LOCUS       sequence-418205-        6452 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SARS-CoV-2 NSP6 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6452)
  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 6452)
  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: NSP6-FR-2Strep
FEATURES             Location/Qualifiers
     source          1..6452
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    916..940
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1822..1831
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1826..1835
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1843..1866
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1903..1926
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1951..1974
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(2005..2023)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2006..2023)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2043..2060)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2049..2273
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2319..2747
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2406..2425)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2616..2637
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2756..2776)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2761..3090
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2941..3076
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3003..3022
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3157..3951
                     /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(3211..3230)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3821..3840
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4125..4246
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4162..4181)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4216..4235
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4295..4311)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4295..4311)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4308..4330)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4319..4335
                     /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(4343..4349,4350..4367,4368..4373))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4388..4409
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4526..4543)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4697..5285)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4777..4796)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5456..6247,6248..6316))
                     /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     6079..6098
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6317..6421)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccatgccaac tttgtacaaa aaagttggca tgagcgccgt gaagagaacc
      961 atcaagggaa cccaccactg gctgctgctg accatcctga cctccctgct ggtgctggtg
     1021 cagtctaccc agtggagcct gttcttcttc ctgtacgaga acgccttcct gcccttcgct
     1081 atgggtatca tcgccatgtc cgccttcgct atgatgttcg tgaagcacaa gcacgctttc
     1141 ctgtgcctgt tcctgctgcc atccctggcc accgtggctt acttcaacat ggtgtacatg
     1201 cccgcctctt gggtcatgcg catcatgacc tggctggaca tggtggacac cagcctgtcc
     1261 ggcttcaagc tgaaggactg cgtgatgtac gcctctgctg tggtgctgct gatcctgatg
     1321 accgctagaa ccgtgtacga tgatggtgct agaagggtgt ggaccctgat gaacgtgctg
     1381 accctggtgt acaaggtgta ctacggcaac gccctggacc aggctatcag catgtgggct
     1441 ctgatcatct ctgtgaccag caactactcc ggagtggtga ccaccgtgat gttcctggcc
     1501 agaggtatcg tgttcatgtg cgtggagtac tgccctatct tcttcatcac cggcaacacc
     1561 ctgcagtgca tcatgctggt gtactgcttc ctgggttact tctgcacctg ctatttcggc
     1621 ctgttctgcc tgctgaacag atacttccgt ctgaccctgg gcgtgtacga ctacctggtg
     1681 tccacccagg aattccgtta catgaactct cagggactgc tgccccctaa gaacagcatc
     1741 gacgctttca agctgaacat caagctgctg ggagtgggcg gaaagccatg catcaaggtg
     1801 gccaccgtgc aggaattcgc cgccaccatg gcggccgcca gctggagcca ccctcagttc
     1861 gagaagggag gaggaagcgg cggaggcagc ggaggaggaa gctggagcca cccgcagttc
     1921 gagaaatgac tcgagcatgc atctagaggc tggagccacc cgcagttcga gaaatgactc
     1981 gagcatgcat ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     2041 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2101 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2161 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2221 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2281 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2341 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2401 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2461 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2521 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2581 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2641 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2701 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     2761 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2821 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2881 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     2941 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3001 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3061 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3121 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3181 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3241 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3301 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3361 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3421 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3481 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3541 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3601 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3661 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     3721 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3781 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3841 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3901 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     3961 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4021 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4081 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4141 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4201 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4261 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4321 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4381 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4441 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4501 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4561 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4621 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4681 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4741 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4801 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4861 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4921 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4981 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5041 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5101 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5161 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5221 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5281 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5341 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5401 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5461 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5521 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5581 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5641 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5701 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5761 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5821 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5881 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5941 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6001 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6061 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6121 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6181 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6241 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6301 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6361 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6421 gcacatttcc ccgaaaagtg ccacctgacg tc
//
