LOCUS       sequence-336870-        6570 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 virus NSP8 with
            N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6570)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 6570)
  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_NSP8
FEATURES             Location/Qualifiers
     source          1..6570
                     /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"
     promoter        1449..1726
                     /label=chicken beta-actin promoter
     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(3699..3723)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3758..3775
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3865..3884)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3930..3985
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3984..4003)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4073..4120
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4133..4838
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4221..4593
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4250..4838)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4847..4880)
                     /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(5048..5635)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5127..5146)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5648..6439,6440..6508))
                     /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     6271..6290
                     /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 gggsgaggcg
     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 agcaggctta atggctattg cttctgaatt
     3121 ttcttctttg ccatcttatg ctgcttttgc tacagctcaa gaagcttatg aacaagctgt
     3181 tgctaatggt gattctgaag ttgttttaaa aaagttgaag aagtccttga acgttgctaa
     3241 atctgaattt gatagagatg ctgctatgca aagaaaatta gaaaaaatgg ctgaccaagc
     3301 tatgacacaa atgtataaac aagctagatc tgaagataag agagctaaag ttacatctgc
     3361 tatgcaaaca atgttattta ctatgttgag gaagttggat aacgatgctt taaataatat
     3421 catcaacaac gctagagacg gttgtgttcc attaaatatt attccattga ccacagctgc
     3481 taaattgatg gttgttattc cagattataa cacttacaag aacacttgtg atggtacaac
     3541 ttttacttat gcttctgctt tgtgggaaat tcaacaagtt gttgatgctg attctaaaat
     3601 tgttcaattg tctgaaatct ccatggataa ttctccaaat ttagcttggc cattgattgt
     3661 tactgcttta agagctaatt ctgctgttaa attgcaaaac ccagctttct tgtacaaagt
     3721 ggtgtaagcg gccgccagct ttctagacca gtttaaacat caccatcacc atcactaagt
     3781 gattaacctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     3841 gctcacaaat accactgaga tcgatctttt tccctctgcc aaaaattatg gggacatcat
     3901 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt
     3961 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa
     4021 acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgt aactagcata
     4081 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaagcat gcggaggaaa
     4141 ttctccttga agtttccctg gtgttcaaag taaaggagtt tgcaccagac gcacctctgt
     4201 tcactggtcc ggcgtattaa aacacgatac attgttatta gtacatttat taagcgctag
     4261 attctgtgcg ttgttgattt acagacaatt gttgtacgta ttttaataat tcattaaatt
     4321 tataatcttt agggtggtat gttagagcga aaatcaaatg attttcagcg tctttatatc
     4381 tgaatttaaa tattaaatcc tcaatagatt tgtaaaatag gtttcgatta gtttcaaaca
     4441 agggttgttt ttccgaaccg atggctggac tatctaatgg attttcgctc aacgccacaa
     4501 aacttgccaa atcttgtagc agcaatctag ctttgtcgat attcgtttgt gttttgtttt
     4561 gtaataaagg ttcgacgtcg ttcaaaatat tatgcgcttt tgtatttctt tcatcactgt
     4621 cgttagtgta caattgactc gacgtaaaca cgttaaatag agcttggaca tatttaacat
     4681 cgggcgtgtt agctttatta ggccgattat cgtcgtcgtc ccaaccctcg tcgttagaag
     4741 ttgcttccga agacgatttt gccatagcca cacgacgcct attaattgtg tcggctaaca
     4801 cgtccgcgat caaatttgta gttgagcttt ttggaattgc gatcgcataa cttcgtatag
     4861 catacattat acgaagttat aagctcggaa cgctgcgctc ggtcgttcgg ctgcggcgag
     4921 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4981 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5041 tggcgttttt ccataggctc cgccccctga cgagcatcac aaaaatcgac gctcaagtca
     5101 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5161 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5221 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5281 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5341 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5401 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5461 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5521 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5581 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5641 tcctttgtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5701 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5761 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5821 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5881 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5941 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6001 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6061 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6121 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6181 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6241 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6301 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6361 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6421 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6481 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6541 atcagggtta ttgtctcatg tccgcgcgtt
//
