LOCUS       sequence-295403-        7535 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 nsp8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7535)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7535)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGBW-m4134039
FEATURES             Location/Qualifiers
     source          1..7535
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             596..616
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             965..982
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    1075..1196
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1086..1105)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1140..1159
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1311..1357
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1387..1523
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1568..1639
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1865..2453)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1945..1964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2953..4162
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3999..4018
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4166..5194
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    5221..5269
                     /note="synthetic polyadenylation signal"
     CDS             join(5535..5603,5604..6395)
                     /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     complement(5753..5772)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6408..6544
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6689..6760
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6765..6811)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6829..6877
                     /note="synthetic polyadenylation signal"
     enhancer        6958..7261
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7263..7465
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7416..7436
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 caccatgata gcctcagagt ttagttccct tccatcatat gcagcttttg ctactgctca
       61 agaagcttat gagcaggctg ttgctaatgg tgattctgaa gttgttctta aaaagttgaa
      121 gaagtctttg aatgtggcta aatctgaatt tgaccgtgat gcagccatgc aacgtaagtt
      181 ggaaaagatg gctgatcaag ctatgaccca aatgtataaa caggctagat ctgaggacaa
      241 gagggcaaaa gttactagtg ctatgcagac aatgcttttc actatgctta gaaagttgga
      301 taatgatgca ctcaacaaca ttatcaacaa tgcaagagat ggttgtgttc ccttgaacat
      361 aatacctctt acaacagcag ccaaactaat ggttgtcata ccagactata acacatataa
      421 aaatacgtgt gatggtacaa catttactta tgcatcagca ttgtgggaaa tccaacaggt
      481 tgtagatgca gatagtaaaa ttgttcaact tagtgaaatt agtatggaca attcacctaa
      541 tttagcatgg cctcttattg taacagcttt aagggccaat tctgctgtca aattagaaaa
      601 cctttacttt cagtcgatga gtgacaaaat cattcatcta accgatgact cctttgatac
      661 agacgtgttg aaggccgatg gtgcgatact ggtagatttc tgggctgaat ggtgtggccc
      721 gtgcaagatg atcgcaccca ttctggacga gatcgccgac gagtatcaag gaaaattaac
      781 tgtcgctaag cttaatatag atcagaaccc agggacggca cctaaatacg gcatcagggg
      841 aatacctacc ctcctgctct ttaagaatgg ggaagttgcc gcgacaaaag tgggagccct
      901 gtctaagggc cagctaaaag agttcttgga cgctaacctg gcaggttcag gctcggggca
      961 catgcaccat caccatcacc atagcagcgg gctcgtgcca cgcggctaag gatccgcagg
     1021 cctctgctag cttgactgac tgagatacag cgtaccttca gctcacagac atgataagat
     1081 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     1141 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     1201 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     1261 gcaagtaaaa cctctacaaa tgtggtattg gcccatctct atcggtatcg tagcataacc
     1321 ccttggggcc tctaaacggg tcttgagggg ttttttgtgc ccctcgggcc ggattgctat
     1381 ctaccggcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac
     1441 atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca tacgtgtcct
     1501 ccttaccaga aatttatcct taaggtcgtc agctatcctg caggcgatct ctcgatttcg
     1561 atcaagacat tcctttaatg gtcttttctg gacaccacta ggggtcagaa gtagttcatc
     1621 aaactttctt ccctccctaa tctcattggt taccttgggc tatcgaaact taattaacca
     1681 gtcaagtcag ctacttggcg agatcgactt gtctgggttt cgactacgct cagaattgcg
     1741 tcagtcaagt tcgatctggt ccttgctatt gcacccgttc tccgattacg agtttcattt
     1801 aaatcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1861 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1921 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1981 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2041 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2101 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2161 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2221 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2281 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     2341 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     2401 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     2461 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     2521 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     2581 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     2641 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgca tttaaatttc
     2701 cgaactctcc aaggccctcg tcggaaaatc ttcaaacctt tcgtccgatc catcttgcag
     2761 gctacctctc gaacgaacta tcgcaagtct cttggccggc cttgcgcctt ggctattgct
     2821 tggcagcgcc tatcgccagg tattactcca atcccgaata tccgagatcg ggatcacccg
     2881 agagaagttc aacctacatc ctcaatcccg atctatccga gatccgagga atatcgaaat
