LOCUS       sequence-335277-        6471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 virus NSP1 with
            N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6471)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 6471)
  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 _NSP1
FEATURES             Location/Qualifiers
     source          1..6471
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     400..422
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     484..503
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     542..561
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        542..560
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             704..727
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             734..1444
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             734..1444
                     /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(1034..1053)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1381..1402
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1445..1468
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    1469..1493
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2039..2063)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2098..2115
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2205..2224)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2270..2325
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2324..2343)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      2413..2460
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     2473..3178
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           2561..2933
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(2590..3178)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(3187..3220)
                     /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(3388..3975)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3467..3486)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3988..4779,4780..4848))
                     /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     4611..4630
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_recomb     5091..5918
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             5091..5215
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(5253..5858)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        6050..6353
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
       61 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      121 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      181 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      241 ttcgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      301 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggctgtccgc
      361 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
      421 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
      481 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg gatcggacgt
      541 ctaatacgac tcactatagg gacatcttaa gtttatttta ttttatttta ttttatttta
      601 ttttatttta ttttatttta ttttatttta tttaaccatg acagtaatgt ataaagtctg
      661 taaagacatt aaacacgtaa gtgaaaccat gggcacctct ggtcaccacc atcaccacca
      721 tcaccacggc acaagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
      781 gttggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
      841 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
      901 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
      961 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     1021 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1081 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     1141 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     1201 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     1261 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     1321 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     1381 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctata
     1441 caagctggag gtgctcttcc agggtccgac aagtttgtac aaaaaagcag gcttaatgga
     1501 atctttagtt ccaggtttta atgaaaagac tcatgttcaa ttgtcattac cagttttgca
     1561 agttagagat gttttagtta gaggttttgg tgactctgtt gaagaagttt tgtcagaagc
     1621 tagacaacat ttgaaagatg gtacatgtgg tttggttgaa gttgaaaaag gtgttttgcc
     1681 acaattagaa caaccatacg tttttattaa gagatcagat gctagaactg caccacatgg
     1741 tcatgttatg gttgaattgg ttgctgaatt agaaggtatt caatatggta gatcaggtga
     1801 aacattgggt gttttagttc cacatgttgg tgaaatccca gttgcataca gaaaggtttt
     1861 gttgagaaag aatggtaata agggtgctgg tggtcattct tacggtgcag atttgaaatc
     1921 atttgatttg ggtgacgaat taggtactga tccatacgaa gatttccaag aaaactggaa
     1981 cactaagcat tcttcaggtg ttacaagaga attgatgaga gaattaaatg gtggttaaac
     2041 ccagctttct tgtacaaagt ggtgtaagcg gccgccagct ttctagacca gtttaaacat
     2101 caccatcacc atcactaagt gattaacctc aggtgcaggc tgcctatcag aaggtggtgg
     2161 ctggtgtggc caatgccctg gctcacaaat accactgaga tcgatctttt tccctctgcc
     2221 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa
     2281 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat
     2341 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat
     2401 gcccatatgt aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2461 ctgaaagcat gcggaggaaa ttctccttga agtttccctg gtgttcaaag taaaggagtt
     2521 tgcaccagac gcacctctgt tcactggtcc ggcgtattaa aacacgatac attgttatta
     2581 gtacatttat taagcgctag attctgtgcg ttgttgattt acagacaatt gttgtacgta
     2641 ttttaataat tcattaaatt tataatcttt agggtggtat gttagagcga aaatcaaatg
     2701 attttcagcg tctttatatc tgaatttaaa tattaaatcc tcaatagatt tgtaaaatag
     2761 gtttcgatta gtttcaaaca agggttgttt ttccgaaccg atggctggac tatctaatgg
     2821 attttcgctc aacgccacaa aacttgccaa atcttgtagc agcaatctag ctttgtcgat
     2881 attcgtttgt gttttgtttt gtaataaagg ttcgacgtcg ttcaaaatat tatgcgcttt
     2941 tgtatttctt tcatcactgt cgttagtgta caattgactc gacgtaaaca cgttaaatag
     3001 agcttggaca tatttaacat cgggcgtgtt agctttatta ggccgattat cgtcgtcgtc
     3061 ccaaccctcg tcgttagaag ttgcttccga agacgatttt gccatagcca cacgacgcct
     3121 attaattgtg tcggctaaca cgtccgcgat caaatttgta gttgagcttt ttggaattgc
     3181 gatcgcataa cttcgtatag catacattat acgaagttat aagctcggaa cgctgcgctc
     3241 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3301 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3361 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgccccctga cgagcatcac
     3421 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3481 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3541 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3601 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3661 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3721 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3781 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     3841 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3901 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3961 aaaaaaggat ctcaagaaga tcctttgtta ccaatgctta atcagtgagg cacctatctc
     4021 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4081 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4141 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4201 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4261 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4321 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4381 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4441 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4501 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4561 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4621 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4681 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4741 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4801 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4861 tcaatattat tgaagcattt atcagggtta ttgtctcatg tccgcgcgtt tcctgcatct
     4921 tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga ggtttatact
     4981 aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat gtttaatcaa
     5041 ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca tgcatctttt
     5101 aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa ctgtcgacaa
     5161 gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt attgaataat
     5221 aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa cgcaacaaga
     5281 acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag gtaatattta
     5341 aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt ttcacataaa
     5401 ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt ctcttcataa
     5461 aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt aaattttttg
     5521 ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg catagttttt
     5581 ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc tttaattatt
     5641 aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt gccggcatag
     5701 tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt aacataactt
     5761 tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc tatactattg
     5821 tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg cacaaactaa
     5881 tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggccg taatgagacg
     5941 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctct agttattaat
     6001 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     6061 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     6121 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     6181 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     6241 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     6301 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atgcatggtc
     6361 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
     6421 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg g
//
