LOCUS       sequence-410541-        6832 bp    DNA     circular SYN 14-APR-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 Apr 14, 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 Nsp3TM
FEATURES             Location/Qualifiers
     source          1..6832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..147
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        164..355
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     312..332
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(357..461)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      488..943
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(575..594)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     785..806
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    950..1071
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(961..980)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1015..1034
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1345..1364
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1345..1363
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1419..1435
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1420..1436
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3992..4015)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      4015..4024
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4059..4077)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(4123..4326)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4152..4172)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4327..4630
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(4805..5393)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4885..4904)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5722..5769
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     5794..5813
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(6001..6795)
                     /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(6001..6795)
                     /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(6112..6131)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6722..6741
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6830..6832,1..355))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
ORIGIN
        1 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga
       61 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
      121 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
      181 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
      241 cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
      301 ggactttcca caccctaact gacacacatt ccacagctgg ttctttccgc ctcaggattc
      361 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
      421 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
      481 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      541 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      601 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      661 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      721 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      781 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
      841 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
      901 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttacgcgtt aagatacatt
      961 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     1021 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     1081 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     1141 taaaacctct acaaatgtgg tatggctgat tatgatcatg aacagactgt gaggactgag
     1201 gggcctgaaa tgagccttgg gactgtgaat ctaaaataca caaacaatta gaatcagtag
     1261 tttaacacat tatacactta aaaattggat ctccattcgc cattcaggct gcgcaactgt
     1321 tgggaagggc gatcgagtga attgtaatac gactcactat agggcgaatt gggtacactt
     1381 acctggtacc ttaattaatt aaggtaccgg gccccccctc gaggtcgacg gtatcgataa
     1441 gcttgatatc gaattcctcg agttagtagg acttccacac gtagtagaag ctggcgaaga
     1501 agatgtacat gcgcaccatg gcagagatgg gagccatctg caccaggttg atgatcagcc
     1561 acatcagcca ggagttgctg atgaagtgca cagcgaagta ggagaagaac agctgcatga
     1621 tagcggccag gcccagcacg tagaagaacc tggtgaacag gatgtaggcc aggaaccact
     1681 cagccaccag accgaaggcg gtcaggtccc acttgaagct agagatggtg atctggatgg
     1741 tttccaggga ggggtaggtg tccaggctgt ccagtccaga caggcacacg gagcatggga
     1801 tgctgccggt gcagtaggtg gcgatggtca cgttggtgct gttcaggtat ccctctctgt
     1861 aaccagtgca gtagctaggc atacccaggt tagacatcag cactcccaga gcggcggtag
     1921 agtagatcag ggaacccagg cacacggaga gcagcaggaa ccagatgatg atgttgatca
     1981 gcttgctgaa gtttggagac ttcaggtagt tgaaggaagc ctccaggcag aactttccca
     2041 cgctcttcac ggtgttcttg gcgatggtgg taggcatgct agccttgata cgggagttgg
     2101 tgcttctggt gaaggtgcac agctgcagca gcagggtgaa gaagtagggc atgtagttgg
     2161 tgcacacgcg gttcagacat ctggtcacga tgttggtggt ggtggacacc accttgttca
     2221 ggaaaggctt ggcgtagtta gcgatggtgt cccatggcac agagttcaca gcagccagac
     2281 cgtgagtagc cagggtcttc agacccagca ccctgctcag ctcgttgggc ttcttgatgg
     2341 tcagggagct gttgtccacg taagcggcca tcaggtcggt gtgacccact tcctcggtga
