LOCUS       sequence-300409-        3806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for producing recombinant proteins by wheat germ-based 
            cell-free translation, contains T7 promoter, FLAG-tag and NotI 
            restriction site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3806)
  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 3806)
  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-FLAGLICNot
FEATURES             Location/Qualifiers
     source          1..3806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             10..33
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             34..54
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(746..762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(746..762)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(759..781)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    770..786
                     /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(794..800,801..818,819..824))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    839..860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(977..994)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1148..1736)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1228..1247)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1907..2698,2699..2767))
                     /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     2530..2549
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2768..2871)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2939..2957
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2995..3017)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3117..3136
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3350..3369
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3659..3678
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3659..3677
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 actagtatgg attataagga cgacgacgac aaagagaacc tgtacttcca atccaatatt
       61 ggaagtggat aacggatcca tatatagggc ccgggttata attacctcag gtcgacgtcc
      121 catggttttg tatagaattt acggctagcg ccggatgcga cgccggtcgc gtcttatccg
      181 gccttcctat atcaggctgt gtttaagacg ccgccgcttc gcccaaatcc ttatgccggt
      241 tcgacgactg gacaaaatac tgtttatctt cccagcgcag gcaggttaat gtaccacccc
      301 agcagcagcc ggtatccagc gcgtatatac cttccggcgt acctttgccc tccagcgatg
      361 cccagtgacc aaaggcgatg ctgtattctt cagcgacagg gccaggaatc gcaaaccacg
      421 gtttcagtgg ggcaggggcc tcttccggcg attcttacta gctagtatgc ataggtgctg
      481 aaatataaag tttgtgtttc taaaacacac gtggtacgta cgataacgta cagtgttttt
      541 ccctccactt aaatcgaagg gtagtgtctt ggagcgcgcg gagtaaacat atatggttca
      601 tatatgtccg taggcacgta aaaaaagcga gggattcgaa ttcccccgga acccccggtt
      661 ggggcccacg cctcgatcga gcaaaaaaaa aaaagaaaaa aaaaaaaaaa aaaagctttc
      721 ccgcggccag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
      781 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
      841 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
      901 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
      961 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgctcgg ctgcggcgag
     1021 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     1081 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1141 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1201 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1261 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1321 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1381 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     1441 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     1501 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     1561 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     1621 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     1681 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     1741 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     1801 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     1861 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     1921 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     1981 ccgtcgtgta gataactacg atacgggagg gcttaccacc tggccccagt gctgcaatga
     2041 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2101 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2161 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2221 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2281 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2341 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     2401 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     2461 actcaaccaa gtcattctga gaatagtgta tgcggccgcc gagttgctct tgcccggcgt
     2521 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     2581 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     2641 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     2701 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     2761 tactcatact cttccttttt caatgttatg aagcatttat cagggttatt gtctcatgag
     2821 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     2881 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     2941 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     3001 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     3061 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     3121 atcagagcag attgtactga gagtgcacca ttcgacgctc tcccttatgc gactcctgca
     3181 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     3241 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     3301 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     3361 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     3421 cgtagaggat ctggctagcg atgaccctgc tgattggttc gctgaccatt tccgggtgcg
     3481 ggacggcgtt accagaaact cagaaggttc gtccaaccaa accgactctg gcggcagttt
     3541 acgagagaga tgatagggtc tgcttcagta agccagatgc tacacaatta ggcttgtaca
     3601 tactgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata cacatacgta
     3661 atacgactca ctatagggta cacggaattc gcgtttttat ttttaatttt ctttcaaata
     3721 cttctagcta gagtattttt acaacaatta ccaacaacaa caacaaacaa caacaacatt
     3781 acattttaca ttctacaact acagat
//
