LOCUS       sequence-299970-        5944 bp    DNA     circular SYN 14-APR-2025
DEFINITION  inducible expression of SARS 3CLpro protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5944)
  AUTHORS   Lee H, Mittal A, Patel K, Gatuz JL, Truong L, Torres J, Mulhearn DC,
            Johnson ME
  TITLE     Identification of novel drug scaffolds for inhibition of SARS-CoV 
            3-Chymotrypsin-like protease using virtual and high-throughput 
            screenings.
  JOURNAL   Bioorg Med Chem. 2014 Jan 1;22(1):167-77. doi: 
            10.1016/j.bmc.2013.11.041. Epub 2013 Dec 1.
  PUBMED    24332657
REFERENCE   2  (bases 1 to 5944)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.bmc.2013.11.041"; journalName: "Bioorg Med Chem"; date: 
            "2014-01-1- 1"; volume: "22"; issue: "1"; pages: "167-77"
COMMENT     Sequence Label: pGEX-6P-1 SARS-CoV 3CLpro
FEATURES             Location/Qualifiers
     source          1..5944
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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)."
     CDS             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..759
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1711..1728
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1812..1834)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1979..1997)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2065..2169
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2170..2238,2239..3030)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2388..2407)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3201..3789
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3690..3709
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3943..3960
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4033..4110
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4111..5193
                     /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(4130..4149)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5242..5247,5248..5265,5266..5272)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5280..5296
                     /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     5304..5320
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5304..5320
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5316..5843
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5336..5353)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5336..5352)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5345..5367)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctgga agttctgttc caggggcccc tgggatccac ctctgcggtc
      781 ctgcaatctg gctttcgtaa aatggcgttc ccgtccggca aagtcgaagg ctgtatggtg
      841 caggttacct gcggtactac caccctgaat ggtctgtggc tggatgacac tgtttattgc
      901 ccgcgccacg ttatttgcac tgcagaagat atgctgaacc caaactatga agatctgctg
      961 attcgcaaat ctaaccactc ctttctggtt caggcgggta acgttcaact gcgcgttatc
     1021 ggtcactcca tgcagaactg cctgctgcgt ctgaaagtgg atacttctaa cccgaaaacc
     1081 cctaaatata agttcgtgcg tatccagcct ggccagacgt tcagcgtact ggcttgctat
     1141 aacggttctc cgtctggcgt gtaccagtgt gcgatgcgtc cgaatcacac tatcaaaggt
     1201 tcttttctga acggctcctg cggctccgtt ggctttaata tcgattacga ttgtgtgagc
     1261 ttctgttaca tgcaccacat ggaactgccg acgggtgttc acgccggcac cgatctggaa
     1321 ggtaagttct acggtccgtt cgtggaccgt cagaccgcac aggccgctgg tacggacacc
     1381 accatcactc tgaacgttct ggcctggctg tacgcagctg ttatcaacgg cgaccgctgg
     1441 ttcctgaacc gtttcaccac taccctgaac gacttcaacc tggtagctat gaaatacaac
     1501 tacgagccgc tgacccaaga tcacgtagac atcctgggcc cgctgtccgc gcagaccggt
     1561 attgcagtgc tggacatgtg tgctgcgctg aaagaactgc tgcagaacgg catgaacggt
     1621 cgtactatcc tgggtagcac cattctggag gacgaattta ccccgttcga cgtagtacgt
     1681 cagtgcagcg gtgtcacgtt ccagggccca catcaccatc atcatcacta agaattcccg
     1741 ggtcgactcg agcggccgca tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat
     1801 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     1861 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     1921 gcagccatga cccagtcacg tagcgatagc ggagtgtata attcttgaag acgaaagggc
     1981 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2041 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2101 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2161 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2221 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2281 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2341 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2401 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2461 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2521 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2581 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2641 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2701 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2761 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2821 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2881 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2941 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3001 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3061 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3121 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3181 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3241 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3301 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3361 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3421 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3481 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3541 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3601 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3661 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3721 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3781 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3841 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3901 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3961 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4021 cgcataaatt ccgacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     4081 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     4141 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     4201 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     4261 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     4321 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     4381 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     4441 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     4501 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     4561 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     4621 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     4681 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     4741 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     4801 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     4861 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     4921 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     4981 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     5041 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccacgcta
     5101 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     5161 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     5221 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     5281 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg gattcactgg
     5341 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     5401 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     5461 cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg gcaccagaag
     5521 cggtgccgga aagctggctg gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct
     5581 caaactggca gatgcacggt tacgatgcgc ccatctacac caacgtgacc tatcccatta
     5641 cggtcaatcc gccgtttgtt cccacggaga atccgacggg ttgttactcg ctcacattta
     5701 atgttgatga aagctggcta caggaaggcc agacgcgaat tatttttgat ggcgttggaa
     5761 ttagcttatc gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg
     5821 tggtatggct gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc
     5881 gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga
     5941 gctg
//
