LOCUS       sequence-295763-        7255 bp    DNA     circular SYN 15-DEC-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 7255)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7255)
  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"
COMMENT     Sequence Label: pGBW-m4133234
FEATURES             Location/Qualifiers
     source          1..7255
                     /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             650..727
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     CDS             755..775
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    1224..1345
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1235..1254)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1289..1308
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1460..1506
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1536..1672
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1717..1788
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2014..2602)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2094..2113)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3102..4311
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4148..4167
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4315..4914
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4315..4334)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4811..4831
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4941..4989
                     /note="synthetic polyadenylation signal"
     CDS             join(5255..5323,5324..6115)
                     /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(5473..5492)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6128..6264
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6409..6480
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6485..6531)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6549..6597
                     /note="synthetic polyadenylation signal"
     enhancer        6678..6981
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6983..7185
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7136..7156
                     /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 cagtcgggac ggcgaatccc ggggctgata aatccctgga aaaggagatg
      661 gaagaagaac tttattgcag tgtctgcggc taaccggttc aaaaagatca gttcaagcgg
      721 cgccctagat tacgacattc ctacgaccgc ctccgagaat ctctatttcc agggtgaatt
      781 tggcttggca caacatgacg aggccgtcga taacaagttt aataaagaac agcagaacgc
      841 tttctatgag atccttcact taccaaatct gaacgaagag caacgcaacg ctttcattca
      901 gagcctgaaa gacgatccct cgcagtctgc aaatctcctg gccgaagcca agaagctgaa
      961 tgacgcacag gctccaaaag ttgataacaa gtttaacaag gagcagcaga atgctttcta
     1021 cgagatattg caccttccta atctgaatga ggaacaaaga aatgcattta tccagtccct
     1081 aaaagacgat ccatcacaga gtgccaacct gcttgcggaa gccaaaaagc tcaatgatgc
     1141 ccaggccccg aaagtagacg cgaaccatca ataagctagc ttgactgact gagatacagc
     1201 gtaccttcag ctcacagaca tgataagata cattgatgag tttggacaaa ccacaactag
     1261 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1321 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     1381 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtattgg
     1441 cccatctcta tcggtatcgt agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1501 tttttgtgcc cctcgggccg gattgctatc taccggcatt ggcgcagaaa aaaatgcctg
     1561 atgcgacgct gcgcgtctta tactcccaca tatgccagat tcagcaacgg atacggcttc
     1621 cccaacttgc ccacttccat acgtgtcctc cttaccagaa atttatcctt aaggtcgtca
     1681 gctatcctgc aggcgatctc tcgatttcga tcaagacatt cctttaatgg tcttttctgg
     1741 acaccactag gggtcagaag tagttcatca aactttcttc cctccctaat ctcattggtt
     1801 accttgggct atcgaaactt aattaaccag tcaagtcagc tacttggcga gatcgacttg
     1861 tctgggtttc gactacgctc agaattgcgt cagtcaagtt cgatctggtc cttgctattg
     1921 cacccgttct ccgattacga gtttcattta aatcatgtga gcaaaaggcc agcaaaaggc
     1981 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2041 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2101 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2161 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2221 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2281 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2341 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2401 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2461 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2521 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2581 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2641 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2701 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2761 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2821 tcgttcatcc atagttgcat ttaaatttcc gaactctcca aggccctcgt cggaaaatct
     2881 tcaaaccttt cgtccgatcc atcttgcagg ctacctctcg aacgaactat cgcaagtctc
     2941 ttggccggcc ttgcgccttg gctattgctt ggcagcgcct atcgccaggt attactccaa
     3001 tcccgaatat ccgagatcgg gatcacccga gagaagttca acctacatcc tcaatcccga
