LOCUS       sequence-441975-        8757 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutated SARS-CoV-2 Spike under CMV with D614G, C-term 19 amino acid 
            truncation, and 3x FLAG. Encodes F486S mutation. For expression and 
            pseudotyping lentiviral particles. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8757)
  AUTHORS   Gunnels TF, Stranford DM, Mitrut RE, Kamat NP, Leonard JN
  TITLE     Elucidating Design Principles for Engineering Cell-Derived Vesicles 
            to Inhibit SARS-CoV-2 Infection.
  JOURNAL   Small. 2022 May;18(19):e2200125. doi: 10.1002/smll.202200125. Epub 
            2022 Apr 7.
  PUBMED    35388947
REFERENCE   2  (bases 1 to 8757)
  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: "Small."; 
            date: "2022-05"; volume: "18(19):e2200125. doi"; pages: " 
            10.1002/smll.202200125. Epub 2022 Apr 7"
COMMENT     Sequence Label: pTFG027_Spike_sACE2-resistant_D614G_F486S_3xFlag
FEATURES             Location/Qualifiers
     source          1..8757
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          954..1429
                     /label=beta-globin intron
                     /note="internally truncated intron from human beta-globin"
     CDS             5274..5339
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(5387..5404)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5393..5617
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5663..6091
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5750..5769)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5960..5981
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6114..6310
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6161..6296
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6223..6242
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    6430..6551
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6467..6486)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6521..6540
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6600..6616)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6600..6616)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6613..6635)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6624..6640
                     /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(6648..6654,6655..6672,6673..6678))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6693..6714
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6831..6848)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7002..7590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7082..7101)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7761..8552,8553..8621))
                     /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     8384..8403
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8622..8726)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagtcccaa gctggctagc
      901 gtttaaactt aagcttacat gtggtaccga gctcggatcc tgagaacttc agggtgagtc
      961 tatgggaccc ttgatgtttt ctttcccctt cttttctatg gttaagttca tgtcatagga
     1021 aggggagaag taacagggta cacatattga ccaaatcagg gtaattttgc atttgtaatt
     1081 ttaaaaaatg ctttcttctt ttaatatact tttttgttta tcttatttct aatactttcc
     1141 ctaatctctt tctttcaggg caataatgat acaatgtatc atgcctcttt gcaccattct
     1201 aaagaataac agtgataatt tctgggttaa ggcaatagca atatttctgc atataaatat
     1261 ttctgcatat aaattgtaac tgatgtaaga ggtttcatat tgctaatagc agctacaatc
     1321 cagctaccat tctgctttta ttttatggtt gggataaggc tggattattc tgagtccaag
     1381 ctaggccctt ttgctaatca tgttcatacc tcttatcttc ctcccacagc tcctgggcaa
     1441 cgtgctggtc tgtgtgctgg cccatcactt tggcaaagca cgtgagatct gaattcgcca
     1501 ccatgtttgt cttcctggtc ctgctgcctc tggtctcgtc tcagtgcgtg aacctgacta
     1561 ctagaaccca gctgcctcct gcctatacta actccttcac ccgcggcgtg tactacccag
     1621 acaaggtgtt ccgcagctcc gtgctgcact ccacccagga tctgttcctg cccttcttca
     1681 gcaacgtgac ctggttccac gccatccacg tgagcggcac caatggcacc aagcggttcg
     1741 acaatcccgt gctgccattc aacgatggcg tgtacttcgc ctccaccgag aagagcaaca
     1801 tcatccgcgg ctggatcttc ggcaccaccc tggactccaa gacccagagc ctgctgatcg
     1861 tgaacaatgc caccaacgtg gtcatcaagg tgtgcgagtt ccagttctgc aatgatccat
     1921 tcctgggcgt gtactaccac aagaacaata agtcctggat ggagagcgag ttccgcgtgt
     1981 acagctccgc caacaattgc accttcgagt acgtgtccca gcccttcctg atggacctgg
     2041 agggcaagca gggcaatttc aagaacctgc gcgagttcgt gttcaagaat atcgatggct
     2101 acttcaagat ctactccaag cacaccccca tcaacctggt gcgcgacctg ccacagggct
     2161 tcagcgccct ggagccactg gtggatctgc caatcggcat caacatcacc aggttccaga
     2221 ccctgctggc cctgcaccgc agctacctga ccccaggcga cagctccagc ggatggaccg
     2281 ctggagctgc tgcctactac gtgggctacc tgcagccccg caccttcctg ctgaagtaca
     2341 acgagaatgg caccatcacc gacgccgtgg attgcgccct ggatccactg tccgagacaa
     2401 agtgcaccct gaagagcttc accgtggaga agggcatcta ccagacctcc aatttccgcg
     2461 tgcagccaac cgagagcatc gtgcgcttcc ccaatatcac caacctgtgc ccattcggcg
     2521 aggtgttcaa cgctaccagg ttcgccagcg tgtacgcttg gaatcgcaag cgcatctcca
     2581 actgcgtggc cgactacagc gtgctgtaca actccgccag cttctccacc ttcaagtgct
     2641 acggcgtgtc ccccaccaag ctgaatgatc tgtgcttcac caacgtgtac gccgatagct
     2701 tcgtgatcag gggcgacgag gtgcgccaga tcgctccagg acagaccggc aagatcgctg
     2761 actacaatta caagctgccc gacgatttca ccggctgcgt gatcgcctgg aactccaaca
     2821 atctggatag caaagtgggc ggcaactaca attacctgta ccgcctgttc cgcaagtcca
     2881 atctgaagcc attcgagcgc gacatctcca ccgagatcta ccaggctgga agcaccccat
     2941 gcaatggagt ggagggcagc aactgctact tccccctgca gagctacggc ttccagccaa
     3001 ccaacggagt gggataccag ccatacaggg tggtggtgct gtcattcgag ctgctgcacg
     3061 ctccagctac cgtgtgcgga ccaaagaaga gcaccaatct ggtgaagaac aagtgcgtga
     3121 acttcaattt caacggcctg accggaaccg gcgtgctgac cgagtccaac aagaagttcc
     3181 tgccattcca gcagttcgga agggacatcg ctgataccac cgacgccgtg cgcgacccac
     3241 agaccctgga gatcctggat atcaccccat gctccttcgg cggcgtgagc gtgatcaccc
     3301 caggaaccaa taccagcaac caggtggccg tgctgtacca gggcgtgaat tgcaccgagg
     3361 tgccagtggc tatccacgct gatcagctga ccccaacctg gcgcgtgtac agcaccggat
