LOCUS       sequence-409785-        6832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of SARS-CoV-2 Nsp3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6832)
  AUTHORS   Yang M, Mariano J, Su R, Smith CE, Das S, Gill C, Andresson T, 
            Loncarek J, Tsai YC, Weissman AM
  TITLE     SARS-CoV-2 papain-like protease plays multiple roles in regulating 
            cellular proteins in the endoplasmic reticulum.
  JOURNAL   J Biol Chem. 2023 Oct 12;299(12):105346. doi: 
            10.1016/j.jbc.2023.105346.
  PUBMED    37838170
REFERENCE   2  (bases 1 to 6832)
  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 Biol 
            Chem."; date: "2023-10-12"; volume: "299(12):105346. doi"; pages: " 
            10.1016/j.jbc.2023.105346"
COMMENT     Sequence Label: pCMV-SARS-CoV-2 Nsp3TMC111A
FEATURES             Location/Qualifiers
     source          1..6832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      13..148
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        165..356
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     313..333
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(358..462)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      489..944
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(576..595)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     786..807
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    951..1072
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(962..981)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1016..1035
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1346..1365
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1346..1364
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1420..1436
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1421..1437
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3993..4016)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      4016..4025
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4060..4078)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(4124..4327)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4153..4173)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4328..4631
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(4806..5394)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4886..4905)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5723..5770
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     5795..5814
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(6002..6796)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(6002..6796)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6113..6132)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6723..6742
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6831..6832,1..356))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
ORIGIN
        1 tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggccg
       61 aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg
      121 cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
      181 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
      241 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
      301 gggactttcc acaccctaac tgacacacat tccacagctg gttctttccg cctcaggatt
      361 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
      421 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
      481 gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
      541 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
      601 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
      661 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
      721 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
      781 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
      841 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
      901 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttacgcgt taagatacat
      961 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     1021 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     1081 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     1141 gtaaaacctc tacaaatgtg gtatggctga ttatgatcat gaacagactg tgaggactga
     1201 ggggcctgaa atgagccttg ggactgtgaa tctaaaatac acaaacaatt agaatcagta
     1261 gtttaacaca ttatacactt aaaaattgga tctccattcg ccattcaggc tgcgcaactg
     1321 ttgggaaggg cgatcgagtg aattgtaata cgactcacta tagggcgaat tgggtacact
     1381 tacctggtac cttaattaat taaggtaccg ggccccccct cgaggtcgac ggtatcgata
     1441 agcttgatat cgaattcctc gagttagtag gacttccaca cgtagtagaa gctggcgaag
     1501 aagatgtaca tgcgcaccat ggcagagatg ggagccatct gcaccaggtt gatgatcagc
     1561 cacatcagcc aggagttgct gatgaagtgc acagcgaagt aggagaagaa cagctgcatg
     1621 atagcggcca ggcccagcac gtagaagaac ctggtgaaca ggatgtaggc caggaaccac
     1681 tcagccacca gaccgaaggc ggtcaggtcc cacttgaagc tagagatggt gatctggatg
     1741 gtttccaggg aggggtaggt gtccaggctg tccagtccag acaggcacac ggagcatggg
     1801 atgctgccgg tgcagtaggt ggcgatggtc acgttggtgc tgttcaggta tccctctctg
     1861 taaccagtgc agtagctagg catacccagg ttagacatca gcactcccag agcggcggta
     1921 gagtagatca gggaacccag gcacacggag agcagcagga accagatgat gatgttgatc
     1981 agcttgctga agtttggaga cttcaggtag ttgaaggaag cctccaggca gaactttccc
     2041 acgctcttca cggtgttctt ggcgatggtg gtaggcatgc tagccttgat acgggagttg
     2101 gtgcttctgg tgaaggtgca cagctgcagc agcagggtga agaagtaggg catgtagttg
     2161 gtgcacacgc ggttcagaca tctggtcacg atgttggtgg tggtggacac caccttgttc
