LOCUS       sequence-418213-        7037 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SARS-CoV-2 NSP14 fused with Flag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7037)
  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 7037)
  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: NSP14-Flag
FEATURES             Location/Qualifiers
     source          1..7037
                     /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"
     regulatory      916..925
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2530..2553
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2590..2608)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2591..2608)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2628..2645)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2634..2858
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2904..3332
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2991..3010)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3201..3222
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3341..3361)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3346..3675
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3526..3661
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3588..3607
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3742..4536
                     /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(3796..3815)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4406..4425
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4710..4831
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4747..4766)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4801..4820
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4880..4896)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4880..4896)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4893..4915)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4904..4920
                     /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(4928..4934,4935..4952,4953..4958))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4973..4994
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5111..5128)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5282..5870)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5362..5381)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6041..6832,6833..6901))
                     /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     6664..6683
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6902..7006)
                     /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 ccgccgccac catggctgaa aatgtaacgg gcttgtttaa agactgtagt
      961 aaagtgatca caggactcca ccccacacaa gcacctaccc acctttccgt agatacgaag
     1021 ttcaaaacgg aaggattgtg tgtggatata ccagggatac caaaggatat gacgtaccga
     1081 aggctgattt ccatgatggg ttttaagatg aattaccaag ttaatggcta ccccaacatg
     1141 ttcatcacca gggaggaggc aattagacac gtaagagcct ggataggctt cgatgttgaa
     1201 ggttgccatg caacccggga agccgtaggc acaaaccttc cgttgcagct tggcttttcc
     1261 acgggcgtca acctcgttgc cgtaccgact ggctatgttg acacgccgaa caacaccgat
     1321 ttctctcgcg taagtgctaa gcctcctccg ggagatcaat tcaagcatct tatacctctc
     1381 atgtacaagg gactgccgtg gaatgtagtt cgcattaaga tcgtgcagat gctctccgat
     1441 actttgaaaa atctgagcga cagggtggtc ttcgttctgt gggcacatgg cttcgaactt
     1501 acttctatga aatactttgt aaaaatcggg cccgagcgga catgttgttt gtgcgaccga
     1561 cgagctactt gttttagcac agcatctgac acctacgcat gctggcacca cagtataggc
     1621 ttcgattacg tctacaatcc ctttatgata gacgtacaac aatggggctt tacgggtaac
     1681 ttgcagagta atcacgatct ttactgccaa gttcatggga acgcacacgt ggcctcctgc
     1741 gatgcgataa tgacgaggtg cttggccgtg cacgagtgct ttgttaagcg ggtcgattgg
     1801 actatagagt atcccataat cggtgacgaa cttaaaatta atgctgcatg caggaaagtg
     1861 cagcacatgg tagtaaaagc agcgctcctc gcggataagt ttcctgtgct ccacgatatc
     1921 ggcaacccta aggccattaa atgcgtcccc caagctgatg tagagtggaa attctatgac
     1981 gctcagccat gtagtgacaa agcatacaag attgaagaat tgttttattc atatgcgaca
     2041 cactccgata agttcactga cggagtttgt ctcttttgga actgtaatgt tgataggtat
     2101 ccggcgaata gcatagtgtg tcgatttgac acgcgcgttc tctctaattt gaacctccct
     2161 ggctgcgacg gggggtcttt gtacgtaaac aagcatgcat ttcatacccc agcttttgac
     2221 aaatccgctt tcgtaaatct gaaacaactt cccttcttct attattcaga tagcccttgc
     2281 gaatctcacg gtaagcaagt ggttagtgac attgactatg taccacttaa aagtgcaacg
     2341 tgcattacac gatgcaattt ggggggcgcg gtttgccgac accatgccaa cgaatacaga
     2401 ttgtacctgg atgcatataa catgatgata agtgcagggt ttagtctttg ggtgtataag
     2461 cagttcgata cgtataatct ctggaacaca tttacccgat tgcagggcgg tggttcagga
     2521 ggaggtagcg actacaagga cgacgatgac aagggtagct agctcgagca tgcatctaga
     2581 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     2641 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2701 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2761 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2821 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2881 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2941 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3001 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3061 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3121 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3181 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3241 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3301 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3361 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3421 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3481 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3541 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3601 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3661 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     3721 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3781 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3841 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3901 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3961 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4021 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4081 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4141 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4201 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4261 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4321 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4381 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4441 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4501 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4561 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4621 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4681 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4741 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4801 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4861 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4921 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4981 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5041 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5101 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5161 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5221 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5281 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5341 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5401 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5461 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5521 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5581 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5641 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5701 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5761 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5821 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5881 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5941 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6001 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6061 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6121 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6181 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6241 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6301 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6361 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6421 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6481 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6541 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6601 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6661 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6721 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6781 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6841 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6901 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6961 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7021 aagtgccacc tgacgtc
//
