LOCUS       sequence-335429-        6561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 mutated RBD 
            domain (N510Y, UK strain) of S protein with N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6561)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 6561)
  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_RBD -N501Y
FEATURES             Location/Qualifiers
     source          1..6561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     406..428
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     490..509
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     548..567
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        548..566
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             710..733
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             740..1450
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             740..1450
                     /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(1040..1059)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1387..1408
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1451..1474
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2184..2201
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2291..2310)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2356..2411
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2410..2429)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      2499..2546
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     2559..3264
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           2647..3019
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(2676..3264)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(3273..3306)
                     /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(3474..4061)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3553..3572)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4074..4865,4866..4934))
                     /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     4697..4716
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_recomb     5177..6004
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             5177..5301
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(5339..5944)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        6136..6439
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg
       61 aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg
      121 gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg
      181 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc
      241 ttctccttcg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct
      301 gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcggggct
      361 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
      421 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
      481 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc
      541 ggacgtctaa tacgactcac tatagggaca tcttaagttt attttatttt attttatttt
      601 attttatttt attttatttt attttatttt attttattta accatgacag taatgtataa
      661 agtctgtaaa gacattaaac acgtaagtga aaccatgggc acctctggtc accaccatca
      721 ccaccatcac cacggcacaa gcaagggcga ggagctgttc accggggtgg tgcccatcct
      781 ggtcgagttg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
      841 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
      901 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
      961 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1021 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     1081 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1141 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     1201 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     1261 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1321 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     1381 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1441 gctatacaag ctggaggtgc tcttccaggg tccgatgcgc gttcaaccaa ccgagagcat
     1501 tgtgcgcttt ccaaatatta caaacctttg tccatttgga gaggttttca atgcgacgcg
     1561 cttcgcaagt gtctatgcct ggaaccgcaa aagaatttcc aactgtgttg ctgattactc
     1621 tgtcctgtat aactctgcaa gtttcagcac ctttaaatgt tacggtgttt cccccactaa
     1681 acttaacgat ttatgtttta ctaacgtgta cgctgactcg ttcgtcataa gaggggatga
     1741 ggtaagacaa atcgctcctg gtcagactgg taaaattgct gactacaatt acaagttacc
     1801 cgatgacttt accggctgtg tcatcgcttg gaattccaac aacttagaca gcaaggtggg
     1861 tggcaactat aattatttgt atcgtttgtt cagaaagtcc aatcttaaac catttgaacg
     1921 ggacatctct acagagatat accaggccgg ttcgacccct tgtaacgggg tcgaagggtt
     1981 caattgttac tttccattac aaagctacgg ctttcaacca acgtatggag tggggtatca
     2041 accgtacaga gtagtagtct taagtttcga gttacttcat gcgcctgcca ccgtatgcgg
     2101 ccccaaaaaa agcacgaatt tagtcaagaa caaatgcgtc aatttttaag taagcggccg
     2161 ccagctttct agaccagttt aaacatcacc atcaccatca ctaagtgatt aacctcaggt
     2221 gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca
     2281 ctgagatcga tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc
     2341 atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt
     2401 tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt
     2461 atttggttta gagtttggca acatatgccc atatgtaact agcataaccc cttggggcct
     2521 ctaaacgggt cttgaggggt tttttgctga aagcatgcgg aggaaattct ccttgaagtt
     2581 tccctggtgt tcaaagtaaa ggagtttgca ccagacgcac ctctgttcac tggtccggcg
     2641 tattaaaaca cgatacattg ttattagtac atttattaag cgctagattc tgtgcgttgt
     2701 tgatttacag acaattgttg tacgtatttt aataattcat taaatttata atctttaggg
     2761 tggtatgtta gagcgaaaat caaatgattt tcagcgtctt tatatctgaa tttaaatatt
     2821 aaatcctcaa tagatttgta aaataggttt cgattagttt caaacaaggg ttgtttttcc
     2881 gaaccgatgg ctggactatc taatggattt tcgctcaacg ccacaaaact tgccaaatct
     2941 tgtagcagca atctagcttt gtcgatattc gtttgtgttt tgttttgtaa taaaggttcg
     3001 acgtcgttca aaatattatg cgcttttgta tttctttcat cactgtcgtt agtgtacaat
     3061 tgactcgacg taaacacgtt aaatagagct tggacatatt taacatcggg cgtgttagct
     3121 ttattaggcc gattatcgtc gtcgtcccaa ccctcgtcgt tagaagttgc ttccgaagac
     3181 gattttgcca tagccacacg acgcctatta attgtgtcgg ctaacacgtc cgcgatcaaa
     3241 tttgtagttg agctttttgg aattgcgatc gcataacttc gtatagcata cattatacga
     3301 agttataagc tcggaacgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3361 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3421 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3481 aggctccgcc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3541 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3601 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3661 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3721 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3781 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3841 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3901 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3961 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4021 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgttaccaa
     4081 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4141 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4201 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4261 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4321 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4381 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4441 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     4501 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     4561 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4621 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     4681 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4741 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4801 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4861 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     4921 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4981 ctcatgtccg cgcgtttcct gcatctttta atcaaatccc aagatgtgta taaacgcgcc
     5041 ggtatgtaca ggaagaggtt tatactaaac tgttacattg caaacgtggt ttcgtgtgcc
     5101 aagtgtgaaa accgatgttt aatcaaggct ctgacgcatt tctacaacca cgactccaag
     5161 tgtgtgggtg aagtcatgca tcttttaatc aaatcccaag atgtgtataa accaccaaac
     5221 tgccaaaaaa tgaaaactgt cgacaagctc tgtccgtttg ctggcaactg caagggtctc
     5281 aatcctattt gtaattattg aataataaaa caattataaa tgtcaaattt gttttttatt
     5341 aacgatacaa accaaacgca acaagaacat ttgtagtatt atctataatt gaaaacgcgt
     5401 agttataatc gctgaggtaa tatttaaaat cattttcaaa tgattcacag ttaatttgcg
     5461 acaatataat tttattttca cataaactag acgccttgtc gtcttcttct tcgtattcct
     5521 tctctttttc atttttctct tcataaaaat taacatagtt attatcgtat ccatatatgt
     5581 atctatcgta tagagtaaat tttttgttgt cataaatata tatgtctttt ttaatggggt
     5641 gtatagtacc gctgcgcata gtttttctgt aatttacaac agtgctattt tctggtagtt
     5701 cttcggagtg tgttgcttta attattaaat ttatataatc aatgaatttg ggatcgtcgg
     5761 ttttgtacaa tatgttgccg gcatagtacg cagcttcttc tagttcaatt acaccatttt
     5821 ttagcagcac cggattaaca taactttcca aaatgttgta cgaaccgtta aacaaaaaca
     5881 gttcacctcc cttttctata ctattgtctg cgagcagttg tttgttgtta aaaataacag
     5941 ccattgtaat gagacgcaca aactaatatc acaaactgga aatgtctatc aatatatagt
     6001 tgctgatggc cggccgtaat gagacgcaca aactaatatc acaaactgga aatgtctatc
     6061 aatatatagt tgctctagtt attaatagta atcaattacg gggtcattag ttcatagccc
     6121 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     6181 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     6241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     6301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     6361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     6421 agtcatcgct attaccatgc atggtcgagg tgagccccac gttctgcttc actctcccca
     6481 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
     6541 cgatgggggc gggggggggg g
//
