LOCUS       sequence-339562-        7385 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for SARS-COV-2 virus NSP4 with
            N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7385)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 7385)
  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_NSP4
FEATURES             Location/Qualifiers
     source          1..7385
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_recomb     550..1377
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             610..1215
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     CDS             complement(1253..1377)
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             join(1620..1688,1689..2480)
                     /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     complement(1838..1857)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2493..3080
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2982..3001
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3248..3281
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_recomb     3290..3995
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     CDS             3290..3878
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     3'UTR           3535..3907
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     terminator      complement(4008..4055)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4125..4144
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    4143..4198
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     4244..4263
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(4353..4370)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    4405..4429
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5936..5960)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(5961..5984)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(5985..6695)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             complement(5985..6695)
                     /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(6027..6048)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6376..6395
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             complement(6702..6725)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(6868..6887)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6869..6887)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6926..6945)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(7007..7029)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 cccgccccca tcgctgcaca aaataattaa aaaataaata aatacaaaat tgggggtggg
       61 gagggggggg agatggggag agtgaagcag aacgtggggc tcacctcgac catgcatggt
      121 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
      181 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
      241 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
      301 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
      361 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
      421 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactaga
      481 gcaactatat attgatagac atttccagtt tgtgatatta gtttgtgcgt ctcattacgg
      541 ccggccatca gcaactatat attgatagac atttccagtt tgtgatatta gtttgtgcgt
      601 ctcattacaa tggctgttat ttttaacaac aaacaactgc tcgcagacaa tagtatagaa
      661 aagggaggtg aactgttttt gtttaacggt tcgtacaaca ttttggaaag ttatgttaat
      721 ccggtgctgc taaaaaatgg tgtaattgaa ctagaagaag ctgcgtacta tgccggcaac
      781 atattgtaca aaaccgacga tcccaaattc attgattata taaatttaat aattaaagca
      841 acacactccg aagaactacc agaaaatagc actgttgtaa attacagaaa aactatgcgc
      901 agcggtacta tacaccccat taaaaaagac atatatattt atgacaacaa aaaatttact
      961 ctatacgata gatacatata tggatacgat aataactatg ttaattttta tgaagagaaa
     1021 aatgaaaaag agaaggaata cgaagaagaa gacgacaagg cgtctagttt atgtgaaaat
     1081 aaaattatat tgtcgcaaat taactgtgaa tcatttgaaa atgattttaa atattacctc
     1141 agcgattata actacgcgtt ttcaattata gataatacta caaatgttct tgttgcgttt
     1201 ggtttgtatc gttaataaaa aacaaatttg acatttataa ttgttttatt attcaataat
     1261 tacaaatagg attgagaccc ttgcagttgc cagcaaacgg acagagcttg tcgacagttt
     1321 tcattttttg gcagtttggt ggtttataca catcttggga tttgattaaa agatgcatga
     1381 cttcacccac acacttggag tcgtggttgt agaaatgcgt cagagccttg attaaacatc
     1441 ggttttcaca cttggcacac gaaaccacgt ttgcaatgta acagtttagt ataaacctct
     1501 tcctgtacat accggcgcgt ttatacacat cttgggattt gattaaaaga tgcaggaaac
     1561 gcgcggacat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1621 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1681 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1741 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1801 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1861 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1921 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1981 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     2041 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2101 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2161 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2221 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2281 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     2341 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2401 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2461 cactgattaa gcattggtaa caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2521 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2581 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     2641 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2701 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2761 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2821 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2881 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2941 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3001 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3061 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3121 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3181 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgttc
