LOCUS       sequence-336889-        8028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 virus N-term 
            fragment of S protein after the furin cleavag with N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8028)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 8028)
  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: pmCell-Free_KA1_SARS-COV-2_S-frag3
FEATURES             Location/Qualifiers
     source          1..8028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1140..1443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2005..2027
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2089..2108
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2147..2166
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2147..2165
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2309..2332
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2339..3049
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2339..3049
                     /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(2639..2658)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2986..3007
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3050..3073
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    3074..3098
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5157..5181)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5216..5233
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5323..5342)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5388..5443
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5442..5461)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      5531..5578
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5591..6296
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5679..6051
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5708..6296)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(6305..6338)
                     /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(6506..7093)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6585..6604)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7106..7897,7898..7966))
                     /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     7729..7748
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggccg
     1021 taatgagacg cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctct
     1081 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     1141 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     1201 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     1261 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     1321 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     1381 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     1441 atgcatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     1501 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     1561 gggggggggg cgcgcgccgg gggggggggg gggggggggg gggsggggsg gggsgrggsg
     1621 grgaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     1681 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1741 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1801 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctccttcgg gctgtaatta
     1861 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
     1921 ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc
     1981 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     2041 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     2101 ttattgtgct gtctcatcat tttggcaaag aattggatcg gacgtctaat acgactcact
     2161 atagggacat cttaagttta ttttatttta ttttatttta ttttatttta ttttatttta
     2221 ttttatttta ttttatttaa ccatgacagt aatgtataaa gtctgtaaag acattaaaca
     2281 cgtaagtgaa accatgggca cctctggtca ccaccatcac caccatcacc acggcacaag
     2341 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagttgg acggcgacgt
     2401 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2461 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2521 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2581 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2641 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2701 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2761 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2821 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2881 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2941 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3001 gttcgtgacc gccgccggga tcactctcgg catggacgag ctatacaagc tggaggtgct
     3061 cttccagggt ccgacaagtt tgtacaaaaa agttggcatg ttcgtgttcc tggtgctgct
     3121 gccactggtg agctcccagt gcgtgaacct gaccaccaga acccagctgc cacctgccta
     3181 caccaactcc ttcaccaggg gcgtgtacta cccagacaag gtgttcagat ccagcgtgct
     3241 gcactctacc caggacctgt tcctgccatt cttctctaac gtgacctggt tccacgccat
     3301 ccacgtgtct ggaaccaacg gcaccaagag attcgacaac ccagtgctgc cattcaacga
     3361 cggcgtgtac ttcgcttcta ccgagaagtc taacatcatc aggggttgga tcttcggaac
     3421 caccctggac tctaagaccc agtctctgct gatcgtgaac aacgctacca acgtggtgat
     3481 caaggtgtgc gagttccagt tctgcaacga cccattcctg ggagtgtact accacaagaa
     3541 caacaagtct tggatggaaa gcgagttcag ggtgtacagc tctgctaaca actgcacctt
