LOCUS       sequence-455394-        6340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  10xHis-SUMO (Smt3) fused, HRV-3C cleavable, GST-tag for rapid 
            cloning/DNA assembly of pulldown targets via NotI restriction site 
            for E. coli based protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6340)
  TITLE     Molecular Toolbox
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6340)
  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: 
            "Unpublished"
COMMENT     Sequence Label: MZB174
FEATURES             Location/Qualifiers
     source          1..6340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     183..202
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(326..373)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     369..387
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(440..457)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(557..580)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(584..1237)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             complement(1538..1567)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1581..1603
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1618..1642
                     /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(1642..1661)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1643..1661)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1745..1764)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1970..2047
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2048..3130
                     /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(2067..2086)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3939..4130
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4124..4146)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4232..4371
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4287..4306
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4386..4403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4557..5145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4637..4656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5256..5275)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5267..6082
                     /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             5267..6082
                     /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(5340..5359)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(6174..6340,1..289))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6311..6332)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
       61 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
      121 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
      181 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
      241 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
      301 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
      361 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      421 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gactaagttg gcagcatcac
      481 ccgacgcact ttgcgccgaa taaataccta agcttgtctt cactcgaggg tacccatgga
      541 tccttattag cggccgcggg ccctgaaaca gcacttccag gccttttgga ggatggtcgc
      601 caccaccaaa cgtggcttgc cagccctgca aaggccatgc tatatacttg ctggatttca
      661 agtacttatc aatttgtggg atagcttcaa tacgtttttt aaaacaaact aattttggga
      721 acgcatccag gcacattggg tccatgtata aaacaacatc aagagcgtca tacaacatga
      781 agtcaggatg ggttacatga tcaccattta aatatgtttt atgacataaa cgatcttcga
      841 acattttcag catttcaggt agcttgctaa gaaaatcaac tttgagagtt tcaaagtctt
      901 tactatatgc aattctcgaa acaccgtatc taatatccaa aaccgctcct tcaagcattg
      961 aaatctctgc acgctctttt ggacaaccac ccaacatgtt gtgcttgtca gctatataac
     1021 gtatgatggc catagactgt gttaatttaa catcaccatc aatataataa ggaagattgg
     1081 gaaactccaa acccaattca aactttttgt ttcgccattt atcaccttca tcgcgctcat
     1141 acaaatgctc ttcatatttt tcttcaagat attccaaaag aagtcgagtg ggttgcacaa
     1201 ggcccttaat tttccaataa cctagtatag gggacatgcc gccaatctgt tcacgatgcg
     1261 cttcaataat atcgttatct tccatatcca gatcttccgg ggtctgatcc gcctgaatac
     1321 gaatgccatc atacagaaaa cgcaggctat ccatttcttt gccctgacgt ttcgcaaacg
     1381 cttccatcag acgacgcagc ggggtggttt ttttaatttt aaaaaaaatt tcgctgctgc
     1441 catcgctcac tttcaggtta atatgggttt ccggtttcac ttccggtttc acttccggtt
     1501 tcgcttcctg gttcacttcg ctatcgctca tgctgccgtg atggtgatgg tgatggtgat
     1561 ggtgatggcc catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga
     1621 attgttatcc gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc
     1681 tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct
     1741 ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg
     1801 agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc
     1861 atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg
     1921 ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga
     1981 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     2041 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     2101 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     2161 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     2221 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     2281 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     2341 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     2401 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     2461 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     2521 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     2581 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     2641 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     2701 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     2761 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     2821 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
     2881 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     2941 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     3001 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     3061 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     3121 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc
     3181 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga
     3241 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg
     3301 cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc
     3361 tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg
     3421 ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc
     3481 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
     3541 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
     3601 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
     3661 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
     3721 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc
     3781 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg
     3841 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta
     3901 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
     3961 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
     4021 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
     4081 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     4141 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     4201 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     4261 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     4321 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     4381 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4441 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4501 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4561 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4621 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4681 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4741 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4801 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4861 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4921 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4981 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5041 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5101 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5161 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5221 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     5281 acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg
     5341 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     5401 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     5461 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     5521 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac
     5581 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     5641 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     5701 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     5761 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     5821 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     5881 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     5941 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     6001 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     6061 gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt
     6121 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     6181 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     6241 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     6301 gagtgttgtt ccagtttgga acaagagtcc actattaaag
//
