LOCUS       Exported                9159 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expression of codon-optimized SARS-CoV2 Nsp12 (RdRp) in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    p3xFLAG SARS-CoV2 RdRp.co
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9159)
  TITLE     Mammalian expression plasmid for production of codon-optimized 
            SARS-CoV2 Nsp12 (RdRp)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9159)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9159
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     29..51
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     43..60
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     44..60
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        221..600
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        601..804
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     755..775
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3661..3726
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3745..3810
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    3821..4443
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4020..4039)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(4467..4487)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4485..4782
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     4714..4733
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4836..5630
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4890..4909)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5500..5519
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6282..6416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6319..6338)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6373..6392
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6421..6440)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6422..6440)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6454..6470)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6454..6470)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6467..6489)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6478..6494
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(6502..6532)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6547..6568
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6685..6702)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6856..7444)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6936..6955)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7615..8475)
                     /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     8238..8257
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8476..8580)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      8607..9062
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8694..8713)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8904..8925
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
       61 gccaagctga tctatacatt gaatcaatat tggcaattag ccatattagt cattggttat
      121 atagcataaa tcaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg
      181 tacatttata ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt
      241 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      301 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      361 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      421 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      481 ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      541 accttacggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      601 gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt
      661 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      721 tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg
      781 tgggaggtct atataagcag agctcgttta gtgaaccgtc agaattaagc ttgcggccgc
      841 gaattcaaca tgtccgcaga tgcgcaatct tttttgaaca gagtttgtgg tgtaagtgca
      901 gcccgtctta caccgtgcgg cacaggcact agtactgatg tcgtctatag ggcgttcgat
      961 atttataacg ataaagtggc gggcttcgca aaattcctta aaactaactg ctgtaggttc
     1021 caggaaaaag acgaagatga caatcttata gacagctatt tcgtcgttaa acgacatact
     1081 tttagcaact accaacatga ggaaacgatc tacaacttgc ttaaagattg tccggcggta
     1141 gcgaagcacg acttcttcaa atttcggatc gatggagata tggttcctca catttccaga
     1201 caacgcctga ctaaatacac tatggcagat cttgtttacg ccttgcgaca ttttgacgag
     1261 ggtaactgcg atactcttaa ggagatcctc gttacctata attgctgcga tgatgattac
     1321 tttaataaga aagactggta cgattttgta gagaaccctg atatcctccg ggtctatgcg
     1381 aatctcgggg agcgagttag acaggcgctc cttaagacag tgcaattctg cgacgcaatg
     1441 cggaatgccg gaatagtggg tgtacttacg cttgataatc aagacctcaa cggtaactgg
     1501 tatgacttcg gagatttcat ccagaccacg cctggatcag gcgtccccgt cgtggattca
     1561 tattactctc tgttgatgcc tattcttacc cttaccagag ctcttacagc tgaatctcat
     1621 gtggatactg atctcactaa accatacatc aagtgggacc tcttgaaata tgattttact
     1681 gaagaaaggc tgaagctgtt cgacaggtac tttaagtatt gggaccaaac atatcatccg
     1741 aactgtgtga attgtcttga tgatcgatgc atactccatt gtgcgaattt taatgtactc
     1801 ttctcaacag tcttcccacc tacaagtttt ggaccgctcg taagaaaaat ttttgtggac
     1861 ggagtccctt tcgtcgtctc tactggctat cactttcgag agttgggtgt cgttcacaac
     1921 caggacgtaa accttcacag tagtcgactt tcctttaaag aactgcttgt ctacgccgcc
     1981 gacccagcta tgcacgcagc cagtggaaac ctcttgctcg ataaacgaac tacctgcttt
     2041 agcgttgcgg ccttgacaaa caacgtcgca ttccagactg tgaagccagg taattttaac
     2101 aaagacttct atgactttgc agttagtaag ggattcttta aggaaggaag ttccgttgaa
     2161 ctcaaacatt tttttttcgc gcaagatggc aatgccgcta tctcagacta cgactattac
     2221 cgatataacc tgcccaccat gtgcgacata cgccaactgc tttttgtggt cgaagttgtg
     2281 gataagtact ttgactgcta tgatgggggt tgcattaatg caaaccaagt cattgttaat
     2341 aaccttgata aatccgctgg cttccccttt aataaatggg ggaaagcacg gttgtattat
     2401 gactctatgt cctacgagga tcaagatgcc ttgtttgcgt ataccaaaag gaacgttata
     2461 cccacaatta cgcagatgaa tctgaagtat gccataagtg cgaaaaatag ggcacgcacc
     2521 gtcgcaggag taagcatatg ttcaacaatg accaataggc aatttcatca aaaactgttg
     2581 aagtcaattg ccgccacgag aggcgctact gtcgtaattg gaacatcaaa attttatggg
     2641 ggctggcata acatgcttaa aacggtgtat tcagatgtcg agaatccgca cctcatgggc
     2701 tgggattacc cgaagtgcga ccgggcaatg ccgaacatgc tcaggatcat ggctagtttg
     2761 gtactggccc ggaaacatac cacatgctgt tcactcagtc atcgatttta tcgacttgcg
     2821 aacgagtgcg cccaagtcct gagcgaaatg gtaatgtgtg gaggctctct ctatgtaaaa
     2881 cccggtggta cctcctcagg agatgcaact accgcgtacg cgaactctgt atttaatatt
     2941 tgccaggcag taactgcgaa tgttaatgct ctgctgagca cagatgggaa taagatagcc
     3001 gataaatacg tgcgcaacct ccaacaccgc ttgtatgagt gtctgtatag aaatcgggac
     3061 gtagataccg actttgtgaa tgaattttac gcgtatctgc ggaagcattt ttctatgatg
