LOCUS       sequence-300453-        4455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for producing recombinant proteins by wheat germ-based 
            cell-free translation, contains T7 promoter, and NotI restriction 
            site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4455)
  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 4455)
  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-His
FEATURES             Location/Qualifiers
     source          1..4455
                     /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..731
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1395..1411)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1395..1411)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1408..1430)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1419..1435
                     /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(1443..1449,1450..1467,1468..1473))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1488..1509
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1626..1643)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1797..2385)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1877..1896)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2556..3347,3348..3416))
                     /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     3179..3198
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3417..3520)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3588..3606
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3644..3666)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3766..3785
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3999..4018
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4308..4327
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4308..4326
                     /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 ccccatcatc
      721 atcatcatca ttgacccggg tgggttataa ttacctcagg tcgacgtccc atggttttgt
      781 atagaattta cggctagcgc cggatgcgac gccggtcgcg tcttatccgg ccttcctata
      841 tcaggctgtg tttaagacgc cgccgcttcg cccaaatcct tatgccggtt cgacgactgg
      901 acaaaatact gtttatcttc ccagcgcagg caggttaatg taccacccca gcagcagccg
      961 gtatccagcg cgtatatacc ttccggcgta cctttgccct ccagcgatgc ccagtgacca
     1021 aaggcgatgc tgtattcttc agcgacaggg ccaggaatcg caaaccacgg tttcagtggg
     1081 gcaggggcct cttccggcga ttcttactag ctagtatgca taggtgctga aatataaagt
     1141 ttgtgtttct aaaacacacg tggtacgtac gataacgtac agtgtttttc cctccactta
     1201 aatcgaaggg tagtgtcttg gagcgcgcgg agtaaacata tatggttcat atatgtccgt
     1261 aggcacgtaa aaaaagcgag ggattcgaat tcccccggaa cccccggttg gggcccacgc
     1321 ctcgatcgag caaaaaaaaa aaagaaaaaa aaaaaaaaaa aaagctttcc cgcggccagc
     1381 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1441 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1501 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1561 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1621 gcttcctcgc tcactgactc gctgcgctcg gtcgctcggc tgcggcgagc ggtatcagct
     1681 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1741 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1801 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1861 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1921 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1981 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2041 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2101 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2161 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2221 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2281 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2341 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2401 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2461 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2521 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2581 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2641 ataactacga tacgggaggg cttaccacct ggccccagtg ctgcaatgat accgcgagac
     2701 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     2761 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     2821 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     2881 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     2941 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3001 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3061 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3121 tcattctgag aatagtgtat gcggccgccg agttgctctt gcccggcgtc aatacgggat
     3181 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3241 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3301 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3361 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3421 ttcctttttc aatgttatga agcatttatc agggttattg tctcatgagc ggatacatat
     3481 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     3541 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     3601 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     3661 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     3721 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     3781 ttgtactgag agtgcaccat tcgacgctct cccttatgcg actcctgcat taggaagcag
     3841 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     3901 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     3961 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     4021 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     4081 tggctagcga tgaccctgct gattggttcg ctgaccattt ccgggtgcgg gacggcgtta
     4141 ccagaaactc agaaggttcg tccaaccaaa ccgactctgg cggcagttta cgagagagat
     4201 gatagggtct gcttcagtaa gccagatgct acacaattag gcttgtacat actgtcgtta
     4261 gaacgcggct acaattaata cataacctta tgtatcatac acatacgtaa tacgactcac
     4321 tatagggtac acggaattcg cgtttttatt tttaattttc tttcaaatac ttctagctag
     4381 agtattttta caacaattac caacaacaac aacaaacaac aacaacatta cattttacat
     4441 tctacaacta cagat
//
