LOCUS       sequence-295735-        9425 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 RNA-dependent RNA 
            polymerase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9425)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 9425)
  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-m4133616
FEATURES             Location/Qualifiers
     source          1..9425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2798..2818
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2819..2896
                     /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    2965..3086
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2976..2995)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3030..3049
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      3201..3247
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3277..3413
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    3458..3529
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(3755..4343)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3835..3854)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4843..6052
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5889..5908
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             6056..7084
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    7111..7159
                     /note="synthetic polyadenylation signal"
     CDS             join(7425..7493,7494..8285)
                     /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(7643..7662)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      8298..8434
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    8579..8650
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(8655..8701)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    8719..8767
                     /note="synthetic polyadenylation signal"
     enhancer        8848..9151
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        9153..9355
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     9306..9326
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(9424..9425,1..8)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 caccatggct gatgcacaat cgtttttaaa ccgggtttgc ggtgtaagtg cagcccgtct
       61 tacaccgtgc ggcacaggca ctagtactga tgtcgtatac agggcttttg acatctacaa
      121 tgataaagta gctggttttg ctaaattcct aaaaactaat tgttgtcgct tccaagaaaa
      181 ggacgaagat gacaatttaa ttgattctta ctttgtagtt aagagacaca ctttctctaa
      241 ctaccaacat gaagaaacaa tttataattt acttaaggat tgtccagctg ttgctaaaca
      301 tgacttcttt aagtttagaa tagacggtga catggtacca catatatcac gtcaacgtct
      361 tactaaatac acaatggcag acctcgtcta tgctttaagg cattttgatg aaggtaattg
      421 tgacacatta aaagaaatac ttgtcacata caattgttgt gatgatgatt atttcaataa
      481 aaaggactgg tatgattttg tagaaaaccc agatatatta cgcgtatacg ccaacttagg
      541 tgaacgtgta cgccaagctt tgttaaaaac agtacaattc tgtgatgcca tgcgaaatgc
      601 tggtattgtt ggtgtactga cattagataa tcaagatctc aatggtaact ggtatgattt
      661 cggtgatttc atacaaacca cgccaggtag tggagttcct gttgtagatt cttattattc
      721 attgttaatg cctatattaa ccttgaccag ggctttaact gcagagtcac atgttgacac
      781 tgacttaaca aagccttaca ttaagtggga tttgttaaaa tatgacttca cggaagagag
      841 gttaaaactc tttgaccgtt attttaaata ttgggatcag acataccacc caaattgtgt
      901 taactgtttg gatgacagat gcattctgca ttgtgcaaac tttaatgttt tattctctac
      961 agtgttccca cctacaagtt ttggaccact agtgagaaaa atatttgttg atggtgttcc
     1021 atttgtagtt tcaactggat accacttcag agagctaggt gttgtacata atcaggatgt
     1081 aaacttacat agctctagac ttagttttaa ggaattactt gtgtatgctg ctgaccctgc
     1141 tatgcacgct gcttctggta atctattact agataaacgc actacgtgct tttcagtagc
     1201 tgcacttact aacaatgttg cttttcaaac tgtcaaaccc ggtaatttta acaaagactt
     1261 ctatgacttt gctgtgtcta agggtttctt taaggaagga agttctgttg aattaaaaca
     1321 cttcttcttt gctcaggatg gtaatgctgc tatcagcgat tatgactact atcgttataa
     1381 tctaccaaca atgtgtgata tcagacaact actatttgta gttgaagttg ttgataagta
     1441 ctttgattgt tacgatggtg gctgtattaa tgctaaccaa gtcatcgtca acaacctaga
     1501 caaatcagct ggttttccat ttaataaatg gggtaaggct agactttatt atgattcaat
     1561 gagttatgag gatcaagatg cacttttcgc atatacaaaa cgtaatgtca tccctactat
     1621 aactcaaatg aatcttaagt atgccattag tgcaaagaat agagctcgca ccgtagctgg
     1681 tgtctctatc tgtagtacta tgaccaatag acagtttcat caaaaattat tgaaatcaat
     1741 agccgccact agaggagcta ctgtagtaat tggaacaagc aaattctatg gtggttggca
     1801 caacatgtta aaaactgttt atagtgatgt agaaaaccct caccttatgg gttgggatta