     3241 cgagcttata acttcgtata atgtatgcta tacgaagtta tgcgatcgca attccaaaaa
     3301 gctcaactac aaatttgatc gcggacgtgt tagccgacac aattaatagg cgtcgtgtgg
     3361 ctatggcaaa atcgtcttcg gaagcaactt ctaacgacga gggttgggac gacgacgata
     3421 atcggcctaa taaagctaac acgcccgatg ttaaatatgt ccaagctcta tttaacgtgt
     3481 ttacgtcgag tcaattgtac actaacgaca gtgatgaaag aaatacaaaa gcgcataata
     3541 ttttgaacga cgtcgaacct ttattacaaa acaaaacaca aacgaatatc gacaaagcta
     3601 gattgctgct acaagatttg gcaagttttg tggcgttgag cgaaaatcca ttagatagtc
     3661 cagccatcgg ttcggaaaaa caacccttgt ttgaaactaa tcgaaaccta ttttacaaat
     3721 ctattgagga tttaatattt aaattcagat ataaagacgc tgaaaatcat ttgattttcg
     3781 ctctaacata ccaccctaaa gattataaat ttaatgaatt attaaaatac gtacaacaat
     3841 tgtctgtaaa tcaacaacgc acagaatcta gcgcttaata aatgtactaa taacaatgta
     3901 tcgtgtttta atacgccgga ccagtgaaca gaggtgcgtc tggtgcaaac tcctttactt
     3961 tgaacaccag ggaaacttca aggagaattt cctccgcatg ctttcagcaa aaaacccctc
     4021 aagacccgtt tagaggcccc aaggggttat gctagttaca tatgggcata tgttgccaaa
     4081 ctctaaacca aatactcatt ctgatgtttt aaatgatttg ccctcccata tgtccttccg
     4141 agtgagagac acaaaaaatt ccaacacact attgcaatga aaataaattt cctttattag
     4201 ccagaagtca gatgctcaag gggcttcatg atgtccccat aatttttggc agagggaaaa
     4261 agatcgatct cagtggtatt tgtgagccag ggcattggcc acaccagcca ccaccttctg
     4321 ataggcagcc tgcacctgag gttaatcact tagtgatggt gatggtgatg tttaaactgg
     4381 tctagaaagc tggcggccgc ttacaccact ttgtacaaga aagctgggtt ttgcaaaaca
     4441 gcagatgtaa tagatgtttg tggtggttga tataaaacat cagaaccaga gttagagaag
     4501 tcatttaaag ctttagctaa atgacaacaa gcagcttctc tataagaagt agtatccata
     4561 gcaccagaga agtacttata cttgttatac aaagccaagt atctgttata ttgtgttaat
     4621 ggcaacaaaa catcagatct taacttcagg tacatctctt tattcaacaa aaaagtacac
     4681 aaagcagctt cttcgaaagt agaaaaagaa acaccgttaa aaacaactct tctcttcaag
     4741 tagttagaga aaaaccaata gaaatgttta gtggaaatgc agatgatata agcaattgta
     4801 atccaaaatg gaaccaatgg tgtaaacata accatccatt gaatatgagc caggaaagaa
     4861 acatcattag tcaaatagaa ggtcaggtac aaataaataa cagaataaac acctggcaag
     4921 aaagaataaa ctggtgttaa acacaaaaca gtgaaagaca tcaagaataa caaggtatta
     4981 aaagcaacaa catgagaata ttcaccaaaa gctcttctga atctcatgaa gtaataagct
     5041 aaacaagtaa caacaatagc aacaatacca ccagcaacaa tagaagcaga aatatctaaa
     5101 gcaccaattg gttgaatcaa tggagtgaac atatttgtca acaaattaac agcatcaaca
     5161 ccacaaaaaa cacctggcag agacctgtag taatcattat ttaaaaccca tctaccagat
     5221 gtagaaacac aaacaccagc ttcagatctt tcacaagtac catgtctaca gtattcagaa
     5281 tcaaatgttg taacaactct gacagaacct tccagataag tatttggaaa ttggatgata
     5341 gaaccatcca ttaaaacata tctagtatct ggtctcaaag attcataagc aacagaacct
     5401 tccaaaacgt tagtatcata acaatatgga actggtttac cagaagcatc cttaaaaatt
     5461 gtacattcag cagctaaaac acaagcagat gtagcgaaat ctgtatattc gatcaattta
     5521 gatggagtgt aacaaatatt accaacagca gaaaaaactc ttggcaaaaa atgcaagaaa
     5581 tcaccatttg tagttctcaa aatagtacct ggcaaacctg gaacaacaaa accaacttct
     5641 cttgtaataa cagcagcgat caatggacaa gctttatcat ttgtataaga accacctctt
     5701 tgagaaaacc aagtatcaaa atcagcgtgt ttattagcaa aacatgtatc tgtagaagca
     5761 atatctcttg taacaccacc atcaatagct ttataaccga tgatctcaga agaaaaatct
     5821 gtatgtttag acatgacatg aactggagta atcagataaa aaatagcagc aacgaacaaa
     5881 aagaccaaag taaccttgat taattgcttt aaccagttgt taacgatctt cattaagcct
     5941 gcttttttgt acaaacttgt cggaccctgg aagagcacct ccagcttgta tagctcgtcc
     6001 atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc
     6061 tcgttggggt ctttgctcag ggcggactgg gtgctcaggt agtggttgtc gggcagcagc
     6121 acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg
     6181 tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc
     6241 atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg
     6301 tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac
     6361 ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg
     6421 acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag
     6481 cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc
     6541 agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg
     6601 ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccaactc gaccaggatg
     6661 ggcaccaccc cggtgaacag ctcctcgccc ttgcttgtgc cgtggtgatg gtggtgatgg
     6721 tggtgaccag aggtgcccat ggtttcactt acgtgtttaa tgtctttaca gactttatac
     6781 attactgtca tggttaaata aaataaaata aaataaaata aaataaaata aaataaaata
     6841 aaataaaata aaataaactt aagatgtccc tatagtgagt cgtattagac gtccgatcca
     6901 attctttgcc aaaatgatga gacagcacaa taaccagcac gttgcccagg agctgtagga
     6961 aaaagaagaa ggcatgaaca tggttagcag aggctctaga gccgccggtc acacgccaga
     7021 agccgaaccc cgccctgccc cgtccccccc gaaggcagcc gtccccccgc ggacagcccc
     7081 gagccgctcc ccccgcacaa agggccctcc cggagcccct caaggctttc acgcagccac
     7141 agaaaagaaa caagccgtca ttaaaccaag cgctaattac agcccgaagg agaagggccg
     7201 tcccgcccgc tcacctgtgg gagtaacgcg gtcagtcaga gccggggcgg gcggcgcgag
     7261 gcggcggcgg agcggggcac ggggcgaagg cagcgcgcag cgactcccgc ccgccgcgcg
     7321 cttcgctttt tatagggccg ccgccgccgc cgcctcgcca taaaaggaaa ctttcggagc
     7381 gcgcc
//
