LOCUS       sequence-300388-        4439 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 NotI site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4439)
  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 4439)
  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
FEATURES             Location/Qualifiers
     source          1..4439
                     /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"
     primer_bind     complement(1379..1395)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1379..1395)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1392..1414)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1403..1419
                     /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(1427..1433,1434..1451,1452..1457))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1472..1493
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1610..1627)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1781..2369)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1861..1880)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2540..3331,3332..3400))
                     /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     3163..3182
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3401..3504)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3572..3590
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3628..3650)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3750..3769
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3983..4002
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4292..4311
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4292..4310
                     /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 ccatatatag
      721 ggcccgggtt ataattacct caggtcgacg tcccatggtt ttgtatagaa tttacggcta
      781 gcgccggatg cgacgccggt cgcgtcttat ccggccttcc tatatcaggc tgtgtttaag
      841 acgccgccgc ttcgcccaaa tccttatgcc ggttcgacga ctggacaaaa tactgtttat
      901 cttcccagcg caggcaggtt aatgtaccac cccagcagca gccggtatcc agcgcgtata
      961 taccttccgg cgtacctttg ccctccagcg atgcccagtg accaaaggcg atgctgtatt
     1021 cttcagcgac agggccagga atcgcaaacc acggtttcag tggggcaggg gcctcttccg
     1081 gcgattctta ctagctagta tgcataggtg ctgaaatata aagtttgtgt ttctaaaaca
     1141 cacgtggtac gtacgataac gtacagtgtt tttccctcca cttaaatcga agggtagtgt
     1201 cttggagcgc gcggagtaaa catatatggt tcatatatgt ccgtaggcac gtaaaaaaag
     1261 cgagggattc gaattccccc ggaacccccg gttggggccc acgcctcgat cgagcaaaaa
     1321 aaaaaaagaa aaaaaaaaaa aaaaaaagct ttcccgcggc cagcttggcg taatcatggt
     1381 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     1441 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     1501 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1561 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     1621 actcgctgcg ctcggtcgct cggctgcggc gagcggtatc agctcactca aaggcggtaa
     1681 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     1741 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     1801 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     1861 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     1921 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     1981 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2041 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2101 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2161 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2221 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     2281 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     2341 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     2401 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     2461 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     2521 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     2581 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     2641 agggcttacc acctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     2701 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     2761 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     2821 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     2881 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     2941 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3001 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     3061 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     3121 gtatgcggcc gccgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     3181 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     3241 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     3301 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     3361 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatgtt
     3421 atgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     3481 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     3541 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     3601 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     3661 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     3721 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     3781 ccattcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     3841 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     3901 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     3961 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     4021 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctggcta gcgatgaccc
     4081 tgctgattgg ttcgctgacc atttccgggt gcgggacggc gttaccagaa actcagaagg
     4141 ttcgtccaac caaaccgact ctggcggcag tttacgagag agatgatagg gtctgcttca
     4201 gtaagccaga tgctacacaa ttaggcttgt acatactgtc gttagaacgc ggctacaatt
     4261 aatacataac cttatgtatc atacacatac gtaatacgac tcactatagg gtacacggaa
     4321 ttcgcgtttt tatttttaat tttctttcaa atacttctag ctagagtatt tttacaacaa
     4381 ttaccaacaa caacaacaaa caacaacaac attacatttt acattctaca actacagat
//
