LOCUS       sequence-295897-        6778 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 6778)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6778)
  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-m4133437
FEATURES             Location/Qualifiers
     source          1..6778
                     /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             617..694
                     /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"
     polyA_signal    747..868
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(758..777)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     812..831
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      983..1029
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1059..1195
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1240..1311
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1537..2125)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1617..1636)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2625..3834
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3671..3690
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3838..4437
                     /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(3838..3857)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4334..4354
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4464..4512
                     /note="synthetic polyadenylation signal"
     CDS             join(4778..4846,4847..5638)
                     /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(4996..5015)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5651..5787
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5932..6003
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6008..6054)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6072..6120
                     /note="synthetic polyadenylation signal"
     enhancer        6201..6504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6506..6708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6659..6679
                     /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 cagtcgaaga ggcgatggaa aaagaatttt atcgcggtga gtgcagccaa
      661 cagattcaaa aagatatcct cgtctggtgc cctctaagct agcttgactg actgagatac
      721 agcgtacctt cagctcacag acatgataag atacattgat gagtttggac aaaccacaac
      781 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
      841 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
      901 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat
      961 tggcccatct ctatcggtat cgtagcataa ccccttgggg cctctaaacg ggtcttgagg
     1021 ggttttttgt gcccctcggg ccggattgct atctaccggc attggcgcag aaaaaaatgc
     1081 ctgatgcgac gctgcgcgtc ttatactccc acatatgcca gattcagcaa cggatacggc
     1141 ttccccaact tgcccacttc catacgtgtc ctccttacca gaaatttatc cttaaggtcg
     1201 tcagctatcc tgcaggcgat ctctcgattt cgatcaagac attcctttaa tggtcttttc
     1261 tggacaccac taggggtcag aagtagttca tcaaactttc ttccctccct aatctcattg
     1321 gttaccttgg gctatcgaaa cttaattaac cagtcaagtc agctacttgg cgagatcgac
     1381 ttgtctgggt ttcgactacg ctcagaattg cgtcagtcaa gttcgatctg gtccttgcta
     1441 ttgcacccgt tctccgatta cgagtttcat ttaaatcatg tgagcaaaag gccagcaaaa
     1501 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1561 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1621 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1681 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1741 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1801 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1861 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1921 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     1981 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2041 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2101 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2161 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2221 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2281 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2341 atttcgttca tccatagttg catttaaatt tccgaactct ccaaggccct cgtcggaaaa
     2401 tcttcaaacc tttcgtccga tccatcttgc aggctacctc tcgaacgaac tatcgcaagt
     2461 ctcttggccg gccttgcgcc ttggctattg cttggcagcg cctatcgcca ggtattactc
     2521 caatcccgaa tatccgagat cgggatcacc cgagagaagt tcaacctaca tcctcaatcc
     2581 cgatctatcc gagatccgag gaatatcgaa atcggggcgc gcctggcctc cgcgccgggt
     2641 tttggcgcct cccgcgggcg cccccctcgt cacggcgagc gctgccacgt cagacgaagg
     2701 gcgcaggagc gtcctgatcc ttccgcccgg acgctcagga cagcggcccg ctgctcataa
     2761 gactcggcct tagaacccca gtatcagcag aaggacattt taggacggga cttgggtgac
     2821 tctagggcac tggttttctt tccagagagc ggaacaggcg aggaaaagta gtcccttctc