     2941 cggggcgcgc ctggcctccg cgccgggttt tggcgcctcc cgcgggcgcc cccctcgtca
     3001 cggcgagcgc tgccacgtca gacgaagggc gcaggagcgt cctgatcctt ccgcccggac
     3061 gctcaggaca gcggcccgct gctcataaga ctcggcctta gaaccccagt atcagcagaa
     3121 ggacatttta ggacgggact tgggtgactc tagggcactg gttttctttc cagagagcgg
     3181 aacaggcgag gaaaagtagt cccttctcgg cgattctgcg gagggatctc cgtggggcgg
     3241 tgaacgccga tgattatata aggacgcgcc gggtgtggca cagctagttc cgtcgcagcc
     3301 gggatttggg tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg
     3361 gctgctgggc tggccggggc tttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt
     3421 ggagagaccg ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg aactgggggt
     3481 tggggggagc gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga cgcttgtgag
     3541 gcgggctgtg aggtcgttga aacaaggtgg ggggcatggt gggcggcaag aacccaaggt
     3601 cttgagccct tcgctaatgc gggaaagctc ttattcgggt gagatgggct gggcaccatc
     3661 tggggaccct gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg
     3721 gggcggcagt tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc
     3781 tcgtcgtgtc gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc
     3841 ggtaggtgtg cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc
     3901 tctcctgaat cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt
     3961 tctttggtcg gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat
     4021 gcgctcgggg ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc
     4081 atttgggtca atatgtaatt ttcagtgtta gacttgtaaa ttgtccgcta aattctggcc
     4141 gtttttggct tttttgttag acaacatggg taaaaagcct gaactcaccg cgacgtctgt
     4201 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     4261 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     4321 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catccgccgc
     4381 gctcccgatt ccggaagtgc ttgacattgg ggagttcagc gagagcctga cctattgcat
     4441 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     4501 tctgcagccg gtcgcggagg caatggatgc catcgctgcc gccgatctta gccagacgag
     4561 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     4621 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     4681 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     4741 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     4801 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     4861 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     4921 gaggcatccg gagcttgcag gatcgccccg gctccgggcg tatatgctcc gcattggtct
     4981 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     5041 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     5101 cagaagcgcc gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     5161 acgccccagc actcgtccga gggcaaagga ataagctagt atgtaagcct agtcttagat
     5221 aataaaatcg ctatccatcg aagatggatg tgtgttggtt ttttgtgtgt gtaacgctag
     5281 gcgcgcctgg tgtaccgaga acgatcctct cagtgcgagt ctcgacgatc catatcgttg
     5341 cttggcagtc agccagtcgg aatccagctt gggacccagg aagtccaatc gtcagatatt
     5401 gtactcaagc ctggtcacgg cagcgtaccg atctgtttaa acctagatat tgatagtctg
     5461 atcggtcaac gtataatcga gtcctagctt ttgcaaacat ctatcaagag acaggatcag
     5521 caggaggctt tcgcatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5581 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5641 atcagttggg tgcgcgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5701 agagttttcg ccccgaagaa cgctttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5761 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5821 ctcagaatga cttggttgag tattcaccag tcacagaaaa gcatcttacg gatggcatga
     5881 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5941 ttctgacaac gattggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6001 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6061 gtgacaccac gatgcctgta gcaatggcaa caaccttgcg taaactatta actggcgaac
     6121 tacttactct agcttcccgg caacagttga tagactggat ggaggcggat aaagttgcag
     6181 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6241 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6301 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6361 ctgagatagg tgcctcactg attaagcatt ggtaaccgat tctaggtgca ttggcgcaga
     6421 aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac
     6481 ggatacggct tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc
     6541 ttaagatcgt ttaaactcga ctctggctct atcgaatctc cgtcgtttcg agcttacgcg
     6601 aacagccgtg gcgctcattt gctcgtcggg catcgaatct cgtcagctat cgtcagctta
     6661 cctttttggc agcgatcgcg gctcccgaca tcttggacca ttagctccac aggtatcttc
     6721 ttccctctag tggtcataac agcagcttca gctacctctc aattcaaaaa acccctcaag
     6781 acccgtttag aggccccaag gggttatgct atcaatcgtt gcgttacaca cacaaaaaac
     6841 caacacacat ccatcttcga tggatagcga ttttattatc taactgctga tcgagtgtag
     6901 ccagatctag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     6961 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     7021 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     7081 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     7141 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     7201 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     7261 gctgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     7321 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     7381 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     7441 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatcagat ctttgtcgat
     7501 cctaccatcc actcgacaca cccgccagcg gccgc
//
