta ttggtcccgg gggtgatcac agaaactcct ccgaaggagc
     5281 agggtgtaat atccaggatt tccagggtct gtggatctct gacggcatct gtggtgtcag
     5341 cgatatcacg gccgaactgc tggaagggga ggaacttttt gttgctttcg gtcagcacgc
     5401 cggtcccggt caggccgttg aagttgaagt tcacgcattt gttcttaacc agattggtgg
     5461 acttcttagg accgcagacg gtggcggggg cgtggagtaa ctcgaaggac agcacgacca
     5521 cccggtatgg ctggtagccg actccattgg taggctggaa gccgtagctt tgcagaggga
     5581 agtaacagtt gaagccttcc accccgttac aaggtgtaga accggcctgg tagatctctg
     5641 tggagatgtc tctctcgaaa ggtttcaggt tagacttgcg aaacagccgg tacaggtagt
     5701 tgtagttgcc gcccaccttg ctatccaggt tgttagagtt ccaggcgatc acgcagcctg
     5761 taaaatcatc aggcagctta tagttgtagt cagcgatctt accggtctgg cctggtgcga
     5821 tctgccgcac ctcatcgcct ctaattacga aggagtcggc gtacacgttg gtgaagcaca
     5881 ggtcattcag ctttgttggg gaaacgccgt agcacttgaa ggtgctgaaa gaggcgctgt
     5941 tatacagcac ggagtagtcg gccacacaat tgctgattct ctttctattc caggcgtaaa
     6001 cggaggcaaa tcttgtggcg ttaaacacct cgccgaaggg gcagaggttg gtaatgtttg
     6061 gaaatcgcac gatgctctct gtaggctgca cccggaaggg gctggtctgg tagatgccct
     6121 tttccacggt gaaggacttc agggtgcact ttgtctcaga gagagggtcc agggcgcagt
     6181 cgacggcgtc tgtgattgtg ccgttctcgt tgtacttcag caggaaggtt ctaggttgca
     6241 ggtagcccac atagtaggct gcagctccgg cggtccatcc gctgctgctg tcgccaggag
     6301 tcaggtagga tctatgcagg gccagcaggg tttggaaccg cgttatgttg atgcctatgg
     6361 gcaggtcgac caggggttcc agggcgctga agccctgagg gaggtcccgg accaggttga
     6421 tgggtgtgtg cttggagtag attttgaagt agccgtcgat gttcttgaac acgaattctc
     6481 tcaggttctt gaagttgcct tgtttcccct caaggtccat cagaaaaggc tgagagacgt
     6541 actcgaatgt gcagttgttg gcgctgctgt agactctgaa ctcgctttcc atccagctct
     6601 tgttgttctt gtggtagtac acacccagga aaggatcatt gcaaaactgg aactcgcaca
     6661 ccttgatcac gacgttcgtg gcattgttca cgatcaggag gctctgtgtt ttgctatcca
     6721 gggtggtgcc gaagatccag ccccggatga tgttactctt ctcggtactg gcgaagtaaa
     6781 ctccatcatt gaatggcagg acggggttat caaaccgctt ggttccgttt gtgccgctca
     6841 cgtggatggc gtggaaccat gtcacgttgc tgaagaacgg caggaagagg tcctgagtgc
     6901 tgtgcaggac gctagatctg aacaccttgt cagggtagta cacgccccgt gtaaagctgt
     6961 tggtgtaggc tggtgggagc tgggttcggg tggtcaggtt cacgcactgg ctgctcacca
     7021 gaggcaggag caccaggaac acgaacatgg tggcggtacc gaactcggcc tgagacggcc
     7081 gcaattcttt gccaaaatga tgagacagca caacaaccag cacgttgccc aggagctgta
     7141 ggaaaaagaa gaaggcatga acatggttag cagaggcgct agagccgccg gtcacacgcc
     7201 agaagccgaa ccccgccctg ccccgtcccc cccgaaggca gccgtccccc cgcggacagc
     7261 cccgaggctg gagagggaga aggggacggc ggcgcggcga cgcacgaagg ccctccccgc
     7321 ccatttcctt cctgccggcg ccgcaccgct tcgccccgcg cccgctagag ggggtgcggc
     7381 ggcgcctccc agatttcggc tccgcacaga tttgggacaa aggaagtccc tgcgccctct
     7441 cgcacgatta ccataaaagg caatggctgc ggctcgccgc gcctcgacag ccgccggcgc
     7501 tccgggggcc gccgcgcccc tcccccgagc cctccccggc ccgaggc
//
