LOCUS       Exported                7084 bp ds-DNA     circular SYN 03-JUN-2021
DEFINITION  To express His-SUMO-nsp12 (SARS-CoV-2) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    pK27Sumo_His-SUMO-nsp12 (SARS-CoV-2)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  AUTHORS   Bertolin AP, Weissmann F, Zeng J, Posse V, Milligan JC, Canal B, 
            Ulferts R, Wu M, Drury LS, Howell M, Beale R, Diffley JFX
  TITLE     Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small 
            Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase
  JOURNAL   bioRxiv https://www.biorxiv.org/content/10.1101/2021.04.07.438807v1
REFERENCE   2  (bases 1 to 7084)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 3, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7084
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     680..698
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        722..766
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    742..758
                     /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)."
     protein_bind    774..790
                     /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)."
     RBS             809..820
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             905..1192
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG"
     terminator      4041..4135
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(4183..4202)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4194..5009
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4267..4286)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      5228..5314
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5407..6489)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6451..6470
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6490..6567)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(6651..6673)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6759..6899
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6814..6833
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6914..6931)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc acctcgagaa
      721 atcataaaaa atttatttgc tttgtgagcg gataacaatt ataatagatt caattgtgag
      781 cggataacaa tttcacacag aattcattaa agaggagaaa ttaaccatga gcaagcatca
      841 ccatcattca ggccatcacc ataccggaca ccaccatcat tcaggcagtc atcaccatac
      901 cggatcggac tcagaagtca atcaagaagc taagccagag gtcaagccag aagtcaagcc
      961 tgagactcac atcaatttaa aggtgtccga tggatcttca gagatcttct tcaagatcaa
     1021 aaagaccact cctttaagaa ggctgatgga agcgttcgct aaaagacagg gtaaggaaat
     1081 ggactcctta agattcttgt acgacggtat tagaattcaa gctgatcaga cccctgaaga
     1141 tttggacatg gaggataacg atattattga ggctcacaga gaacagattg gtggcagcgc
     1201 agatgcacag agctttctga atcgtgtttg tggtgttagc gcagcacgtc tgaccccgtg
     1261 tggcaccggc accagcaccg atgttgttta tcgtgcattt gatatctaca acgataaagt
     1321 tgccggtttc gccaaatttc tgaaaaccaa ttgttgccgc ttccaagaga aagatgaaga
     1381 tgataatctg atcgacagct actttgtggt taaacgtcat acctttagca actatcagca
     1441 cgaggaaacc atttacaatc tgctgaaaga ttgtccggca gttgccaaac atgatttttt
     1501 caaatttcgc atcgatggcg atatggttcc gcatattagc cgtcagcgtc tgaccaaata
     1561 taccatggca gatctggttt atgcactgcg tcattttgat gaaggtaatt gcgataccct
     1621 gaaagaaatt ctggtgacct ataattgttg cgacgatgac tacttcaaca agaaagattg
     1681 gtatgatttc gtggaaaacc cggatattct gcgtgtttat gcaaatctgg gtgaacgtgt
     1741 tcgtcaggca ctgctgaaaa ccgttcagtt ttgtgatgca atgcgtaatg caggtattgt
     1801 tggtgttctg accctggata atcaggatct gaatggtaat tggtacgatt ttggcgattt
     1861 tattcagacc acacctggta gcggtgttcc ggttgttgat agctattata gcctgctgat
     1921 gccgattctg acactgaccc gtgcactgac cgcagaaagc catgttgata ccgatctgac
     1981 aaaaccgtac attaaatggg acctgctgaa atatgatttt accgaagaac gcctgaaact
     2041 gttcgatcgc tatttcaaat attgggacca gacctatcat ccgaattgtg ttaattgtct
     2101 ggatgatcgt tgtattctgc attgcgccaa ttttaacgtt ctgtttagca ccgtttttcc
     2161 gcctaccagc tttggtccgc tggttcgtaa aatctttgtt gatggtgtgc cgtttgttgt
     2221 tagcaccggt tatcattttc gtgaactggg tgttgttcat aaccaggatg ttaatctgca
     2281 tagcagccgt ctgagcttta aagaactgct ggtgtatgca gcagatccgg caatgcatgc
     2341 agcaagcggt aatctgctgc tggataaacg taccacctgt tttagcgttg cagcactgac
     2401 caataatgtt gcatttcaga ccgttaaacc gggtaatttc aacaaggatt tctatgattt
     2461 tgccgtgagc aagggctttt tcaaagaagg tagcagcgtt gaactgaagc attttttctt
     2521 tgcacaggat ggtaatgcag ccatcagcga ttatgattat tatcgctata atctgccgac
     2581 catgtgtgat attcgtcagc tgctgtttgt tgtggaagtt gtggataaat acttcgattg
     2641 ctatgatggc ggttgcatta acgcaaatca ggtgattgtt aataacctgg acaaaagcgc
     2701 aggtttcccg tttaacaaat ggggtaaagc ccgtctgtat tatgatagca tgagctatga
     2761 agatcaggat gcactgtttg catataccaa acgtaatgtg attccgacca ttacgcagat
     2821 gaatctgaaa tacgcaatta gcgcaaaaaa tcgtgcacgt accgttgccg gtgttagcat
