LOCUS       sequence-335424-        7509 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 virus 
            C-terminal ectodomain of S protein from the furin cleavag with 
            N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7509)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 7509)
  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"
COMMENT     Sequence Label: pmCellFree_KA1_SARS-COV-2_S-frag5
FEATURES             Location/Qualifiers
     source          1..7509
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     394..416
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     478..497
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     536..555
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        536..554
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             698..721
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             728..1438
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             728..1438
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(1028..1047)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1375..1396
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1439..1462
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    1463..1487
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3072..3096)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3131..3148
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3238..3257)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3303..3358
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3357..3376)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3446..3493
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3506..4211
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3594..3966
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3623..4211)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4220..4253)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(4421..5008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4500..4519)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5021..5812,5813..5881))
                     /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     5644..5663
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_recomb     6124..6951
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             6124..6248
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6286..6891)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        7083..7386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
       61 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt
      121 cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
      181 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctccttcggg
      241 ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct
      301 tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggctgt ccgcgggggg
      361 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
      421 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
      481 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attggatcgg acgtctaata
      541 cgactcacta tagggacatc ttaagtttat tttattttat tttattttat tttattttat
      601 tttattttat tttattttat tttatttaac catgacagta atgtataaag tctgtaaaga
      661 cattaaacac gtaagtgaaa ccatgggcac ctctggtcac caccatcacc accatcacca
      721 cggcacaagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagttgga
      781 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
      841 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      901 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
      961 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1021 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1081 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1141 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1201 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1261 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1321 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1381 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tatacaagct
     1441 ggaggtgctc ttccagggtc cgacaagttt gtacaaaaaa gttggcatgg tggcctctca
     1501 gagcatcatc gcctacacca tgagcctggg cgctgaaaac agcgtggctt acagcaacaa
     1561 ctctatcgcc atcccaacca acttcaccat ctctgtgacc accgaaatcc tgccagtgag
     1621 catgaccaag accagcgtgg actgcaccat gtacatctgc ggagactcta ccgaatgctc
     1681 taacctgctg ctgcagtacg gcagcttctg cacccagctg aaccgcgctc tgaccggaat
     1741 cgctgtggag caggacaaga acacccagga agtgttcgct caggtgaagc agatatacaa
     1801 gaccccacct atcaaggact tcggaggatt caacttctct cagatcctgc ccgacccatc
     1861 taagccctct aagaggtctt tcatcgaaga cctgctgttc aacaaggtga ccctggccga
     1921 cgccggtttc atcaagcagt acggtgactg cctgggagac atcgccgcta gagacctgat
     1981 ctgcgcccag aagttcaacg gtctgaccgt gctgccacca ctgctgaccg acgagatgat
     2041 cgcccagtac acctctgccc tgctggctgg aaccatcacc tctggttgga ccttcggagc
     2101 tggcgccgcc ctgcagatcc cattcgctat gcagatggcc taccgtttca acggtatcgg
     2161 agtgacccag aacgtgctgt acgaaaacca gaagctgatc gctaaccagt tcaactctgc
     2221 tatcggaaag atccaggact ccctgtcctc taccgcttct gccctgggaa agctgcagga
     2281 cgtggtgaac cagaacgccc aggccctgaa caccctggtg aagcagctgt cttctaactt
     2341 cggagccatc tcctctgtgc tgaacgacat cctgtctagg ctggacaagg tggaagccga
     2401 ggtgcagatc gacagactga tcaccggaag actgcagtct ctgcagacct acgtgaccca
     2461 gcagctgatc agggccgctg aaatcagagc ttctgccaac ctggctgcca ccaagatgtc
     2521 cgaatgcgtg ctgggccagt ctaagagggt ggacttctgc ggaaagggat accacctgat
     2581 gtccttccca cagtctgctc cacacggagt ggtgttcctg cacgtgacct acgtgcccgc
