LOCUS       sequence-328453-        7557 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 7557)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 7557)
  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..7557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     184..1011
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             184..308
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(346..951)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1143..1446
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1452..1729
                     /label=chicken beta-actin promoter
     primer_bind     2008..2030
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2092..2111
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2150..2169
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2150..2168
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2312..2335
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2342..3052
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2342..3052
                     /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(2381..2402)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2642..2661)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2989..3010
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3053..3076
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3077..3101
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4686..4710)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4745..4762
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4852..4871)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4917..4972
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4971..4990)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      5060..5107
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5120..5825
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5208..5580
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5237..5825)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5834..5867)
                     /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(6035..6622)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6114..6133)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6635..7426,7427..7495))
                     /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     7258..7277
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctctcctgca tcttttaatc aaatcccaag atgtgtataa acgcgccggt atgtacagga
       61 agaggtttat actaaactgt tacattgcaa acgtggtttc gtgtgccaag tgtgaaaacc
      121 gatgtttaat caaggctctg acgcatttct acaaccacga ctccaagtgt gtgggtgaag
      181 tcatgcatct tttaatcaaa tcccaagatg tgtataaacc accaaactgc caaaaaatga
      241 aaactgtcga caagctctgt ccgtttgctg gcaactgcaa gggtctcaat cctatttgta
      301 attattgaat aataaaacaa ttataaatgt caaatttgtt ttttattaac gatacaaacc
      361 aaacgcaaca agaacatttg tagtattatc tataattgaa aacgcgtagt tataatcgct
      421 gaggtaatat ttaaaatcat tttcaaatga ttcacagtta atttgcgaca atataatttt
      481 attttcacat aaactagacg ccttgtcgtc ttcttcttcg tattccttct ctttttcatt
      541 tttctcttca taaaaattaa catagttatt atcgtatcca tatatgtatc tatcgtatag
      601 agtaaatttt ttgttgtcat aaatatatat gtctttttta atggggtgta tagtaccgct
      661 gcgcatagtt tttctgtaat ttacaacagt gctattttct ggtagttctt cggagtgtgt
      721 tgctttaatt attaaattta tataatcaat gaatttggga tcgtcggttt tgtacaatat
      781 gttgccggca tagtacgcag cttcttctag ttcaattaca ccatttttta gcagcaccgg
      841 attaacataa ctttccaaaa tgttgtacga accgttaaac aaaaacagtt cacctccctt
      901 ttctatacta ttgtctgcga gcagttgttt gttgttaaaa ataacagcca ttgtaatgag
      961 acgcacaaac taatatcaca aactggaaat gtctatcaat atatagttgc tgatggccgg
     1021 ccgtaatgag acgcacaaac taatatcaca aactggaaat gtctatcaat atatagttgc
     1081 tctagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     1141 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     1201 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     1261 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     1321 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     1381 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     1441 accatgcatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
     1501 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg
     1561 gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag
     1621 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
     1681 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
     1741 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
     1801 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctt cgggctgtaa
     1861 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
     1921 gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg ggggacggct
     1981 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     2041 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     2101 tggttattgt gctgtctcat cattttggca aagaattgga tcggacgtct aatacgactc
     2161 actataggga catcttaagt ttattttatt ttattttatt ttattttatt ttattttatt
     2221 ttattttatt ttattttatt taaccatgac agtaatgtat aaagtctgta aagacattaa
     2281 acacgtaagt gaaaccatgg gcacctctgg tcaccaccat caccaccatc accacggcac
     2341 aagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2401 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2461 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2521 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     2581 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     2641 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2701 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2761 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     2821 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     2881 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2941 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     3001 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctataca agctggaggt
