LOCUS       sequence-399653-        6000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressess an optimized construct of human rhinovirus 14 3C protease
            (NT*-HRV3CP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6000)
  AUTHORS   Abdelkader EH, Otting G
  TITLE     NT*-HRV3CP: an optimized construct of human rhinovirus 14 3C 
            protease for high-yield expression and fast affinity-tag cleavage.
  JOURNAL   J Biotechnol. 2020 Nov 6. pii: S0168-1656(20)30306-0. doi: 
            10.1016/j.jbiotec.2020.11.005.
  PUBMED    33166527
REFERENCE   2  (bases 1 to 6000)
  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"; journalName: "J 
            Biotechnol. 2020 Nov 6. pii: S0168-1656(20)30306-0. doi: 
            10.1016/j.jbiotec.2020.11.005."
COMMENT     Sequence Label: pET-NT*-HRV3CP
FEATURES             Location/Qualifiers
     source          1..6000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1029..1220
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1214..1236)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1322..1461
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1377..1396
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1476..1493)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1647..2235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1727..1746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2346..2365)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2357..3172
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2357..3172
                     /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(2430..2449)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3264..3719)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3401..3422)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3613..3632
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3756..3803)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3799..3817
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4477..4875)
                     /codon_start=1
                     /product="solubility tag derived from the N-terminal domain
                     of a spidroin (Kronqvist et al., 2017)"
                     /label=NT* tag
                     /translation="SHTTPWTNPGLAENFMNSFMQGLSSMPGFTASQLDKMSTIAQSMV
                     QSIQSLAAQGRTSPNDLQALNMAFASSMAEIAASEEGGGSLSTKTSSIASAMSNAFLQT
                     TGVVNQPFINEITQLVSMFAQAGMNDVSA"
     CDS             complement(4876..4893)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4904..4926
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4941..4965
                     /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(4965..4984)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4966..4984)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5072..5091)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5297..5374
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5375..6000,1..457)
                     /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(5394..5413)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
       61 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
      121 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggacatctc
      181 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
      241 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
      301 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
      361 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
      421 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
      481 agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca
      541 gctccttccg gtgggcgcgg ggcatgacta gcatgatcgt gctcctgtcg ttgaggaccc
      601 ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa
      661 cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg
      721 gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc
      781 ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga
      841 agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt
      901 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga
      961 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg
     1021 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc
     1081 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct
     1141 gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg
     1201 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     1261 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     1321 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     1381 atcagagcag attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta
     1441 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     1501 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     1561 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1621 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1681 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1741 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1801 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1861 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1921 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1981 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2041 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     2101 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2161 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2221 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2281 gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag
     2341 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt
     2401 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     2461 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa
     2521 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     2581 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     2641 gttactcacc actgcgatcc ccggcaaaac agcattccag gtattagaag aatatcctga
     2701 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     2761 tgtttgtaat tgtcctttta acagtgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     2821 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     2881 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     2941 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     3001 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     3061 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     3121 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat
     3181 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3241 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     3301 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     3361 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     3421 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     3481 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     3541 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     3601 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     3661 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     3721 ccattcgcca atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta
     3781 gaggccccaa ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc
     3841 tttcgggctt tgttagcagc cggatctatc gatgcatgcc atggtacccg ggagctcgaa
     3901 ttcttactgc ttttccacga aatactgttt tttcagctgt gcgctaaaac cctgacgacc
     3961 attgccacca acatgaatgc caaagatttt accggttgca cacagaacac caccacactg
     4021 accggttttg gttgcataat cataacgaat catacgattg gtcggggtac tgctcagatt
     4081 aatcagacct gccatggtaa ccggaccaac ttccaggatg gtgttggtaa agttattgct
     4141 atgaacaacc agggttgcat caacaccttc cagatcttcg ctgataaagc cacggatatc
     4201 acgaaatttt tcattacgat ccagggtcag aacggtcagt tccagattaa tgttttccgg
     4261 atccaccagt ttatacttgt ctttcacgcg aattttctga ccattaacca gaacgtcatc
     4321 acccggttgt gcatgtgtcg gaataacaca aacacgatca tgaataccca gaccggtaaa
     4381 ttcgcctttg gaggtggtaa tggtcataat gtttttacgc agcaggctca gtgcaaattc
     4441 ggtattcgga ccgctaccac caccaccgct attacctgcg ctaacatcat tcatacctgc
     4501 ctgtgcaaac atgctaacca gctgggtaat ttcgttaata aacggctgat taacaacacc
     4561 ggttgtctgc agaaatgcat tgctcattgc gcttgcaatg ctgctggttt tggtgctcag
     4621 gctaccgcca ccttcttcgc ttgctgcaat ttctgccatg ctgcttgcaa atgccatatt
     4681 cagtgcctgc agatcattcg gactggtacg accctgtgct gccaggctct gaatgctctg
     4741 aaccatgctc tgtgcaatgg tgctcatttt gtccagctgg cttgcggtaa aaccaggcat
     4801 gctgctcaga ccctgcataa agctattcat aaagttttct gccagaccag gattggtcca
     4861 cggtgtggta tggctgtggt gatgatgatg atgcatatgt atatctcctt cttaaagtta
     4921 aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt
     4981 attaatttcg cgggatcgag atcgatctcg atcctctacg ccggacgcat cgtggccggc
     5041 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
     5101 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
     5161 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
     5221 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
     5281 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
     5341 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     5401 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     5461 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     5521 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     5581 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     5641 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     5701 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     5761 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     5821 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     5881 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     5941 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
//