     2881 ttgtagcacc atgaccaatc gtcagtttca tcagaaactg ctgaagagta ttgcagcaac
     2941 ccgtggtgca accgttgtta ttggtacaag caaattctat ggtggctggc ataatatgct
     3001 taagaccgtg tattcagatg tggaaaatcc gcatctgatg ggttgggatt atccgaaatg
     3061 tgatcgtgca atgccgaata tgctgcgtat tatggcaagc ctggttctgg cacgtaaaca
     3121 taccacatgt tgcagcctga gccatcgttt ttatcgtctg gcaaatgaat gtgcacaggt
     3181 tctgagcgaa atggttatgt gtggtggtag cctgtatgtt aaacctggtg gcaccagctc
     3241 aggtgatgca accaccgcct atgcaaatag cgtttttaac atttgtcagg ccgttaccgc
     3301 caatgttaat gccctgctga gtaccgatgg caacaaaatt gcagataaat atgtgcgcaa
     3361 tctgcagcat cgtctgtatg aatgtctgta tcgtaatcgt gatgtggata ccgactttgt
     3421 gaatgagttt tatgcctatc tgcgcaaaca cttcagcatg atgattctga gtgatgatgc
     3481 cgttgtttgc tttaatagca cctatgcaag ccaaggtctg gttgcaagca tcaaaaactt
     3541 taaaagcgtg ctgtactacc agaacaatgt gtttatgagc gaagcaaaat gttggaccga
     3601 aaccgacctg accaaaggtc cgcatgaatt ttgtagccag cataccatgc tggttaaaca
     3661 gggtgatgat tatgtttatc tgccgtatcc tgatccgagc cgtattttag gtgcaggttg
     3721 ttttgtggat gacatcgtta aaaccgatgg tacgctgatg attgaacgtt ttgttagcct
     3781 ggcaattgat gcatatccgc tgaccaaaca tccgaatcaa gaatatgcag atgtgttcca
     3841 tctgtacctg cagtatattc gtaaactgca tgatgaactg acaggtcaca tgctggatat
     3901 gtatagcgtt atgctgacca atgataatac cagccgttat tgggaacccg aattttatga
     3961 agcaatgtat acaccgcata ccgtgctgca gtaataagcg gccgcacatc accatcacta
     4021 agcttaatta gctgagcttg gactcctgtt gatagatcca gtaatgacct cagaactcca
     4081 tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatccaag
     4141 ctagccatga aaataaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     4201 atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt
     4261 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     4321 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     4381 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     4441 ccatcaagca ttttatccgt actcctgatg atgcttggtt actcacgact gcgatccccg
     4501 gcaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     4561 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     4621 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     4681 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     4741 acaaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     4801 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     4861 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     4921 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     4981 ttcatttgat gctcgatgag tttttctaag aattaattca tgggcaaata ttatacgcaa
     5041 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc
     5101 catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg
     5161 taattttttt aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt
     5221 gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     5281 tttgtcggtg aacgctctcc tgagtaggac aaatccgccc tctagattac gtgcagtcga
     5341 tgataagctg tcaaacatga gaattgtgcc taatgagtga gctaacttac attaattgcg
     5401 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5461 ggccaacgcg cggggagagg cggtttgcgt attgggggcc agggtggttt ttcttttcac
     5521 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     5581 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     5641 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     5701 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     5761 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     5821 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     5881 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     5941 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     6001 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     6061 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     6121 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     6181 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     6241 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     6301 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     6361 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     6421 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     6481 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     6541 aaaggttttg caccattcga tggtgtcgga atttcgggca gcgttgggtc ctggccacgg
     6601 gtgcgcatga tctagagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     6661 atgcagctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag cagacaagcc
     6721 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     6781 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     6841 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     6901 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     6961 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     7021 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     7081 cgtt
//
