LOCUS       sequence-295560-        6751 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 6751)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6751)
  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-m4134164
FEATURES             Location/Qualifiers
     source          1..6751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             602..622
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             623..667
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    720..841
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(731..750)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     785..804
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      956..1002
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1032..1168
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1213..1284
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1510..2098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1590..1609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2598..3807
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3644..3663
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3811..4410
                     /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(3811..3830)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4307..4327
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4437..4485
                     /note="synthetic polyadenylation signal"
     CDS             join(4751..4819,4820..5611)
                     /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(4969..4988)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5624..5760
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5905..5976
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(5981..6027)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6045..6093
                     /note="synthetic polyadenylation signal"
     enhancer        6174..6477
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6479..6681
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6632..6652
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(6750..6751,1..8)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 caccatggca attgcctcag aatttagttc cctcccctcg tacgctgcgt tcgctaccgc
       61 ccaagaggca tatgagcagg ccgtagccaa cggggacagc gaagtggttc ttaagaaact
      121 gaagaaatct ttgaatgtgg caaagagcga gtttgatcgg gacgcggcaa tgcagaggaa
      181 gctggaaaaa atggctgatc aggctatgac acagatgtac aaacaagccc gatccgagga
      241 caagagagct aaagtcactt ctgccatgca aacgatgcta ttcaccatgt tacgtaagct
      301 ggataacgac gcactcaata acataatcaa taacgcccgc gatggttgtg tgccattgaa
      361 tatcattcct cttactacag cggccaagct gatggtcgtt atcccggact ataacacata
      421 caaaaatacc tgcgacggca ccactttcac gtatgctagc gccctgtggg aaatacagca
      481 ggtagtggat gccgattcaa agattgtgca gcttagtgag atctccatgg acaacagtcc
      541 caacttggct tggcctttaa tcgtcacagc tctcagagcc aatagcgcag ttaaactgca
      601 agaaaacctt tactttcagt cgaaagaaac cgcggcagct aagttcgaga ggcagcacat
      661 ggacagttaa gctagcttga ctgactgaga tacagcgtac cttcagctca cagacatgat
      721 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
      781 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
      841 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
      901 ttaaagcaag taaaacctct acaaatgtgg tattggccca tctctatcgg tatcgtagca
      961 taaccccttg gggcctctaa acgggtcttg aggggttttt tgtgcccctc gggccggatt
     1021 gctatctacc ggcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact
     1081 cccacatatg ccagattcag caacggatac ggcttcccca acttgcccac ttccatacgt
     1141 gtcctcctta ccagaaattt atccttaagg tcgtcagcta tcctgcaggc gatctctcga
     1201 tttcgatcaa gacattcctt taatggtctt ttctggacac cactaggggt cagaagtagt
     1261 tcatcaaact ttcttccctc cctaatctca ttggttacct tgggctatcg aaacttaatt
     1321 aaccagtcaa gtcagctact tggcgagatc gacttgtctg ggtttcgact acgctcagaa
     1381 ttgcgtcagt caagttcgat ctggtccttg ctattgcacc cgttctccga ttacgagttt
     1441 catttaaatc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     1501 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     1561 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     1621 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     1681 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     1741 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     1801 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     1861 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     1921 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     1981 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2041 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2101 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     2161 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     2221 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     2281 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcatttaa
     2341 atttccgaac tctccaaggc cctcgtcgga aaatcttcaa acctttcgtc cgatccatct
     2401 tgcaggctac ctctcgaacg aactatcgca agtctcttgg ccggccttgc gccttggcta
     2461 ttgcttggca gcgcctatcg ccaggtatta ctccaatccc gaatatccga gatcgggatc