     2881 ggcgattctg cggagggatc tccgtggggc ggtgaacgcc gatgattata taaggacgcg
     2941 ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg ggtcgcggtt cttgtttgtg
     3001 gatcgctgtg atcgtcactt ggtgagtagc gggctgctgg gctggccggg gctttcgtgg
     3061 ccgccgggcc gctcggtggg acggaagcgt gtggagagac cgccaagggc tgtagtctgg
     3121 gtccgcgagc aaggttgccc tgaactgggg gttgggggga gcgcagcaaa atggcggctg
     3181 ttcccgagtc ttgaatggaa gacgcttgtg aggcgggctg tgaggtcgtt gaaacaaggt
     3241 ggggggcatg gtgggcggca agaacccaag gtcttgagcc cttcgctaat gcgggaaagc
     3301 tcttattcgg gtgagatggg ctgggcacca tctggggacc ctgacgtgaa gtttgtcact
     3361 gactggagaa ctcggtttgt cgtctgttgc gggggcggca gttatggcgg tgccgttggg
     3421 cagtgcaccc gtacctttgg gagcgcgcgc cctcgtcgtg tcgtgacgtc acccgttctg
     3481 ttggcttata atgcagggtg gggccacctg ccggtaggtg tgcggtaggc ttttctccgt
     3541 cgcaggacgc agggttcggg cctagggtag gctctcctga atcgacaggc gccggacctc
     3601 tggtgagggg agggataagt gaggcgtcag tttctttggt cggttttatg tacctatctt
     3661 cttaagtagc tgaagctccg gttttgaact atgcgctcgg ggttggcgag tgtgttttgt
     3721 gaagtttttt aggcaccttt tgaaatgtaa tcatttgggt caatatgtaa ttttcagtgt
     3781 tagacttgta aattgtccgc taaattctgg ccgtttttgg cttttttgtt agacaacatg
     3841 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     3901 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     3961 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     4021 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     4081 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     4141 cggctggccg cgcagcaaca gatggagggc ctcctggcgc cgcaccggcc caaggagccc
     4201 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     4261 gccgtcgtgc tccccggagt ggaggccgcc gagcgcgccg gggtgcccgc cttcctggag
     4321 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     4381 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctaggct
     4441 agtatgtaag cctagtctta gataataaaa tcgctatcca tcgaagatgg atgtgtgttg
     4501 gttttttgtg tgtgtaacgc taggcgcgcc tggtgtaccg agaacgatcc tctcagtgcg
     4561 agtctcgacg atccatatcg ttgcttggca gtcagccagt cggaatccag cttgggaccc
     4621 aggaagtcca atcgtcagat attgtactca agcctggtca cggcagcgta ccgatctgtt
     4681 taaacctaga tattgatagt ctgatcggtc aacgtataat cgagtcctag cttttgcaaa
     4741 catctatcaa gagacaggat cagcaggagg ctttcgcatg agtattcaac atttccgtgt
     4801 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4861 ggtgaaagta aaagatgctg aagatcagtt gggtgcgcga gtgggttaca tcgaactgga
     4921 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgctttc caatgatgag
     4981 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     5041 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtattcac cagtcacaga
     5101 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     5161 tgataacact gcggccaact tacttctgac aacgattgga ggaccgaagg agctaaccgc
     5221 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     5281 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacctt
     5341 gcgtaaacta ttaactggcg aactacttac tctagcttcc cggcaacagt tgatagactg
     5401 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5461 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5521 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5581 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaacc
     5641 gattctaggt gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc
     5701 ccacatatgc cagattcagc aacggatacg gcttccccaa cttgcccact tccatacgtg
     5761 tcctccttac cagaaattta tccttaagat cgtttaaact cgactctggc tctatcgaat
     5821 ctccgtcgtt tcgagcttac gcgaacagcc gtggcgctca tttgctcgtc gggcatcgaa
     5881 tctcgtcagc tatcgtcagc ttaccttttt ggcagcgatc gcggctcccg acatcttgga
     5941 ccattagctc cacaggtatc ttcttccctc tagtggtcat aacagcagct tcagctacct
     6001 ctcaattcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctatcaatc
     6061 gttgcgttac acacacaaaa aaccaacaca catccatctt cgatggatag cgattttatt
     6121 atctaactgc tgatcgagtg tagccagatc tagtaatcaa ttacggggtc attagttcat
     6181 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     6241 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     6301 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     6361 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     6421 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     6481 gtattagtca tcgctattac catgctgatg cggttttggc agtacatcaa tgggcgtgga
     6541 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     6601 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     6661 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac
     6721 cgtcagatca gatctttgtc gatcctacca tccactcgac acacccgcca gcggccgc
//