     3601 cgagtacgtg tctcagcctt tcctgatgga cctggaagga aagcagggaa acttcaagaa
     3661 cctgagagag ttcgtgttca agaacatcga cggttacttc aagatataca gcaagcacac
     3721 cccaatcaac ctggtgaggg acctgccaca gggtttctct gctctggagc ccctggtgga
     3781 cctgccaatc ggcatcaaca tcaccaggtt ccagaccctg ctggctctgc acaggtctta
     3841 cctgacccca ggcgacagca gctctggatg gaccgccggc gccgctgctt actacgtggg
     3901 atacctgcag ccaaggacct tcctgctgaa gtacaacgaa aacggcacca tcaccgacgc
     3961 cgtggactgc gctctggacc cactgtctga gaccaagtgc accctgaagt ctttcaccgt
     4021 ggaaaaggga atctaccaga ccagcaactt ccgcgtgcag cccaccgagt ctatcgtgag
     4081 attcccaaac atcaccaacc tgtgcccatt cggagaagtg ttcaacgcta ccaggttcgc
     4141 cagcgtgtac gcttggaaca ggaagagaat cagcaactgc gtggccgact actctgtgct
     4201 gtacaactct gcctccttct ccaccttcaa gtgctacggc gtgtccccaa ccaagctgaa
     4261 cgacctgtgc ttcaccaacg tgtacgctga ctccttcgtg atcagaggag acgaagtgag
     4321 acagatcgct cccggtcaga ccggaaagat cgccgactac aactacaagc tgccagacga
     4381 cttcaccggt tgcgtgatcg cttggaactc taacaacctg gactctaagg tgggaggcaa
     4441 ctacaactac ctgtacaggc tgttccgcaa gagcaacctg aagcctttcg aaagggacat
     4501 cagcaccgaa atctaccagg ctggatctac cccatgcaac ggagtggaag gattcaactg
     4561 ctacttccct ctgcagtctt acggattcca gccaaccaac ggagtgggat accagccata
     4621 cagagtggtg gtgctgtctt tcgagctgct gcacgctcct gctaccgtgt gcggaccaaa
     4681 gaagtctacc aacctggtga agaacaagtg cgtgaacttc aacttcaacg gactgaccgg
     4741 aaccggagtg ctgaccgaat ccaacaagaa gttcctgcct ttccagcagt tcggtagaga
     4801 catcgccgac accaccgacg ctgtgagaga cccacagacc ctggaaatcc tggacatcac
     4861 cccctgctct ttcggaggag tgagcgtgat caccccagga accaacacct ccaaccaggt
     4921 ggccgtgctg taccaggacg tgaactgcac cgaagtgcca gtggccatcc acgctgacca
     4981 gctgacccca acctggagag tgtacagcac cggatctaac gtgttccaga cccgtgccgg
     5041 ctgcctgatc ggagccgagc acgtgaacaa ctcctacgaa tgcgacatcc caatcggagc
     5101 tggcatctgc gcttcttacc agacccagac caacagccca agaagggcca gatcctaccc
     5161 aactttcttg tacaaagtgg tgtaagcggc cgccagcttt ctagaccagt ttaaacatca
     5221 ccatcaccat cactaagtga ttaacctcag gtgcaggctg cctatcagaa ggtggtggct
     5281 ggtgtggcca atgccctggc tcacaaatac cactgagatc gatctttttc cctctgccaa
     5341 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt
     5401 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg
     5461 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc
     5521 ccatatgtaa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     5581 gaaagcatgc ggaggaaatt ctccttgaag tttccctggt gttcaaagta aaggagtttg
     5641 caccagacgc acctctgttc actggtccgg cgtattaaaa cacgatacat tgttattagt
     5701 acatttatta agcgctagat tctgtgcgtt gttgatttac agacaattgt tgtacgtatt
     5761 ttaataattc attaaattta taatctttag ggtggtatgt tagagcgaaa atcaaatgat
     5821 tttcagcgtc tttatatctg aatttaaata ttaaatcctc aatagatttg taaaataggt
     5881 ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat ggctggacta tctaatggat
     5941 tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag caatctagct ttgtcgatat
     6001 tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt caaaatatta tgcgcttttg
     6061 tatttctttc atcactgtcg ttagtgtaca attgactcga cgtaaacacg ttaaatagag
     6121 cttggacata tttaacatcg ggcgtgttag ctttattagg ccgattatcg tcgtcgtccc
     6181 aaccctcgtc gttagaagtt gcttccgaag acgattttgc catagccaca cgacgcctat
     6241 taattgtgtc ggctaacacg tccgcgatca aatttgtagt tgagcttttt ggaattgcga
     6301 tcgcataact tcgtatagca tacattatac gaagttataa gctcggaacg ctgcgctcgg
     6361 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     6421 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     6481 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg ccccctgacg agcatcacaa
     6541 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6601 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6661 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6721 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6781 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6841 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6901 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6961 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     7021 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     7081 aaaaggatct caagaagatc ctttgttacc aatgcttaat cagtgaggca cctatctcag
     7141 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     7201 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     7261 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     7321 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     7381 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     7441 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     7501 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7561 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7621 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7681 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7741 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7801 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7861 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7921 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7981 aatattattg aagcatttat cagggttatt gtctcatgtc cgcgcgtt
//