     3061 gctcttccag ggtccgacaa gtttgtacaa aaaagttggc atggtggcct ctcagagcat
     3121 catcgcctac accatgagcc tgggcgctga aaacagcgtg gcttacagca acaactctat
     3181 cgccatccca accaacttca ccatctctgt gaccaccgaa atcctgccag tgagcatgac
     3241 caagaccagc gtggactgca ccatgtacat ctgcggagac tctaccgaat gctctaacct
     3301 gctgctgcag tacggcagct tctgcaccca gctgaaccgc gctctgaccg gaatcgctgt
     3361 ggagcaggac aagaacaccc aggaagtgtt cgctcaggtg aagcagatat acaagacccc
     3421 acctatcaag gacttcggag gattcaactt ctctcagatc ctgcccgacc catctaagcc
     3481 ctctaagagg tctttcatcg aagacctgct gttcaacaag gtgaccctgg ccgacgccgg
     3541 tttcatcaag cagtacggtg actgcctggg agacatcgcc gctagagacc tgatctgcgc
     3601 ccagaagttc aacggtctga ccgtgctgcc accactgctg accgacgaga tgatcgccca
     3661 gtacacctct gccctgctgg ctggaaccat cacctctggt tggaccttcg gagctggcgc
     3721 cgccctgcag atcccattcg ctatgcagat ggcctaccgt ttcaacggta tcggagtgac
     3781 ccagaacgtg ctgtacgaaa accagaagct gatcgctaac cagttcaact ctgctatcgg
     3841 aaagatccag gactccctgt cctctaccgc ttctgccctg ggaaagctgc aggacgtggt
     3901 gaaccagaac gcccaggccc tgaacaccct ggtgaagcag ctgtcttcta acttcggagc
     3961 catctcctct gtgctgaacg acatcctgtc taggctggac aaggtggaag ccgaggtgca
     4021 gatcgacaga ctgatcaccg gaagactgca gtctctgcag acctacgtga cccagcagct
     4081 gatcagggcc gctgaaatca gagcttctgc caacctggct gccaccaaga tgtccgaatg
     4141 cgtgctgggc cagtctaaga gggtggactt ctgcggaaag ggataccacc tgatgtcctt
     4201 cccacagtct gctccacacg gagtggtgtt cctgcacgtg acctacgtgc ccgcccagga
     4261 aaagaacttc accaccgccc cagctatctg ccacgacgga aaggctcact tccccaggga
     4321 gggagtgttc gtgtctaacg gaacccactg gttcgtgacc cagaggaact tctacgagcc
     4381 acagatcatc accaccgaca acaccttcgt gtctggaaac tgcgacgtgg tgatcggaat
     4441 cgtgaacaac accgtgtacg acccactgca gccagaactg gactctttca aggaggaact
     4501 ggacaagtac ttcaagaacc acacctctcc agacgtggac ctgggagaca tctccggaat
     4561 caacgcctct gtggtgaaca tccagaagga aatcgacaga ctgaacgagg tggccaagaa
     4621 cctgaacgaa tctctgatcg acctgcagga actgggaaag tacgagcagt acatcaagtg
     4681 gccctaccca actttcttgt acaaagtggt gtaagcggcc gccagctttc tagaccagtt
     4741 taaacatcac catcaccatc actaagtgat taacctcagg tgcaggctgc ctatcagaag
     4801 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatcg atctttttcc
     4861 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     4921 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     4981 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     5041 aacatatgcc catatgtaac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     5101 ttttttgctg aaagcatgcg gaggaaattc tccttgaagt ttccctggtg ttcaaagtaa
     5161 aggagtttgc accagacgca cctctgttca ctggtccggc gtattaaaac acgatacatt
     5221 gttattagta catttattaa gcgctagatt ctgtgcgttg ttgatttaca gacaattgtt
     5281 gtacgtattt taataattca ttaaatttat aatctttagg gtggtatgtt agagcgaaaa
     5341 tcaaatgatt ttcagcgtct ttatatctga atttaaatat taaatcctca atagatttgt
     5401 aaaataggtt tcgattagtt tcaaacaagg gttgtttttc cgaaccgatg gctggactat
     5461 ctaatggatt ttcgctcaac gccacaaaac ttgccaaatc ttgtagcagc aatctagctt
     5521 tgtcgatatt cgtttgtgtt ttgttttgta ataaaggttc gacgtcgttc aaaatattat
     5581 gcgcttttgt atttctttca tcactgtcgt tagtgtacaa ttgactcgac gtaaacacgt
     5641 taaatagagc ttggacatat ttaacatcgg gcgtgttagc tttattaggc cgattatcgt
     5701 cgtcgtccca accctcgtcg ttagaagttg cttccgaaga cgattttgcc atagccacac
     5761 gacgcctatt aattgtgtcg gctaacacgt ccgcgatcaa atttgtagtt gagctttttg
     5821 gaattgcgat cgcataactt cgtatagcat acattatacg aagttataag ctcggaacgc
     5881 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5941 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6001 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc cccctgacga
     6061 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6121 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6181 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg
     6241 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6301 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6361 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6421 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6481 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6541 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6601 gcgcagaaaa aaaggatctc aagaagatcc tttgttacca atgcttaatc agtgaggcac
     6661 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6721 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6781 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6841 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6901 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6961 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7021 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7081 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7141 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7201 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7261 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7321 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7381 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7441 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7501 tcctttttca atattattga agcatttatc agggttattg tctcatgtcc gcgcgtt
//
