LOCUS       sequence-386203-        6807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein expression of the GST-tagged catalytic domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6807)
  TITLE     Knapp lab plasmids
REFERENCE   2  (bases 1 to 6807)
  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: pGTVL2-SENP5
FEATURES             Location/Qualifiers
     source          1..6807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..63
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(57..248)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1057..2139)
                     /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     2101..2120
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2140..2217)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2423..2442
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2526..2545
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2526..2544
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2545..2569
                     /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             2584..2606
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2617..2634
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2641..3294
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGADHPPK"
     CDS             3322..3342
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4197..4214
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4267..4285)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4281..4328
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4365..4820
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4452..4471)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4662..4683
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4912..5727)
                     /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(4912..5727)
                     /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     5635..5654
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5719..5738
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5849..6437
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6338..6357
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6591..6608
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6623..6762
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6688..6707)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
       61 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
      121 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
      181 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
      241 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
      301 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
      361 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
      421 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
      481 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
      541 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
      601 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
      661 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
      721 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
      781 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
      841 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
      901 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
      961 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     1021 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     1081 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1141 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     1201 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     1261 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     1321 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     1381 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     1441 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     1501 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     1561 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     1621 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     1681 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     1741 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     1801 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     1861 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     1921 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     1981 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     2041 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     2101 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     2161 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     2221 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     2281 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     2341 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     2401 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     2461 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     2521 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     2581 aattttgttt aactttaaga aggagatata catatgcacc atcatcatca tcattcttct
     2641 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
     2701 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
     2761 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat
     2821 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
     2881 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg
     2941 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt
     3001 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
     3061 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat
     3121 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
     3181 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca
     3241 tggcctttgc agggctggca agccacgttt ggtggtgccg accatcctcc aaaatcgagc
     3301 tcaggtgtag atctgggtac cgagaacctg tacttccaat ccatgagtcc ggtggatgac
     3361 gaacagctgt ccgtttgcct gtcaggcttt ctggatgaag tgatgaaaaa atatggttct
     3421 ctggtcccgc tgagtgaaaa agaagtgctg ggtcgcctga aagatgtttt taacgaagac
     3481 ttcagcaatc gcaaaccgtt tattaaccgt gaaatcacca attatcgcgc gcgtcatcag
     3541 aaatgtaact tccgtatttt ctacaacaaa cacatgctgg atatggatga cctggccacc
     3601 ctggatggcc aaaactggct gaatgaccag gttattaaca tgtatggtga actgatcatg
     3661 gatgcggtgc cggacaaagt tcatttcttt aacagctttt tccaccgcca gctggtcacg
     3721 aaaggttaca atggcgtgaa acgttggacc aaaaaagttg acctgtttaa aaaatctctg
     3781 ctgctgattc cgatccatct ggaagtccac tggtccctga ttaccgtgac gctgtcaaac
     3841 cgcattatct cgttctatga tagccagggt atccatttca aattctgcgt ggaaaacatc
     3901 cgtaaatacc tgctgaccga agcgcgtgag aaaaaccgtc cggaatttct gcagggctgg
     3961 caaaccgctg ttacgaaatg catcccgcag cagaaaaacg atagcgactg tggtgttttc
     4021 gtcctgcagt actgcaaatg tctggccctg gaacagccgt ttcaattctc acaggaagat
     4081 atgccgcgtg tgcgtaaacg tatctacaaa gaactgtgtg aatgtcgcct gatggactaa
     4141 aggtggatac ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc
     4201 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     4261 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     4321 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     4381 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4441 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4501 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4561 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4621 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4681 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4741 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4801 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4861 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     4921 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     4981 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     5041 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     5101 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     5161 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     5221 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     5281 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     5341 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     5401 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     5461 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     5521 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     5581 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     5641 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     5701 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     5761 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5821 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5881 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5941 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     6001 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6061 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6121 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6181 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6241 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6301 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6361 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6421 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6481 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6541 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     6601 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     6661 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     6721 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     6781 cccgctgacg cgccctgacg ggcttgt
//