     2221 aggaaaggct tggcgtagtt agcgatggtg tcccatggca cagagttcac agcagccaga
     2281 ccgtgagtag ccagggtctt cagacccagc accctgctca gctcgttggg cttcttgatg
     2341 gtcagggagc tgttgtccac gtaagcggcc atcaggtcgg tgtgacccac ttcctcggtg
     2401 atcttcaggc tgttgttagc tggcttcagg atgatgtctc ccaccacctc ggtggtcttc
     2461 acgttgcatt ccagcacgtc cttctggatg gttgggttct ccaccacttc ctcgctcacg
     2521 ggcttcaggt cttcgcaggc caggttgtcc atgccctgag cgtcctcgct cttcagcacg
     2581 tcgaaagagt tggaggtttc cactggcttg gtagaccaca ggcaacggat gcaccaggtg
     2641 ttaggcttgt aggtggcctt gttggtagcg ttgttcacgt gccacacgat gggcttgtgc
     2701 agcagcttgg ctcccttctt gaagcttggg gtgtagtgct tgtagtcgat agccaccacg
     2761 tcgccgttca ggtctgggaa gaaggtcacc ttcagctctc tagaggcagg cttcttgtat
     2821 ccggtcagct ggttcaggtc gtcagcgaac ttgatgttgt cgcacacgaa cttgaagttg
     2881 tcgaagctgg cgttagggta gggctggttt ggcaccaggt cgataggctg ctcggtgaag
     2941 taagagttgt ccttcttgta gtagttgtcc agcttagggt cgatttcggt gcacaccacg
     3001 ccgtccagct tgtaggtcac gggcttgatg gtggtggtgt agctgttctc cttgtagaac
     3061 acgtcggtga tgggaccctt gtattcagag gacttggtca gcagggctcc gtcgatgcag
     3121 tacagggtct ccttagaggt gatgtgcttg tagtgtccgc actggtagtt gccggtgtac
     3181 tcggaagcgc aggtgaaggt gccgtgcttc agttcgtact gggctggagg agcggacatc
     3241 atcacgaatg ggctttcctg ctgcaccagg tacttggtgg cctgctttcc gcaggtgcaa
     3301 gggatctgca cgcccttctt gaactgctcg taggacaggg tacccatgta catcacagct
     3361 tccactccct tcagggtggt ctgctgctgg ccgcaggtct tgcacaccac gttcagcaca
     3421 cgcttgcagg agtccaggtt ggcgtgctgg aacaggtagc tcatggtctc tctcacgtct
     3481 cccagttcgc ccacggtctt gttgcagtag gccaggatca gagcgcagaa gttagcggcc
     3541 tcgccagcac gggctctgta gtaagcgtcc tgcagagcgg gtgggttgaa cttcagttcg
     3601 atctgctgca gggtcagcag agcggtggcc aggtaagcgt tgttgtcggc ccacttgatg
     3661 ctggtcagtc cgttcacctg ggggtacttc cacttcttgg tgtggttcag ggcagacatg
     3721 tacctgccca ggaaggatgg gtcggtggtg tggtagtatt cgaaagcctc cactctcagg
     3781 gtgtcatcgt taggcagcac gtagaaggtc ttgccctcgt gggagttgtg tggcttgatc
     3841 ttggtcacgt cagctccgtc caggtaggta ggaccgaact gctgtccgta ggtcatgctc
     3901 atgtccacca cctgggtgtg caggttgatg ttgtccacgg tggtgaacac cttgatggtc
     3961 ctcacctcgc gcagagagga tccgcccggg ctcttatcgt cgtcatcctt gtaatccatg
     4021 gtggcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaatttc
     4081 gagcttggcg taatcgctag cggatctgac ggttcactaa accagctctg cttatataga
     4141 cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt
     4201 ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca atggggtgga
     4261 gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac tgccaaaacc
     4321 gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc
     4381 ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag
     4441 ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat
     4501 actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca
     4561 ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa
     4621 gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact
     4681 attaataact aatgcatggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4741 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4801 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4861 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4921 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4981 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5041 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5101 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5161 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5221 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5281 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5341 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5401 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5461 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5521 tttaaatcaa tctaaagtat atatgagtaa cctgaggcta tggcagggcc tgccgccccg
     5581 acgttggctg cgagccctgg gccttcaccc gaacttgggg ggtggggtgg ggaaaaggaa
     5641 gaaacgcggg cgtattggcc ccaatggggt ctcggtgggg tatcgacaga gtgccagccc
     5701 tgggaccgaa ccccgcgttt atgaacaaac gacccaacac cgtgcgtttt attctgtctt
     5761 tttattgccg tcatagcgcg ggttccttcc ggtattgtct ccttccgtgt ttcagttagc
     5821 ctccccctag ggtgggcgaa gaactccagc atgagatccc cgcgctggag gatcatccag
     5881 ccggcgtccc ggaaaacgat tccgaagccc aacctttcat agaaggcggc ggtggaatcg
     5941 aaatctcgtg atggcaggtt gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg
     6001 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     6061 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagttcttca gcaatatcac
     6121 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     6181 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     6241 cgacgagatc ctcgccgtcg ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg
     6301 cgagcccctg atgttcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
     6361 tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     6421 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     6481 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     6541 cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     6601 gcgctgcctc gtcttgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     6661 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     6721 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     6781 catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcgatc tt
//
