LOCUS       sequence-417460-        7178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the C terminal of SARS-CoV-2 NSP3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7178)
  AUTHORS   Li J, Gui Q, Liang FX, Sall J, Zhang Q, Duan Y, Dhabaria A, Askenazi
            M, Ueberheide B, Stapleford KA, Pagano M
  TITLE     The REEP5/TRAM1 complex binds SARS-CoV-2 NSP3 and promotes virus 
            replication.
  JOURNAL   J Virol. 2023 Sep 28:e0050723. doi: 10.1128/jvi.00507-23.
  PUBMED    37768083
REFERENCE   2  (bases 1 to 7178)
  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"; journalName: "J Virol. 
            2023 Sep 28:e0050723. doi: 10.1128/jvi.00507-23."
COMMENT     Sequence Label: NSP3C-FR-2Strep
FEATURES             Location/Qualifiers
     source          1..7178
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(26..45)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        217..596
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        597..800
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     751..771
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     845..864
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        845..863
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    898..922
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2530..2539
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2534..2543
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2551..2574
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2611..2634
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2659..2682
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(2713..2731)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2714..2731)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2751..2768)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2757..2981
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3027..3455
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3114..3133)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3324..3345
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3464..3484)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3469..3798
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3649..3784
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3711..3730
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3865..4659
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3919..3938)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4529..4548
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4833..4954
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4870..4889)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4924..4943
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5003..5019)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5003..5019)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5016..5038)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5027..5043
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5051..5057,5058..5075,5076..5081))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5096..5117
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5234..5251)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5405..5993)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5485..5504)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6164..6955,6956..7024))
                     /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     6787..6806
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7025..7129)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
       61 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
      121 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
      181 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
      241 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      301 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      361 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      421 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      481 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      541 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      601 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      661 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      721 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      781 aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg
      841 aaattaatac gactcactat agggagaccc aagcttggta ccgagctcgg atccatgcca
      901 actttgtaca aaaaagttgg catgtggttc ctgctgctct ccgtgtgcct gggttccctg
      961 atctactcta ccgccgctct gggagtgctg atgtctaacc tgggtatgcc tagctactgc
     1021 actggttaca gagagggata cctgaacagc accaacgtga ccatcgccac ctactgcacc
     1081 ggcagcatcc catgctccgt gtgcctgtct ggactggaca gcctggacac ctacccctcc
     1141 ctggaaacca tccagatcac catctctagc ttcaagtggg acctgaccgc cttcggtctg
     1201 gtggctgagt ggttcctggc ctacatcctg ttcaccaggt tcttctacgt gctgggcctg
     1261 gccgctatca tgcagctgtt cttctcctac ttcgctgtgc acttcatcag caactcctgg
     1321 ctgatgtggc tgatcatcaa cctggtgcag atggctccca tctctgccat ggtgcgcatg
     1381 tacatcttct tcgccagctt ctactacgtg tggaagtcct acgtgcacgt ggtggacggt
     1441 tgcaactcct ctacctgcat gatgtgctac aagcgcaaca gggctaccag ggtggagtgc
     1501 accaccatcg tgaacggcgt gcgcaggtcc ttctacgtgt acgccaacgg cggcaaggga
     1561 ttctgcaagc tgcacaactg gaactgcgtg aactgcgaca ccttctgcgc tggctccacc
     1621 ttcatctctg acgaggtggc cagagacctg agcctgcagt tcaagcgtcc aatcaacccc
     1681 accgaccaga gctcctacat cgtggactct gtgaccgtga agaacggtag catccacctg
     1741 tacttcgaca aggctggcca gaagacctac gagcgccact ctctgagcca cttcgtgaac
     1801 ctggacaacc tgagggccaa caacaccaag ggcagcctgc ctatcaacgt gatcgtgttc
     1861 gacggaaagt ccaagtgcga ggaatctagc gccaagtccg ccagcgtgta ctacagccag
     1921 ctgatgtgcc agccaatcct gctgctggac caggctctgg tgtctgacgt gggagacagc
     1981 gccgaagtgg ccgtgaagat gttcgacgcc tacgtgaaca ccttctcctc taccttcaac
     2041 gtgcctatgg agaagctgaa gaccctggtg gctactgctg aggctgaact ggctaagaac
     2101 gtgtccctgg acaacgtgct gagcaccttc atctccgccg ctaggcaggg tttcgtggac
     2161 tctgacgtgg aaaccaaaga cgtggtggag tgcctgaagc tgtctcacca gagcgacatc
     2221 gaagtgaccg gcgactcctg caacaactac atgctgacct acaacaaggt ggagaacatg
     2281 accccaagag acctgggagc ttgcatcgac tgctccgccc gtcacatcaa cgctcaggtg
     2341 gccaagtctc acaacatcgc cctgatctgg aacgtgaagg acttcatgag cctgtccgag
     2401 cagctgcgca agcagatcag gagcgccgct aagaagaaca acctgccctt caagctgacc
     2461 tgcgctacca ccagacaggt ggtgaacgtg gtgaccacca agatcgccct gaagggcgga
     2521 gaattcgccg ccaccatggc ggccgccagc tggagccacc ctcagttcga gaagggagga
     2581 ggaagcggcg gaggcagcgg aggaggaagc tggagccacc cgcagttcga gaaatgactc
     2641 gagcatgcat ctagaggctg gagccacccg cagttcgaga aatgactcga gcatgcatct
     2701 agagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt
     2761 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2821 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2881 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2941 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3001 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3061 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3121 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3181 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3241 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3301 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3361 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3421 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3481 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3541 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3601 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3661 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3721 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3781 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     3841 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3901 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3961 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4021 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4081 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4141 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4201 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4261 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4321 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4381 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4441 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4501 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4561 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4621 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4681 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4741 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4801 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4861 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4921 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4981 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5041 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5101 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5161 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5221 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5281 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5341 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5401 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5461 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5521 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5581 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5641 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5701 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5761 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5821 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5881 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5941 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6001 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6061 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6121 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6181 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6241 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6301 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6361 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6421 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6481 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6541 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6601 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6661 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6721 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6781 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6841 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6901 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6961 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7021 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7081 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7141 gaaaagtgcc acctgacgtc gacggatcgg gagatctc
//