     2401 tcttcaggct gttgttagct ggcttcagga tgatgtctcc caccacctcg gtggtcttca
     2461 cgttgcattc cagcacgtcc ttctggatgg ttgggttctc caccacttcc tcgctcacgg
     2521 gcttcaggtc ttcgcaggcc aggttgtcca tgccctgagc gtcctcgctc ttcagcacgt
     2581 cgaaagagtt ggaggtttcc actggcttgg tagaccacag gcaacggatg caccaggtgt
     2641 taggcttgta ggtggccttg ttggtagcgt tgttcacgtg ccacacgatg ggcttgtgca
     2701 gcagcttggc tcccttcttg aagcttgggg tgtagtgctt gtagtcgata gccaccacgt
     2761 cgccgttcag gtctgggaag aaggtcacct tcagctctct agaggcaggc ttcttgtatc
     2821 cggtcagctg gttcaggtcg tcagcgaact tgatgttgtc gcacacgaac ttgaagttgt
     2881 cgaagctggc gttagggtag ggctggtttg gcaccaggtc gataggctgc tcggtgaagt
     2941 aagagttgtc cttcttgtag tagttgtcca gcttagggtc gatttcggtg cacaccacgc
     3001 cgtccagctt gtaggtcacg ggcttgatgg tggtggtgta gctgttctcc ttgtagaaca
     3061 cgtcggtgat gggacccttg tattcagagg acttggtcag cagggctccg tcgatgcagt
     3121 acagggtctc cttagaggtg atgtgcttgt agtgtccgca ctggtagttg ccggtgtact
     3181 cggaagcgca ggtgaaggtg ccgtgcttca gttcgtactg ggctggagga gcggacatca
     3241 tcacgaatgg gctttcctgc tgcaccaggt acttggtggc ctgctttccg caggtgcaag
     3301 ggatctgcac gcccttcttg aactgctcgt aggacagggt acccatgtac atcacagctt
     3361 ccactccctt cagggtggtc tgctgctggc cgcaggtctt gcacaccacg ttcagcacac
     3421 gcttgcagga gtccaggttg gcgtgctgga acaggtagct catggtctct ctcacgtctc
     3481 ccagttcgcc cacggtcttg ttgcagtagg ccaggatcag agcgcagaag ttagcggcct
     3541 cgccagcacg ggctctgtag taagcgtcct gcagagcggg tgggttgaac ttcagttcga
     3601 tctgctgcag ggtcagcaga gcggtggcca ggtagcagtt gttgtcggcc cacttgatgc
     3661 tggtcagtcc gttcacctgg gggtacttcc acttcttggt gtggttcagg gcagacatgt
     3721 acctgcccag gaaggatggg tcggtggtgt ggtagtattc gaaagcctcc actctcaggg
     3781 tgtcatcgtt aggcagcacg tagaaggtct tgccctcgtg ggagttgtgt ggcttgatct
     3841 tggtcacgtc agctccgtcc aggtaggtag gaccgaactg ctgtccgtag gtcatgctca
     3901 tgtccaccac ctgggtgtgc aggttgatgt tgtccacggt ggtgaacacc ttgatggtcc
     3961 tcacctcgcg cagagaggat ccgcccgggc tcttatcgtc gtcatccttg taatccatgg
     4021 tggcggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaatttcg
     4081 agcttggcgt aatcgctagc ggatctgacg gttcactaaa ccagctctgc ttatatagac
     4141 ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt
     4201 tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag
     4261 acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg
     4321 catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca
     4381 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     4441 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     4501 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
     4561 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     4621 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     4681 ttaataacta atgcatggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4741 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4801 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4861 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4921 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4981 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5041 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5101 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5161 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5221 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5281 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5341 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5401 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5461 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5521 ttaaatcaat ctaaagtata tatgagtaac ctgaggctat ggcagggcct gccgccccga
     5581 cgttggctgc gagccctggg ccttcacccg aacttggggg gtggggtggg gaaaaggaag
     5641 aaacgcgggc gtattggccc caatggggtc tcggtggggt atcgacagag tgccagccct
     5701 gggaccgaac cccgcgttta tgaacaaacg acccaacacc gtgcgtttta ttctgtcttt
     5761 ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc
     5821 tccccctagg gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc
     5881 cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga
     5941 aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc
     6001 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
     6061 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agttcttcag caatatcacg
     6121 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
     6181 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
     6241 gacgagatcc tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc
     6301 gagcccctga tgttcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
     6361 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
     6421 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
     6481 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
     6541 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
     6601 cgctgcctcg tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
     6661 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
     6721 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
     6781 atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcgatct tt
//