     1861 tcctaaatgt gatagagcca tgcctaacat gcttagaatt atggcctcac ttgttcttgc
     1921 tcgcaaacat acaacgtgtt gtagcttgtc acaccgtttc tatagattag ctaatgagtg
     1981 tgctcaagta ttgagtgaaa tggtcatgtg tggcggttca ctatatgtta aaccaggtgg
     2041 aacctcatca ggagatgcca caactgctta tgctaatagt gtttttaaca tttgtcaagc
     2101 tgtcacggcc aatgttaatg cacttttatc tactgatggt aacaaaattg ccgataagta
     2161 tgtccgcaat ttacaacaca gactttatga gtgtctctat agaaatagag atgttgacac
     2221 agactttgtg aatgagtttt acgcatattt gcgtaaacat ttctcaatga tgatactctc
     2281 tgacgatgct gttgtgtgtt tcaatagcac ttatgcatct caaggtctag tggctagcat
     2341 aaagaacttt aagtcagttc tttattatca aaacaatgtt tttatgtctg aagcaaaatg
     2401 ttggactgag actgacctta ctaaaggacc tcatgaattt tgctctcaac atacaatgct
     2461 agttaaacag ggtgatgatt atgtgtacct tccttaccca gatccatcaa gaatcctagg
     2521 ggccggctgt tttgtagatg atatcgtaaa aacagatggt acacttatga ttgaacggtt
     2581 cgtgtcttta gctatagatg cttacccact tactaaacat cctaatcagg agtatgctga
     2641 tgtctttcat ttgtacttac aatacataag aaagctacat gatgagttaa caggacacat
     2701 gttagacatg tattctgtta tgcttactaa tgataacact tcaaggtatt gggaacctga
     2761 gttttatgag gctatgtaca caccgcatac agtcttagaa aacctttact ttcagtcgaa
     2821 gaggcgatgg aaaaagaatt ttatcgcggt gagtgcagcc aacagattca aaaagatatc
     2881 ctcgtctggt gccctctaag gatccgcagg cctctgctag cttgactgac tgagatacag
     2941 cgtaccttca gctcacagac atgataagat acattgatga gtttggacaa accacaacta
     3001 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     3061 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     3121 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtattg
     3181 gcccatctct atcggtatcg tagcataacc ccttggggcc tctaaacggg tcttgagggg
     3241 ttttttgtgc ccctcgggcc ggattgctat ctaccggcat tggcgcagaa aaaaatgcct
     3301 gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg gatacggctt
     3361 ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct taaggtcgtc
     3421 agctatcctg caggcgatct ctcgatttcg atcaagacat tcctttaatg gtcttttctg
     3481 gacaccacta ggggtcagaa gtagttcatc aaactttctt ccctccctaa tctcattggt
     3541 taccttgggc tatcgaaact taattaacca gtcaagtcag ctacttggcg agatcgactt
     3601 gtctgggttt cgactacgct cagaattgcg tcagtcaagt tcgatctggt ccttgctatt
     3661 gcacccgttc tccgattacg agtttcattt aaatcatgtg agcaaaaggc cagcaaaagg
     3721 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3781 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3841 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3901 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3961 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4021 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4081 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4141 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4201 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4261 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4321 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4381 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4441 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4501 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4561 ttcgttcatc catagttgca tttaaatttc cgaactctcc aaggccctcg tcggaaaatc
     4621 ttcaaacctt tcgtccgatc catcttgcag gctacctctc gaacgaacta tcgcaagtct
     4681 cttggccggc cttgcgcctt ggctattgct tggcagcgcc tatcgccagg tattactcca
     4741 atcccgaata tccgagatcg ggatcacccg agagaagttc aacctacatc ctcaatcccg
     4801 atctatccga gatccgagga atatcgaaat cggggcgcgc ctggcctccg cgccgggttt
     4861 tggcgcctcc cgcgggcgcc cccctcgtca cggcgagcgc tgccacgtca gacgaagggc
     4921 gcaggagcgt cctgatcctt ccgcccggac gctcaggaca gcggcccgct gctcataaga
     4981 ctcggcctta gaaccccagt atcagcagaa ggacatttta ggacgggact tgggtgactc
     5041 tagggcactg gttttctttc cagagagcgg aacaggcgag gaaaagtagt cccttctcgg
     5101 cgattctgcg gagggatctc cgtggggcgg tgaacgccga tgattatata aggacgcgcc
     5161 gggtgtggca cagctagttc cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga
     5221 tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc tggccggggc tttcgtggcc
     5281 gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt
     5341 ccgcgagcaa ggttgccctg aactgggggt tggggggagc gcagcaaaat ggcggctgtt
     5401 cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg
     5461 ggggcatggt gggcggcaag aacccaaggt cttgagccct tcgctaatgc gggaaagctc
     5521 ttattcgggt gagatgggct gggcaccatc tggggaccct gacgtgaagt ttgtcactga
     5581 ctggagaact cggtttgtcg tctgttgcgg gggcggcagt tatggcggtg ccgttgggca
