LOCUS       sequence-368607-        6510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminal GST-(His)6-Thrombin cleavage site fusion of 
            human gene or portion of gene with point mutation in bacterial 
            strains. pET based vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6510)
  AUTHORS   Rajiv C, Jackson SR, Cocklin S, Eisenmesser EZ, Davis TL
  TITLE     The spliceosomal proteins PPIH and PRPF4 exhibit bi-partite binding.
  JOURNAL   Biochem J. 2017 Oct 25;474(21):3689-3704. doi: 10.1042/BCJ20170366.
  PUBMED    28935721
REFERENCE   2  (bases 1 to 6510)
  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"; doi: "10"; journalName: 
            "Biochem J"; date: "2017-10-25- 25"; volume: "474"; issue: "21"; 
            pages: "3689-3704"
COMMENT     Sequence Label: ppih.001.177.W133A
FEATURES             Location/Qualifiers
     source          1..6510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             89..742
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             770..787
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             797..814
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1375..1392
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1445..1463)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1459..1506
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1543..1998
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1630..1649)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1840..1861
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2090..2905)
                     /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(2090..2905)
                     /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     2813..2832
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2897..2916
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3027..3615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3516..3535
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3769..3786
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3801..3940
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3866..3885)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4026..4048
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4042..4233)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5042..6124)
                     /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     6086..6105
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6125..6202)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6408..6427
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gtcccctata ctaggttatt ggaaaattaa
      121 gggccttgtg caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca
      181 tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga
      241 gtttcccaat cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat
      301 catacgttat atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga
      361 gatttcaatg cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata
      421 tagtaaagac tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa
      481 aatgttcgaa gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc
      541 tgacttcatg ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga
      601 tgcgttccca aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa
      661 gtacttgaaa tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg
      721 tggtggcgac catcctccaa aatcggatgg ttcatccatg ggcagcagcc atcatcatca
      781 tcatcacagc agcggcctgg ttccgcgtgg tagtatggcg gtggcaaatt caagtcctgt
      841 taaccccgtg gtgttctttg atgtcagtat tggcggtcag gaagttggcc gcatgaagat
      901 cgagctcttt gcagacgttg tgcctaagac ggccgagaac tttaggcagt tctgcaccgg
      961 agaattcagg aaagatgggg ttccaatagg atacaaagga agcaccttcc acagggtcat
     1021 aaaggatttc atgattcagg gtggagattt tgttaatgga gatggtactg gagtcgccag
     1081 tatttaccgg gggccatttg cagatgaaaa ttttaaactt agacactcag ctccaggcct
     1141 gctttccatg gcgaacagtg gtccaagtac aaatggctgt cagttcttta tcacctgctc
     1201 taagtgcgat gcgctggatg ggaagcatgt ggtgtttgga aaaatcatcg atggacttct
     1261 agtgatgaga aagattgaga atgttcccac aggccccaac aataagccca agctacctgt
     1321 ggtgatctcg cagtgtgggg agatgtgatg acgaagcttg cggccgcact cgagcaccac
     1381 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     1441 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1501 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     1561 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1621 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1681 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1741 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1801 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1861 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1921 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1981 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2041 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     2101 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     2161 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     2221 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     2281 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     2341 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     2401 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     2461 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     2521 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     2581 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     2641 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     2701 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     2761 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     2821 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     2881 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     2941 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3001 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3061 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3121 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3181 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3241 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3301 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3361 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3421 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3481 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3541 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3601 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3661 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3721 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3781 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3841 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3901 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3961 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4021 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4081 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4141 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4201 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4261 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     4321 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4381 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4441 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4501 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4561 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4621 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4681 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     4741 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     4801 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     4861 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     4921 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4981 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5041 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5101 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     5161 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     5221 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     5281 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     5341 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     5401 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     5461 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     5521 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     5581 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     5641 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     5701 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     5761 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     5821 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     5881 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     5941 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6001 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     6061 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     6121 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     6181 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     6241 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     6301 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     6361 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     6421 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     6481 tagaggatcg agatctcgat cccgcgaaat
//
