LOCUS       sequence-295543-        6742 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 6742)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6742)
  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-m4133954
FEATURES             Location/Qualifiers
     source          1..6742
                     /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..658
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    711..832
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(722..741)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     776..795
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      947..993
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1023..1159
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1204..1275
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1501..2089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1581..1600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2589..3798
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3635..3654
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3802..4401
                     /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(3802..3821)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4298..4318
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4428..4476
                     /note="synthetic polyadenylation signal"
     CDS             join(4742..4810,4811..5602)
                     /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(4960..4979)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5615..5751
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5896..5967
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(5972..6018)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6036..6084
                     /note="synthetic polyadenylation signal"
     enhancer        6165..6468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6470..6672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6623..6643
                     /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 cagtcgggga aacctatacc caatccactt ctcggcttag attctaccta
      661 agctagcttg actgactgag atacagcgta ccttcagctc acagacatga taagatacat
      721 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
      781 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
      841 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
      901 gtaaaacctc tacaaatgtg gtattggccc atctctatcg gtatcgtagc ataacccctt
      961 ggggcctcta aacgggtctt gaggggtttt ttgtgcccct cgggccggat tgctatctac
     1021 cggcattggc gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac tcccacatat
     1081 gccagattca gcaacggata cggcttcccc aacttgccca cttccatacg tgtcctcctt
     1141 accagaaatt tatccttaag gtcgtcagct atcctgcagg cgatctctcg atttcgatca
     1201 agacattcct ttaatggtct tttctggaca ccactagggg tcagaagtag ttcatcaaac
     1261 tttcttccct ccctaatctc attggttacc ttgggctatc gaaacttaat taaccagtca
     1321 agtcagctac ttggcgagat cgacttgtct gggtttcgac tacgctcaga attgcgtcag
     1381 tcaagttcga tctggtcctt gctattgcac ccgttctccg attacgagtt tcatttaaat
     1441 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1501 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     1561 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     1621 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     1681 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     1741 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     1801 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     1861 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     1921 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     1981 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2041 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2101 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2161 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2221 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     2281 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcattta aatttccgaa
     2341 ctctccaagg ccctcgtcgg aaaatcttca aacctttcgt ccgatccatc ttgcaggcta
     2401 cctctcgaac gaactatcgc aagtctcttg gccggccttg cgccttggct attgcttggc
     2461 agcgcctatc gccaggtatt actccaatcc cgaatatccg agatcgggat cacccgagag
     2521 aagttcaacc tacatcctca atcccgatct atccgagatc cgaggaatat cgaaatcggg
     2581 gcgcgcctgg cctccgcgcc gggttttggc gcctcccgcg ggcgcccccc tcgtcacggc
     2641 gagcgctgcc acgtcagacg aagggcgcag gagcgtcctg atccttccgc ccggacgctc
     2701 aggacagcgg cccgctgctc ataagactcg gccttagaac cccagtatca gcagaaggac
     2761 attttaggac gggacttggg tgactctagg gcactggttt tctttccaga gagcggaaca
     2821 ggcgaggaaa agtagtccct tctcggcgat tctgcggagg gatctccgtg gggcggtgaa
     2881 cgccgatgat tatataagga cgcgccgggt gtggcacagc tagttccgtc gcagccggga
     2941 tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc acttggtgag tagcgggctg
     3001 ctgggctggc cggggctttc gtggccgccg ggccgctcgg tgggacggaa gcgtgtggag
     3061 agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt gccctgaact gggggttggg
     3121 gggagcgcag caaaatggcg gctgttcccg agtcttgaat ggaagacgct tgtgaggcgg
     3181 gctgtgaggt cgttgaaaca aggtgggggg catggtgggc ggcaagaacc caaggtcttg
     3241 agcccttcgc taatgcggga aagctcttat tcgggtgaga tgggctgggc accatctggg
     3301 gaccctgacg tgaagtttgt cactgactgg agaactcggt ttgtcgtctg ttgcgggggc
     3361 ggcagttatg gcggtgccgt tgggcagtgc acccgtacct ttgggagcgc gcgccctcgt
     3421 cgtgtcgtga cgtcacccgt tctgttggct tataatgcag ggtggggcca cctgccggta
     3481 ggtgtgcggt aggcttttct ccgtcgcagg acgcagggtt cgggcctagg gtaggctctc
     3541 ctgaatcgac aggcgccgga cctctggtga ggggagggat aagtgaggcg tcagtttctt
     3601 tggtcggttt tatgtaccta tcttcttaag tagctgaagc tccggttttg aactatgcgc
     3661 tcggggttgg cgagtgtgtt ttgtgaagtt ttttaggcac cttttgaaat gtaatcattt
     3721 gggtcaatat gtaattttca gtgttagact tgtaaattgt ccgctaaatt ctggccgttt
     3781 ttggcttttt tgttagacaa catgaccgag tacaagccca cggtgcgcct cgccacccgc
     3841 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     3901 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     3961 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     4021 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     4081 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga gggcctcctg
     4141 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     4201 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc cgccgagcgc
     4261 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     4321 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     4381 acccgcaagc ccggtgccta ggctagtatg taagcctagt cttagataat aaaatcgcta
     4441 tccatcgaag atggatgtgt gttggttttt tgtgtgtgta acgctaggcg cgcctggtgt
     4501 accgagaacg atcctctcag tgcgagtctc gacgatccat atcgttgctt ggcagtcagc
     4561 cagtcggaat ccagcttggg acccaggaag tccaatcgtc agatattgta ctcaagcctg
     4621 gtcacggcag cgtaccgatc tgtttaaacc tagatattga tagtctgatc ggtcaacgta
     4681 taatcgagtc ctagcttttg caaacatcta tcaagagaca ggatcagcag gaggctttcg
     4741 catgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4801 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4861 gcgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4921 cgaagaacgc tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4981 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5041 ggttgagtat tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5101 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5161 tggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5221 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5281 gcctgtagca atggcaacaa ccttgcgtaa actattaact ggcgaactac ttactctagc
     5341 ttcccggcaa cagttgatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5401 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5461 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5521 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5581 ctcactgatt aagcattggt aaccgattct aggtgcattg gcgcagaaaa aaatgcctga
     5641 tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga tacggcttcc
     5701 ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta agatcgttta
     5761 aactcgactc tggctctatc gaatctccgt cgtttcgagc ttacgcgaac agccgtggcg
     5821 ctcatttgct cgtcgggcat cgaatctcgt cagctatcgt cagcttacct ttttggcagc
     5881 gatcgcggct cccgacatct tggaccatta gctccacagg tatcttcttc cctctagtgg
     5941 tcataacagc agcttcagct acctctcaat tcaaaaaacc cctcaagacc cgtttagagg
     6001 ccccaagggg ttatgctatc aatcgttgcg ttacacacac aaaaaaccaa cacacatcca
     6061 tcttcgatgg atagcgattt tattatctaa ctgctgatcg agtgtagcca gatctagtaa
     6121 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     6181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     6241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     6301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     6361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     6421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct gatgcggttt
     6481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     6541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     6601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     6661 ataagcagag ctggtttagt gaaccgtcag atcagatctt tgtcgatcct accatccact
     6721 cgacacaccc gccagcggcc gc
//
