LOCUS       Exported                7143 bp ds-DNA     circular SYN 10-JAN-2022
DEFINITION  Expresses the N-terminal His6-tagged SARS-CoV2 helicase (NSP13) in E
            coli..
ACCESSION   .
VERSION     .
KEYWORDS    pET28a-SARS-CoV-2 NSP13
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7143)
  AUTHORS   White MA, Lin W, Cheng X
  TITLE     Discovery of COVID-19 Inhibitors Targeting the SARS-CoV-2 Nsp13 
            Helicase.
  JOURNAL   J Phys Chem Lett. 2020 Nov 5;11(21):9144-9151. doi: 
            10.1021/acs.jpclett.0c02421. Epub 2020 Oct 14.
  PUBMED    33052685
REFERENCE   2  (bases 1 to 7143)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 10, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7143
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      26..73
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1985..2017)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             complement(2021..2038)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2048..2065)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2084..2106
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2121..2145
                     /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)."
     primer_bind     complement(2145..2164)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2146..2164)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2248..2267)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2473..2550
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2551..3633
                     /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     complement(2570..2589)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4442..4633
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4627..4649)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4735..4874
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4790..4809
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4889..4906)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5060..5648)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5140..5159)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5759..5778)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5770..6585
                     /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(5843..5862)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6677..7132)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6814..6835)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7026..7045
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtcattgt aatgtagcca
      181 cattgcgacg tggtatttct agacttgtaa attgcagttt gtcataaaga tctctatcag
      241 acattatgca caaaatgcca acttttgccc ttgtgatagc cacattgaag cggttgacat
      301 tacaagagtg tgctgtttca gtagtttgtg tgaatatgac atagtcatat tcagaaccct
      361 gtgatgaatc aacagtctgc gtaggcaatc ctaagatttt tgaagctacg gcgttctgtg
      421 aattataagg tgagataaaa acagcttttc tccaagcagg attgcgtgta agaaattctc
      481 ttacaacgcc tatttgaggt ctgttgattg cagatgaaac atcatgtgta ataacacctt
      541 tgtagaacat tttgaagcat tgagctgact tatccttgtg tgcttttagc ttattgtcat
      601 aaactaaagc actcacagtg tcaacaattt cagcaggaca acggcgacaa gttccaagga
      661 acatgtctgg acctattgtt ttcataagtc tgcacactga attaaaatat tctggttcta
      721 gtgtgccttt agtcagcaat gtgcgggggg ctggtaattg agcaggatcg ccaatataga
      781 cgtagtgttt tgcacgaagt ctagcattga caacactcaa gtcataatta gtagccatag
      841 agatttcatc aaagactaca atgtcagcag ttgtttctgg caatgcattt acagtgcaga
      901 aaacatactg ttctagtgtt gaattcactt tgaatttatc aaaacactct acgcgcgcac
      961 gcgcaggtat gattctacta catttatcta tgggcaaata ttttaatgcc ttttcacata
     1021 gggcatcaac agctgcatga gagcatgccg tatacactat gcgagcagat gggtaataga
     1081 gagcaagtcc gatggcaaaa tgactcttac cagtaccagg tggtccttgg agtgtagagt
     1141 acttttgcat gccgaccttt tgataatttg caacattgct agaaaactca tctgagatgt
     1201 tgagtgttgg gtacaagcca gtaattctca catagtgctc ttgtggcact agagtaggtg
     1261 cactaagtgg cattacagtg tgagatgtca acacaaagta atcaccaaca ttcaacttgt
     1321 atgtcgtagt acctctgtac acaacagcat caccatagtc acctttttca aaggtgtact
     1381 ctccaatctg tactttacta tttttagtta cacggtaacc agtaaagaca tagtttctgt
     1441 tcaatggtgg tctaggtttt ccaacctccc atgaaagatg caattctctg tcagagagta
     1501 cttcgcgtac agtggcaata ccatatgaca gcttaaatgt ttcctcagtg gctttgagcg
     1561 tttctgctgc gaaaagcttg agtctctcag tacaagtgtt ggcaagtatg taatcgccag
     1621 cattagtcca atcacatgtt gctatcgcat tgaagtcagt gacattgtca ctgcctacac
     1681 atgtgttttt gtataaacca aaaacctgac cattagcaca taatggaaaa ctaatgggag
     1741 gcttatgtga cttgcaataa tagctcatac ctcctagata cagttgtgtc acatcagtga
     1801 catcacaacc cggggcattg caaacatagg gattaacaga caacactaat ttgtgtgatg
     1861 ttgaaatgac atggtcatag cagcacttgc aacataggaa tggtctccta atacaggcac
     1921 cgcaacgaag tgaagtctgt gaattgcaca atacacaagc acctacagcc atggcggatc
     1981 cgcgacccat ttgctgtcca ccagtcatgc tagccatatg gctgccgcgc ggcaccaggc
     2041 cgctgctgtg atgatgatga tgatggctgc tgcccatggt atatctcctt cttaaagtta
     2101 aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt
     2161 attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca
     2221 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     2281 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     2341 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     2401 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     2461 gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     2521 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     2581 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     2641 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     2701 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     2761 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     2821 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     2881 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     2941 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     3001 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     3061 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     3121 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     3181 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     3241 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     3301 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     3361 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     3421 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     3481 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     3541 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     3601 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     3661 cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc
     3721 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     3781 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     3841 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     3901 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     3961 cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
     4021 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
     4081 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
     4141 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
     4201 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     4261 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     4321 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     4381 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     4441 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     4501 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     4561 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     4621 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     4681 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     4741 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     4801 cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa
     4861 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4921 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4981 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5041 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5101 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5161 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5221 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5281 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5341 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5401 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5461 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     5521 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5581 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5641 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5701 cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa cagtaataca
     5761 aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg attaaattcc
     5821 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     5881 gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     5941 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     6001 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     6061 accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt
     6121 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     6181 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     6241 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     6301 gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt
     6361 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     6421 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     6481 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     6541 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt aattcatgag
     6601 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6661 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     6721 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     6781 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     6841 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     6901 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     6961 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     7021 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     7081 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     7141 cca
//