     3421 ccaacgtgtt ccagacccgc gccggatgcc tgatcggagc tgagcacgtg aacaattcct
     3481 acgagtgcga catcccaatc ggagctggaa tctgcgccag ctaccagacc cagaccaact
     3541 ccccaaggag ggctcgcagc gtggccagcc agtccatcat cgcctacacc atgtccctgg
     3601 gcgccgagaa tagcgtggcc tacagcaaca attccatcgc catcccaacc aacttcacca
     3661 tctccgtgac caccgagatc ctgcccgtgt ccatgaccaa gaccagcgtg gactgcacca
     3721 tgtacatctg cggcgattcc accgagtgca gcaacctgct gctgcagtac ggcagcttct
     3781 gcacccagct gaatcgcgcc ctgaccggaa tcgctgtgga gcaggataag aacacccagg
     3841 aggtgttcgc ccaggtgaag cagatctaca agaccccccc aatcaaggac ttcggcggct
     3901 tcaatttcag ccagatcctg cccgatccaa gcaagccctc caagcgcagc ttcatcgagg
     3961 acctgctgtt caacaaggtg accctggccg atgccggctt catcaagcag tacggcgatt
     4021 gcctgggcga catcgctgcc cgcgacctga tctgcgccca gaagttcaat ggcctgaccg
     4081 tgctgccacc actgctgacc gatgagatga tcgctcagta cacctccgcc ctgctggccg
     4141 gaaccatcac cagcggatgg accttcggcg ctggagccgc cctgcagatc cccttcgcca
     4201 tgcagatggc ctaccgcttc aacggcatcg gcgtgaccca gaatgtgctg tacgagaacc
     4261 agaagctgat cgccaatcag ttcaactccg ccatcggcaa gatccaggac tccctgtcca
     4321 gcaccgccag cgccctgggc aagctgcagg atgtggtgaa tcagaacgcc caggccctga
     4381 ataccctggt gaagcagctg tccagcaact tcggcgccat ctccagcgtg ctgaatgata
     4441 tcctgagccg cctggacaag gtggaggctg aggtgcagat cgataggctg atcaccggcc
     4501 gcctgcagtc cctgcagacc tacgtgaccc agcagctgat cagggctgct gagatcaggg
     4561 ccagcgccaa tctggctgct accaagatgt ccgagtgcgt gctgggacag agcaagaggg
     4621 tggacttctg cggcaagggc taccacctga tgtccttccc acagagcgcc ccacacggag
     4681 tggtgttcct gcacgtgacc tacgtgccag cccaggagaa gaacttcacc accgctccag
     4741 ctatctgcca cgatggcaag gctcacttcc cacgcgaggg cgtgttcgtg tccaacggca
     4801 cccactggtt cgtgacccag cgcaatttct acgagcccca gatcatcacc accgacaata
     4861 ccttcgtgag cggcaactgc gacgtggtca tcggaatcgt gaacaatacc gtgtacgatc
     4921 ccctgcagcc agagctggac tccttcaagg aggagctgga taagtacttc aagaatcaca
     4981 ccagccccga cgtggatctg ggcgacatct ccggcatcaa tgccagcgtg gtgaacatcc
     5041 agaaggagat cgaccgcctg aacgaggtgg ccaagaatct gaacgagtcc ctgatcgatc
     5101 tgcaggagct gggcaagtac gagcagtaca tcaagtggcc atggtacatc tggctgggct
     5161 tcatcgccgg cctgatcgcc atcgtgatgg tgaccatcat gctgtgctgc atgacctcct
     5221 gctgcagctg cctgaagggc tgctgctcct gcggcagctg ctgcggcacc ggtgattaca
     5281 aggaccacga tggcgactat aaggatcacg acatcgacta caaggacgat gacgacaagt
     5341 aagcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     5401 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     5461 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     5521 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagtc
     5581 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     5641 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5701 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     5761 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5821 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5881 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5941 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     6001 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     6061 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttaggct
     6121 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     6181 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     6241 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     6301 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggaggttggg cttcggaatc
     6361 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     6421 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     6481 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     6541 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     6601 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     6661 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     6721 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6781 ggccaacgcg cggggagagg cggtttgcgt attgggsgct cttccgcttc ctcgctcact
     6841 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6901 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6961 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     7021 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     7081 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     7141 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     7201 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     7261 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     7321 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     7381 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     7441 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     7501 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     7561 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     7621 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     7681 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     7741 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7801 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7861 gagggcttac catctggccc cagtgctgca atgataccgc gagayccacg ctcaccggct
     7921 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7981 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     8041 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     8101 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     8161 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     8221 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     8281 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     8341 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     8401 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     8461 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     8521 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     8581 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     8641 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     8701 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
