LOCUS       sequence-296575-        7329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression/GST pull-down.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7329)
  TITLE     Opher Gileadi SGC expression vectors
REFERENCE   2  (bases 1 to 7329)
  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: TBXTA-c025
FEATURES             Location/Qualifiers
     source          1..7329
                     /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             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             116..769
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGADHPPK"
     CDS             797..817
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2194..2211
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2264..2282)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2278..2325
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2362..2817
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2449..2468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2659..2680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2909..3724)
                     /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(2909..3724)
                     /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     3632..3651
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3716..3735
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3846..4434
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4335..4354
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4588..4605
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4620..4759
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4685..4704)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4845..4867
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4861..5052)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5861..6943)
                     /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     6905..6924
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6944..7021)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7227..7246
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctatgtc
      121 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga
      181 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg
      241 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga
      301 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt
      361 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat
      421 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt
      481 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata
      541 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt
      601 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg
      661 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc
      721 tttgcagggc tggcaagcca cgtttggtgg tgccgaccat cctccaaaat cgagctcagg
      781 tgtagatctg ggtaccgaga acctgtactt ccaatccatg tccaatagca gccctggcac
      841 cgaaagcgcc ggtaagagcc tacagtaccg tgtggaccat ttactgagcg ccgtggagaa
      901 cgaactacag gcaggcagcg aaaaaggtga cccgaccgaa cgcgaactgc gtgtgggtct
      961 ggaagaaagc gaactgtggc tgcgctttaa agagctgacc aacgaaatga tcgtgaccaa
     1021 aaacggccgc cgcatgtttc ctgtgctgaa agttaacgtg agcggcctgg acccgaacgc
     1081 catgtacagc ttcctgctgg atttcgttgc cgcagataac caccgctgga agtatgtgaa
     1141 cggcgaatgg gtgccgggtg gcaaaccgga accgcaggca cctagctgcg tgtatatcca
     1201 cccggatagc cctaacttcg gtgcccattg gatgaaagcc ccggtgagct tcagcaaggt
     1261 gaagctgacc aacaaactga acggcggcgg ccaaatcatg ctgaacagct tacataaata
     1321 tgaacctcgc atccacattg tgcgtgtggg cgacccgcaa cgtatgatca ccagccattg
     1381 cttcccggag acacagttca ttgccgttac cgcctaccag aacgaggaaa ttaccgcact
     1441 gaaaattaaa tacaaccctt ttgcaaaagc cttcttagat gccaaagagc gcagcgacca
     1501 caaagaaatg atggaggaac cgggcgacag ccagcagcct ggttatagcc aatggggctg
     1561 gctgctgccg ggtaccagta cactgtgccc gccggcaaac ccgcatcctc agtttggtgg
     1621 cgccctgagc ttaccgagca cccacagttg tgatcgctat cctaccctgc gtagccatcg
     1681 tagcagtccg tacccgagcc cgtatgcaca tcgtaacaac agcccgacat atagcgacaa
     1741 cagcccggcc tgtctgagca tgctacagag ccatgataac tggagcagcc tgggcatgcc
     1801 ggcacatccg agcatgctgc cggtgagtca caatgccagc ccgccgacca gtagcagcca
     1861 gtatcctagc ctgtggagcg ttagtaacgg tgcagtgacc ccgggtagcc aggcagcagc
     1921 agttagcaat ggtctgggcg cccagttttt tcgcggcagt ccggcacatt acaccccgct
     1981 gacccatcct gtgagtgccc cgagcagtag tggtagcccg ctgtatgaag gtgccgcagc
     2041 cgccaccgac attgttgaca gccagtatga tgccgccgca cagggtcgcc tgattgcaag
     2101 ctggacccct gtgagtccgc cgagcatgtg acagtaaagg tggatacgga tccgaattcg
     2161 agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
     2221 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     2281 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     2341 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2401 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2461 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2521 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2581 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2641 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2701 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2761 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     2821 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2881 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     2941 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     3001 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     3061 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     3121 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     3181 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     3241 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     3301 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     3361 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     3421 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     3481 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     3541 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     3601 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     3661 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     3721 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     3781 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3841 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3901 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3961 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4021 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4081 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4141 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4201 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4261 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4321 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4381 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4441 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4501 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4561 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4621 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     4681 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     4741 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4801 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4861 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     4921 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     4981 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     5041 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     5101 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     5161 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     5221 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     5281 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     5341 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     5401 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     5461 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     5521 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     5581 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     5641 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     5701 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     5761 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     5821 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5881 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5941 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     6001 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     6061 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     6121 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     6181 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     6241 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     6301 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     6361 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     6421 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     6481 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     6541 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     6601 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     6661 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     6721 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     6781 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     6841 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     6901 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     6961 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     7021 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     7081 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     7141 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     7201 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     7261 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     7321 ccgcgaaat
//