     2641 ccaggaaaag aacttcacca ccgccccagc tatctgccac gacggaaagg ctcacttccc
     2701 cagggaggga gtgttcgtgt ctaacggaac ccactggttc gtgacccaga ggaacttcta
     2761 cgagccacag atcatcacca ccgacaacac cttcgtgtct ggaaactgcg acgtggtgat
     2821 cggaatcgtg aacaacaccg tgtacgaccc actgcagcca gaactggact ctttcaagga
     2881 ggaactggac aagtacttca agaaccacac ctctccagac gtggacctgg gagacatctc
     2941 cggaatcaac gcctctgtgg tgaacatcca gaaggaaatc gacagactga acgaggtggc
     3001 caagaacctg aacgaatctc tgatcgacct gcaggaactg ggaaagtacg agcagtacat
     3061 caagtggccc tacccaactt tcttgtacaa agtggtgtaa gcggccgcca gctttctaga
     3121 ccagtttaaa catcaccatc accatcacta agtgattaac ctcaggtgca ggctgcctat
     3181 cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcgatct
     3241 ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg
     3301 ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact
     3361 cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag
     3421 tttggcaaca tatgcccata tgtaactagc ataacccctt ggggcctcta aacgggtctt
     3481 gaggggtttt ttgctgaaag catgcggagg aaattctcct tgaagtttcc ctggtgttca
     3541 aagtaaagga gtttgcacca gacgcacctc tgttcactgg tccggcgtat taaaacacga
     3601 tacattgtta ttagtacatt tattaagcgc tagattctgt gcgttgttga tttacagaca
     3661 attgttgtac gtattttaat aattcattaa atttataatc tttagggtgg tatgttagag
     3721 cgaaaatcaa atgattttca gcgtctttat atctgaattt aaatattaaa tcctcaatag
     3781 atttgtaaaa taggtttcga ttagtttcaa acaagggttg tttttccgaa ccgatggctg
     3841 gactatctaa tggattttcg ctcaacgcca caaaacttgc caaatcttgt agcagcaatc
     3901 tagctttgtc gatattcgtt tgtgttttgt tttgtaataa aggttcgacg tcgttcaaaa
     3961 tattatgcgc ttttgtattt ctttcatcac tgtcgttagt gtacaattga ctcgacgtaa
     4021 acacgttaaa tagagcttgg acatatttaa catcgggcgt gttagcttta ttaggccgat
     4081 tatcgtcgtc gtcccaaccc tcgtcgttag aagttgcttc cgaagacgat tttgccatag
     4141 ccacacgacg cctattaatt gtgtcggcta acacgtccgc gatcaaattt gtagttgagc
     4201 tttttggaat tgcgatcgca taacttcgta tagcatacat tatacgaagt tataagctcg
     4261 gaacgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4321 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4381 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4441 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4501 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4561 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4621 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4681 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4741 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4801 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4861 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4921 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4981 gattacgcgc agaaaaaaag gatctcaaga agatcctttg ttaccaatgc ttaatcagtg
     5041 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5101 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5161 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5221 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5281 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5341 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5401 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5461 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5521 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5581 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5641 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5701 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5761 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5821 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5881 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgtccgcgc
     5941 gtttcctgca tcttttaatc aaatcccaag atgtgtataa acgcgccggt atgtacagga
     6001 agaggtttat actaaactgt tacattgcaa acgtggtttc gtgtgccaag tgtgaaaacc
     6061 gatgtttaat caaggctctg acgcatttct acaaccacga ctccaagtgt gtgggtgaag
     6121 tcatgcatct tttaatcaaa tcccaagatg tgtataaacc accaaactgc caaaaaatga
     6181 aaactgtcga caagctctgt ccgtttgctg gcaactgcaa gggtctcaat cctatttgta
     6241 attattgaat aataaaacaa ttataaatgt caaatttgtt ttttattaac gatacaaacc
     6301 aaacgcaaca agaacatttg tagtattatc tataattgaa aacgcgtagt tataatcgct
     6361 gaggtaatat ttaaaatcat tttcaaatga ttcacagtta atttgcgaca atataatttt
     6421 attttcacat aaactagacg ccttgtcgtc ttcttcttcg tattccttct ctttttcatt
     6481 tttctcttca taaaaattaa catagttatt atcgtatcca tatatgtatc tatcgtatag
     6541 agtaaatttt ttgttgtcat aaatatatat gtctttttta atggggtgta tagtaccgct
     6601 gcgcatagtt tttctgtaat ttacaacagt gctattttct ggtagttctt cggagtgtgt
     6661 tgctttaatt attaaattta tataatcaat gaatttggga tcgtcggttt tgtacaatat
     6721 gttgccggca tagtacgcag cttcttctag ttcaattaca ccatttttta gcagcaccgg
     6781 attaacataa ctttccaaaa tgttgtacga accgttaaac aaaaacagtt cacctccctt
     6841 ttctatacta ttgtctgcga gcagttgttt gttgttaaaa ataacagcca ttgtaatgag
     6901 acgcacaaac taatatcaca aactggaaat gtctatcaat atatagttgc tgatggccgg
     6961 ccgtaatgag acgcacaaac taatatcaca aactggaaat gtctatcaat atatagttgc
     7021 tctagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     7081 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     7141 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     7201 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     7261 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     7321 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     7381 accatgcatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
     7441 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg
     7501 ggggggggg
//