     3061 tctatccgag atccgaggaa tatcgaaatc ggggcgcgcc tggcctccgc gccgggtttt
     3121 ggcgcctccc gcgggcgccc ccctcgtcac ggcgagcgct gccacgtcag acgaagggcg
     3181 caggagcgtc ctgatccttc cgcccggacg ctcaggacag cggcccgctg ctcataagac
     3241 tcggccttag aaccccagta tcagcagaag gacattttag gacgggactt gggtgactct
     3301 agggcactgg ttttctttcc agagagcgga acaggcgagg aaaagtagtc ccttctcggc
     3361 gattctgcgg agggatctcc gtggggcggt gaacgccgat gattatataa ggacgcgccg
     3421 ggtgtggcac agctagttcc gtcgcagccg ggatttgggt cgcggttctt gtttgtggat
     3481 cgctgtgatc gtcacttggt gagtagcggg ctgctgggct ggccggggct ttcgtggccg
     3541 ccgggccgct cggtgggacg gaagcgtgtg gagagaccgc caagggctgt agtctgggtc
     3601 cgcgagcaag gttgccctga actgggggtt ggggggagcg cagcaaaatg gcggctgttc
     3661 ccgagtcttg aatggaagac gcttgtgagg cgggctgtga ggtcgttgaa acaaggtggg
     3721 gggcatggtg ggcggcaaga acccaaggtc ttgagccctt cgctaatgcg ggaaagctct
     3781 tattcgggtg agatgggctg ggcaccatct ggggaccctg acgtgaagtt tgtcactgac
     3841 tggagaactc ggtttgtcgt ctgttgcggg ggcggcagtt atggcggtgc cgttgggcag
     3901 tgcacccgta cctttgggag cgcgcgccct cgtcgtgtcg tgacgtcacc cgttctgttg
     3961 gcttataatg cagggtgggg ccacctgccg gtaggtgtgc ggtaggcttt tctccgtcgc
     4021 aggacgcagg gttcgggcct agggtaggct ctcctgaatc gacaggcgcc ggacctctgg
     4081 tgaggggagg gataagtgag gcgtcagttt ctttggtcgg ttttatgtac ctatcttctt
     4141 aagtagctga agctccggtt ttgaactatg cgctcggggt tggcgagtgt gttttgtgaa
     4201 gttttttagg caccttttga aatgtaatca tttgggtcaa tatgtaattt tcagtgttag
     4261 acttgtaaat tgtccgctaa attctggccg tttttggctt ttttgttaga caacatgacc
     4321 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     4381 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     4441 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     4501 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     4561 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     4621 ctggccgcgc agcaacagat ggagggcctc ctggcgccgc accggcccaa ggagcccgcg
     4681 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     4741 gtcgtgctcc ccggagtgga ggccgccgag cgcgccgggg tgcccgcctt cctggagacc
     4801 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     4861 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctaggctagt
     4921 atgtaagcct agtcttagat aataaaatcg ctatccatcg aagatggatg tgtgttggtt
     4981 ttttgtgtgt gtaacgctag gcgcgcctgg tgtaccgaga acgatcctct cagtgcgagt
     5041 ctcgacgatc catatcgttg cttggcagtc agccagtcgg aatccagctt gggacccagg
     5101 aagtccaatc gtcagatatt gtactcaagc ctggtcacgg cagcgtaccg atctgtttaa
     5161 acctagatat tgatagtctg atcggtcaac gtataatcga gtcctagctt ttgcaaacat
     5221 ctatcaagag acaggatcag caggaggctt tcgcatgagt attcaacatt tccgtgtcgc
     5281 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5341 gaaagtaaaa gatgctgaag atcagttggg tgcgcgagtg ggttacatcg aactggatct
     5401 caacagcggt aagatccttg agagttttcg ccccgaagaa cgctttccaa tgatgagcac
     5461 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5521 cggtcgccgc atacactatt ctcagaatga cttggttgag tattcaccag tcacagaaaa
     5581 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5641 taacactgcg gccaacttac ttctgacaac gattggagga ccgaaggagc taaccgcttt
     5701 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5761 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caaccttgcg
     5821 taaactatta actggcgaac tacttactct agcttcccgg caacagttga tagactggat
     5881 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5941 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6001 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6061 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaaccgat
     6121 tctaggtgca ttggcgcaga aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca
     6181 catatgccag attcagcaac ggatacggct tccccaactt gcccacttcc atacgtgtcc
     6241 tccttaccag aaatttatcc ttaagatcgt ttaaactcga ctctggctct atcgaatctc
     6301 cgtcgtttcg agcttacgcg aacagccgtg gcgctcattt gctcgtcggg catcgaatct
     6361 cgtcagctat cgtcagctta cctttttggc agcgatcgcg gctcccgaca tcttggacca
     6421 ttagctccac aggtatcttc ttccctctag tggtcataac agcagcttca gctacctctc
     6481 aattcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct atcaatcgtt
     6541 gcgttacaca cacaaaaaac caacacacat ccatcttcga tggatagcga ttttattatc
     6601 taactgctga tcgagtgtag ccagatctag taatcaatta cggggtcatt agttcatagc
     6661 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     6721 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     6781 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     6841 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     6901 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     6961 ttagtcatcg ctattaccat gctgatgcgg ttttggcagt acatcaatgg gcgtggatag
     7021 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     7081 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     7141 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
     7201 cagatcagat ctttgtcgat cctaccatcc actcgacaca cccgccagcg gccgc
//