     3121 attctgtccg acgatgcagt agtctgcttc aattcaactt atgcttccca aggcttggtc
     3181 gcctctataa aaaattttaa gtcagtcctg tattatcaaa ataatgtatt tatgtcagaa
     3241 gctaagtgtt ggacggagac cgatctgacg aaaggtcccc atgagttctg ctcccaacat
     3301 acaatgctgg tgaagcaagg tgatgattac gtttacctcc catacccaga tccgtcccgg
     3361 attcttgggg ctgggtgttt cgtggatgat attgtgaaaa ctgatggcac actcatgata
     3421 gagcggtttg tttccctggc tattgatgca taccctctga cgaagcaccc aaatcaagag
     3481 tacgcggacg tgtttcactt gtatctgcag tacattcgga agctgcacga cgaactgaca
     3541 ggacacatgc ttgatatgta cagtgtcatg ctcactaatg ataacacttc ccgctattgg
     3601 gagccggagt tttacgaagc gatgtataca ccccatactg tgcttcaggg gtcccgggcg
     3661 gactataagg accatgatgg ggattacaaa gatcatgaca tcgattataa agatgatgat
     3721 gacaaatgat aaggatcccg ggctgactac aaagaccatg acggtgatta taaagatcat
     3781 gacatcgact acaaggatga cgatgacaag tagtgatccc gggtggcatc cctgtgaccc
     3841 ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct
     3901 aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg
     3961 gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt
     4021 ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc
     4081 ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat
     4141 gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag
     4201 gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg
     4261 attacaggcg tgaaccactg ctcccttccc tgtccttctg attttaaaat aactatacca
     4321 gcaggaggac gtccagacac agcataggct acctggccat gcccaaccgg tgggacattt
     4381 gagttgcttg cttggcactg tcctctcatg cgttgggtcc actcagtaga tgcctgttga
     4441 attgggtacg cggccagctt ggctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     4501 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     4561 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4621 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4681 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4741 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggaattg atcagcttgg
     4801 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     4861 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     4921 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     4981 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     5041 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     5101 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     5161 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     5221 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     5281 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     5341 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     5401 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     5461 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     5521 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     5581 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     5641 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     5701 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     5761 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccgggctcg atcccctcgc
     5821 gagttggttc agctgctgcc tgaggctgga cgacctcgcg gagttctacc ggcagtgcaa
     5881 atccgtcggc atccaggaaa ccagcagcgg ctatccgcgc atccatgccc ccgaactgca
     5941 ggagtgggga ggcacgatgg ccgctttggt cgacccggac gggacgctcc tgcgcctgat
     6001 acagaacgaa ttgcttgcag gcatctcatg agtgtgtctt cccgttttcc gcctgaggtc
     6061 actgcgtgga tggagcgctg gcgcctgctg cgcgacggcg agctgctcac cacccactcg
     6121 ccaagctgga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgccgatca
     6181 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     6241 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     6301 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     6361 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcaatt
     6421 ccctatagtg agtcgtatta aattcgtaat catgtcatag ctgtttcctg tgtgaaattg
     6481 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     6541 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     6601 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6661 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     6721 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     6781 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     6841 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     6901 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6961 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     7021 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     7081 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     7141 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     7201 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     7261 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     7321 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     7381 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     7441 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     7501 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     7561 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     7621 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     7681 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     7741 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     7801 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     7861 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     7921 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     7981 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     8041 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     8101 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     8161 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     8221 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     8281 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     8341 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     8401 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     8461 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     8521 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     8581 cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg
     8641 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     8701 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     8761 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     8821 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     8881 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     8941 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     9001 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     9061 ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     9121 ctattacgcc agctggcgaa agggggatgt gctgcaagg
//
