LOCUS       sequence-288552-        6214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HsRck_296-472-HsPatL1_1-398.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6214)
  TITLE     Izaurralde lab plasmids
REFERENCE   2  (bases 1 to 6214)
  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"
COMMENT     Sequence Label: pRSFDuetG-HsRCK_296-472-HsPatL1_1-398 (MCSII)_D
FEATURES             Location/Qualifiers
     source          1..6214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             110..133
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        742..760
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    761..785
                     /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             831..1484
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1442..1464
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1491..1514
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2751..2795
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2833..2851)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2847..2894
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3124..3939)
                     /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             complement(3124..3939)
                     /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     3847..3866
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3940..4031)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4047..4796)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(4057..4074)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4297..4316)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4992..6074)
                     /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     6036..6055
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6075..6152)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(6198..6214,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agcgcgggtc tggaagttct
      121 gttccagggg ccccaggatc cgaagggagt aacccagtac tacgcatatg taactgagcg
      181 ccaaaaagta cactgcctca acacactttt ctccaggctt cagataaacc agtcgatcat
      241 tttctgtaac tcctctcagc gagttgaatt gctagccaag aagatttctc aactgggtta
      301 ttcttgcttc tatattcatg ctaaaatgag gcaggaacat cgaaatcgtg tatttcatga
      361 tttccgaaat ggcttatgcc gcaatcttgt ttgcactgat ctgtttaccc gaggtattga
      421 tatacaagct gtgaatgtgg taataaactt tgatttccca aagctggcag agacctatct
      481 ccatcgtatt ggaagatcag gtcgctttgg tcatcttggc ttagccatca acttgatcac
      541 atatgatgat cgcttcaacc tgaaaagtat tgaggagcag ctgggaacag aaattaaacc
      601 tattccgagc aacattgata agagcctgta tgtggcagaa taccacagcg agcctgtaga
      661 agatgagaaa ccttaagcgg ccgcataatg cttaagtcga acagaaagta atcgtattgt
      721 acacggccgc ataatcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
      781 attccccatc ttagtatatt agttaagtat aagaaggaga tatacataat atgtccccta
      841 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      901 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      961 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
     1021 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
     1081 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
     1141 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
     1201 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
     1261 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
     1321 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
     1381 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
     1441 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
     1501 tgttccaggg gccccatatg gcagatctca attggatgtt ccgctacgag tctttggagg
     1561 attgtcctct ggatgaagat gaagatgcat ttcagggact gggagaagaa gatgaagaga
     1621 ttgatcaatt caatgatgat acatttgggt caggtgcagt tgatgatgat tggcaggaag
     1681 cacatgagcg cctggctgaa ttggaagaaa agctaccagt ggcagttaat gaacaaacag
     1741 gcaatggaga gagagatgaa atggacttgt tgggtgacca tgaggagaat ctggcagaaa
     1801 ggctcagtaa gatggtgatt gaaaatgaac tagaagatcc agctattatg agggcagtgc
     1861 agaccaggcc agttttacaa ccccaaccag gaagtctgaa ttccagtatc tgggatggat
     1921 ctgaagttct gaggcgaatc cgaggaccac tgcttgctca ggaaatgcct acagtgtctg
     1981 tattagaata tgctttgcct cagaggcccc cccagggtcc agaagatgat cgggaccttt
     2041 ctgaacgagc attaccaagg cggtcaactt cacctatcat tggcagtcct cctgttagag
     2101 ctgtccccat aggcacccca cctaaacaga tggctgtacc cagcttcacc caacagattc
     2161 tgtgtccgaa gcctgtccat gttcggcccc caatgccacc tcgttatcct gctccctatg
     2221 gtgagaggat gtctccaaac cagctctgca gtgtcccgaa ctcttccctc ctgggtcacc
     2281 cttttcctcc tagtgttcct cctgttctca gccccctcca gagagcacag cttcttggag
     2341 gagcacagct acagcctgga cggatgtctc ccagccagtt tgcacgggtc cctggatttg
     2401 ttggtagtcc acttgctgcc atgaatccca agttgctaca agggcgagtt gggcagatgc
     2461 ttcccccagc accaggcttc cgtgccttct ttagtgctcc accctccgct acaccacctc
     2521 cacagcagca ccctcctggc ccaggacccc acctgcaaaa cctaagatct caggccccaa
     2581 tgtttagacc ggacacaact cacctccatc cacagcaccg tcgactcttg catcagagac
     2641 agcaacagaa tagaagtcag catcggaatc tcaatggtgc gggagataga ggaagtcacc
     2701 ggagcagtca tcaagatcat ctccgaaagt agggtaccct cgagtctggt aaagaaaccg
     2761 ctgctgcgaa atttgaacgc cagcacatgg actcgtctac tagcgcagct taattaacct
     2821 aggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     2881 tgaggggttt tttgctgaaa cctcaggcat ttgagaagca cacggtcaca ctgcttccgg
     2941 tagtcaataa accggtaaac cagcaataga cataagcggc tatttaacga ccctgccctg
     3001 aaccgacgac aagctgacga ccgggtctcc gcaagtggca cttttcgggg aaatgtgcgc
     3061 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     3121 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     3181 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     3241 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     3301 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     3361 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     3421 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     3481 ttgcgcctga gcgagacgaa atacgcggtc gctgttaaaa ggacaattac aaacaggaat
     3541 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     3601 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     3661 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     3721 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     3781 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     3841 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     3901 cggcctagag caagacgttt cccgttgaat atggctcata ctcttccttt ttcaatatta
     3961 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4021 aaataaacaa ataggcatgc agcgctcttc cgcttcctcg ctcactgact cgctacgctc
     4081 ggtcgttcga ctgcggcgag cggtgtcagc tcactcaaaa gcggtaatac ggttatccac
     4141 agaatcaggg gataaagccg gaaagaacat gtgagcaaaa agcaaagcac cggaagaagc
     4201 caacgccgca ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4261 gctcaagcca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4321 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4381 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgttggtat ctcagttcgg
     4441 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4501 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4561 tggcagcagc cattggtaac tgatttagag gactttgtct tgaagttatg cacctgttaa
     4621 ggctaaactg aaagaacaga ttttggtgag tgcggtcctc caacccactt accttggttc
     4681 aaagagttgg tagctcagcg aaccttgaga aaaccaccgt tggtagcggt ggtttttctt
     4741 tatttatgag atgatgaatc aatcggtcta tcaagtcaac gaacagctat tccgttactc
     4801 tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat
     4861 aagttgtaat tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     4921 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     4981 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5041 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     5101 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     5161 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     5221 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca
     5281 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     5341 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     5401 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5461 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5521 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5581 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5641 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5701 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5761 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5821 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     5881 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     5941 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     6001 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     6061 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     6121 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     6181 ctcctgcatt aggaaattaa tacgactcac tata
//