     5641 gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt
     5701 ggcttataat gcagggtggg gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg
     5761 caggacgcag ggttcgggcc tagggtaggc tctcctgaat cgacaggcgc cggacctctg
     5821 gtgaggggag ggataagtga ggcgtcagtt tctttggtcg gttttatgta cctatcttct
     5881 taagtagctg aagctccggt tttgaactat gcgctcgggg ttggcgagtg tgttttgtga
     5941 agttttttag gcaccttttg aaatgtaatc atttgggtca atatgtaatt ttcagtgtta
     6001 gacttgtaaa ttgtccgcta aattctggcc gtttttggct tttttgttag acaacatggg
     6061 taaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
     6121 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
     6181 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
     6241 ttatgtttat cggcactttg catccgccgc gctcccgatt ccggaagtgc ttgacattgg
     6301 ggagttcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
     6361 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg caatggatgc
     6421 catcgctgcc gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
     6481 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
     6541 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
     6601 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
     6661 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
     6721 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
     6781 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccccg
     6841 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     6901 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     6961 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcc gccgtctgga ccgatggctg
     7021 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     7081 ataagctagt atgtaagcct agtcttagat aataaaatcg ctatccatcg aagatggatg
     7141 tgtgttggtt ttttgtgtgt gtaacgctag gcgcgcctgg tgtaccgaga acgatcctct
     7201 cagtgcgagt ctcgacgatc catatcgttg cttggcagtc agccagtcgg aatccagctt
     7261 gggacccagg aagtccaatc gtcagatatt gtactcaagc ctggtcacgg cagcgtaccg
     7321 atctgtttaa acctagatat tgatagtctg atcggtcaac gtataatcga gtcctagctt
     7381 ttgcaaacat ctatcaagag acaggatcag caggaggctt tcgcatgagt attcaacatt
     7441 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     7501 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcgcgagtg ggttacatcg
     7561 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgctttccaa
     7621 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     7681 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tattcaccag
     7741 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     7801 ccatgagtga taacactgcg gccaacttac ttctgacaac gattggagga ccgaaggagc
     7861 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     7921 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     7981 caaccttgcg taaactatta actggcgaac tacttactct agcttcccgg caacagttga
     8041 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     8101 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     8161 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     8221 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     8281 ggtaaccgat tctaggtgca ttggcgcaga aaaaaatgcc tgatgcgacg ctgcgcgtct
     8341 tatactccca catatgccag attcagcaac ggatacggct tccccaactt gcccacttcc
     8401 atacgtgtcc tccttaccag aaatttatcc ttaagatcgt ttaaactcga ctctggctct
     8461 atcgaatctc cgtcgtttcg agcttacgcg aacagccgtg gcgctcattt gctcgtcggg
     8521 catcgaatct cgtcagctat cgtcagctta cctttttggc agcgatcgcg gctcccgaca
     8581 tcttggacca ttagctccac aggtatcttc ttccctctag tggtcataac agcagcttca
     8641 gctacctctc aattcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct
     8701 atcaatcgtt gcgttacaca cacaaaaaac caacacacat ccatcttcga tggatagcga
     8761 ttttattatc taactgctga tcgagtgtag ccagatctag taatcaatta cggggtcatt
     8821 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     8881 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     8941 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     9001 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     9061 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     9121 catctacgta ttagtcatcg ctattaccat gctgatgcgg ttttggcagt acatcaatgg
     9181 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     9241 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     9301 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt
     9361 agtgaaccgt cagatcagat ctttgtcgat cctaccatcc actcgacaca cccgccagcg
     9421 gccgc
//