     2521 acccgagaga agttcaacct acatcctcaa tcccgatcta tccgagatcc gaggaatatc
     2581 gaaatcgggg cgcgcctggc ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct
     2641 cgtcacggcg agcgctgcca cgtcagacga agggcgcagg agcgtcctga tccttccgcc
     2701 cggacgctca ggacagcggc ccgctgctca taagactcgg ccttagaacc ccagtatcag
     2761 cagaaggaca ttttaggacg ggacttgggt gactctaggg cactggtttt ctttccagag
     2821 agcggaacag gcgaggaaaa gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg
     2881 ggcggtgaac gccgatgatt atataaggac gcgccgggtg tggcacagct agttccgtcg
     2941 cagccgggat ttgggtcgcg gttcttgttt gtggatcgct gtgatcgtca cttggtgagt
     3001 agcgggctgc tgggctggcc ggggctttcg tggccgccgg gccgctcggt gggacggaag
     3061 cgtgtggaga gaccgccaag ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg
     3121 ggggttgggg ggagcgcagc aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt
     3181 gtgaggcggg ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc
     3241 aaggtcttga gcccttcgct aatgcgggaa agctcttatt cgggtgagat gggctgggca
     3301 ccatctgggg accctgacgt gaagtttgtc actgactgga gaactcggtt tgtcgtctgt
     3361 tgcgggggcg gcagttatgg cggtgccgtt gggcagtgca cccgtacctt tgggagcgcg
     3421 cgccctcgtc gtgtcgtgac gtcacccgtt ctgttggctt ataatgcagg gtggggccac
     3481 ctgccggtag gtgtgcggta ggcttttctc cgtcgcagga cgcagggttc gggcctaggg
     3541 taggctctcc tgaatcgaca ggcgccggac ctctggtgag gggagggata agtgaggcgt
     3601 cagtttcttt ggtcggtttt atgtacctat cttcttaagt agctgaagct ccggttttga
     3661 actatgcgct cggggttggc gagtgtgttt tgtgaagttt tttaggcacc ttttgaaatg
     3721 taatcatttg ggtcaatatg taattttcag tgttagactt gtaaattgtc cgctaaattc
     3781 tggccgtttt tggctttttt gttagacaac atgaccgagt acaagcccac ggtgcgcctc
     3841 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     3901 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     3961 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     4021 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     4081 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggag
     4141 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     4201 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcc
     4261 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     4321 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     4381 tggtgcatga cccgcaagcc cggtgcctag gctagtatgt aagcctagtc ttagataata
     4441 aaatcgctat ccatcgaaga tggatgtgtg ttggtttttt gtgtgtgtaa cgctaggcgc
     4501 gcctggtgta ccgagaacga tcctctcagt gcgagtctcg acgatccata tcgttgcttg
     4561 gcagtcagcc agtcggaatc cagcttggga cccaggaagt ccaatcgtca gatattgtac
     4621 tcaagcctgg tcacggcagc gtaccgatct gtttaaacct agatattgat agtctgatcg
     4681 gtcaacgtat aatcgagtcc tagcttttgc aaacatctat caagagacag gatcagcagg
     4741 aggctttcgc atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4801 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4861 gttgggtgcg cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4921 ttttcgcccc gaagaacgct ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4981 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5041 gaatgacttg gttgagtatt caccagtcac agaaaagcat cttacggatg gcatgacagt
     5101 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5161 gacaacgatt ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5221 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5281 caccacgatg cctgtagcaa tggcaacaac cttgcgtaaa ctattaactg gcgaactact
     5341 tactctagct tcccggcaac agttgataga ctggatggag gcggataaag ttgcaggacc
     5401 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5461 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5521 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5581 gataggtgcc tcactgatta agcattggta accgattcta ggtgcattgg cgcagaaaaa
     5641 aatgcctgat gcgacgctgc gcgtcttata ctcccacata tgccagattc agcaacggat
     5701 acggcttccc caacttgccc acttccatac gtgtcctcct taccagaaat ttatccttaa
     5761 gatcgtttaa actcgactct ggctctatcg aatctccgtc gtttcgagct tacgcgaaca
     5821 gccgtggcgc tcatttgctc gtcgggcatc gaatctcgtc agctatcgtc agcttacctt
     5881 tttggcagcg atcgcggctc ccgacatctt ggaccattag ctccacaggt atcttcttcc
     5941 ctctagtggt cataacagca gcttcagcta cctctcaatt caaaaaaccc ctcaagaccc
     6001 gtttagaggc cccaaggggt tatgctatca atcgttgcgt tacacacaca aaaaaccaac
     6061 acacatccat cttcgatgga tagcgatttt attatctaac tgctgatcga gtgtagccag
     6121 atctagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     6181 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     6241 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     6301 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     6361 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     6421 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatgctg
     6481 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     6541 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     6601 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     6661 gaggtctata taagcagagc tggtttagtg aaccgtcaga tcagatcttt gtcgatccta
     6721 ccatccactc gacacacccg ccagcggccg c
//
