LOCUS       sequence-300441-        4482 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for producing recombinant proteins by wheat germ-based 
            cell-free translation, contains T7 promoter, GST-tag and 
            biotinylation site, and NotI restriction site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4482)
  AUTHORS   Nagy SK, Kallai BM, Andras J, Meszaros T
  TITLE     A novel family of expression vectors with multiple affinity tags for
            wheat germ cell-free protein expression.
  JOURNAL   BMC Biotechnol. 2020 Mar 14;20(1):17. doi: 
            10.1186/s12896-020-00610-5.
  PUBMED    32169064
REFERENCE   2  (bases 1 to 4482)
  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: "BMC 
            Biotechnol."; date: "2020-03-14"; volume: "20(1):17. doi"; pages: " 
            10.1186/s12896-020-00610-5"
COMMENT     Sequence Label: pEU3-NII-GLICNot-C-BIOT
FEATURES             Location/Qualifiers
     source          1..4482
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..660
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     618..640
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             667..687
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             714..758
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     primer_bind     complement(1422..1438)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1422..1438)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1435..1457)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1446..1462
                     /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)."
     promoter        complement(join(1470..1476,1477..1494,1495..1500))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1515..1536
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1653..1670)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1824..2412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1904..1923)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2583..3374,3375..3443))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGGRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPGGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3206..3225
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3444..3547)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3615..3633
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3671..3693)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3793..3812
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4026..4045
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4335..4354
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4335..4353
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 actagtatgt cccctatact aggttattgg aaaattaagg gccttgtgca acccactcga
       61 cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt
      121 gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct tccttattat
      181 attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat agctgacaag
      241 cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg
      301 gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt tgaaactctc
      361 aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt
      421 cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt gtatgacgct
      481 cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa attagtttgt
      541 tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc cagcaagtat
      601 atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa
      661 tcggatgaga acctgtactt ccaatccaat attggaagtg gataacggat cccggcctca
      721 acgacatctt cgaggcccag aagatcgagt ggcacgagtg acccgggtgg gttataatta
      781 cctcaggtcg acgtcccatg gttttgtata gaatttacgg ctagcgccgg atgcgacgcc
      841 ggtcgcgtct tatccggcct tcctatatca ggctgtgttt aagacgccgc cgcttcgccc
      901 aaatccttat gccggttcga cgactggaca aaatactgtt tatcttccca gcgcaggcag
      961 gttaatgtac caccccagca gcagccggta tccagcgcgt atataccttc cggcgtacct
     1021 ttgccctcca gcgatgccca gtgaccaaag gcgatgctgt attcttcagc gacagggcca
     1081 ggaatcgcaa accacggttt cagtggggca ggggcctctt ccggcgattc ttactagcta
     1141 gtatgcatag gtgctgaaat ataaagtttg tgtttctaaa acacacgtgg tacgtacgat
     1201 aacgtacagt gtttttccct ccacttaaat cgaagggtag tgtcttggag cgcgcggagt
     1261 aaacatatat ggttcatata tgtccgtagg cacgtaaaaa aagcgaggga ttcgaattcc
     1321 cccggaaccc ccggttgggg cccacgcctc gatcgagcaa aaaaaaaaaa gaaaaaaaaa
     1381 aaaaaaaaaa gctttcccgc ggccagcttg gcgtaatcat ggtcatagct gtttcctgtg
     1441 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     1501 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     1561 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     1621 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1681 gctcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1741 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1801 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1861 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1921 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1981 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     2041 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     2101 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     2161 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     2221 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     2281 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2341 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2401 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2461 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2521 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2581 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2641 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accacctggc
     2701 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     2761 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     2821 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     2881 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     2941 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     3001 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     3061 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     3121 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gccgccgagt
     3181 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     3241 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3301 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3361 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3421 acacggaaat gttgaatact catactcttc ctttttcaat gttatgaagc atttatcagg
     3481 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     3541 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     3601 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     3661 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     3721 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     3781 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattcg acgctctccc
     3841 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     3901 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     3961 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     4021 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     4081 gccacgatgc gtccggcgta gaggatctgg ctagcgatga ccctgctgat tggttcgctg
     4141 accatttccg ggtgcgggac ggcgttacca gaaactcaga aggttcgtcc aaccaaaccg
     4201 actctggcgg cagtttacga gagagatgat agggtctgct tcagtaagcc agatgctaca
     4261 caattaggct tgtacatact gtcgttagaa cgcggctaca attaatacat aaccttatgt
     4321 atcatacaca tacgtaatac gactcactat agggtacacg gaattcgcgt ttttattttt
     4381 aattttcttt caaatacttc tagctagagt atttttacaa caattaccaa caacaacaac
     4441 aaacaacaac aacattacat tttacattct acaactacag at
//
