LOCUS       sequence-301957-        3800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for producing recombinant proteins by wheat germ-based 
            cell-free translation, contains T7 promoter, 6x His-tag and NotI 
            restriction site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3800)
  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 3800)
  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-HLICNot
FEATURES             Location/Qualifiers
     source          1..3800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             9..26
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             27..47
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(739..755)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(739..755)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(752..774)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    763..779
                     /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(787..793,794..811,812..817))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    832..853
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(970..987)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1141..1729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1221..1240)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1900..2691,2692..2760))
                     /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     2523..2542
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2761..2864)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2932..2950
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2988..3010)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3110..3129
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3343..3362
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3652..3671
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3652..3670
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ctagtatgca tcatcatcat catcatgaga acctgtactt ccaatccaat attggaagtg
       61 gataacggat ccatatatag ggcccgggtt ataattacct caggtcgacg tcccatggtt
      121 ttgtatagaa tttacggcta gcgccggatg cgacgccggt cgcgtcttat ccggccttcc
      181 tatatcaggc tgtgtttaag acgccgccgc ttcgcccaaa tccttatgcc ggttcgacga
      241 ctggacaaaa tactgtttat cttcccagcg caggcaggtt aatgtaccac cccagcagca
      301 gccggtatcc agcgcgtata taccttccgg cgtacctttg ccctccagcg atgcccagtg
      361 accaaaggcg atgctgtatt cttcagcgac agggccagga atcgcaaacc acggtttcag
      421 tggggcaggg gcctcttccg gcgattctta ctagctagta tgcataggtg ctgaaatata
      481 aagtttgtgt ttctaaaaca cacgtggtac gtacgataac gtacagtgtt tttccctcca
      541 cttaaatcga agggtagtgt cttggagcgc gcggagtaaa catatatggt tcatatatgt
      601 ccgtaggcac gtaaaaaaag cgagggattc gaattccccc ggaacccccg gttggggccc
      661 acgcctcgat cgagcaaaaa aaaaaaagaa aaaaaaaaaa aaaaaaagct ttcccgcggc
      721 cagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
      781 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
      841 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
      901 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
      961 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgct cggctgcggc gagcggtatc
     1021 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     1081 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1141 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     1201 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     1261 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     1321 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     1381 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     1441 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     1501 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     1561 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     1621 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     1681 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1741 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     1801 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     1861 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1921 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     1981 gtagataact acgatacggg agggcttacc acctggcccc agtgctgcaa tgataccgcg
     2041 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     2101 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     2161 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     2221 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     2281 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     2341 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     2401 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     2461 caagtcattc tgagaatagt gtatgcggcc gccgagttgc tcttgcccgg cgtcaatacg
     2521 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     2581 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     2641 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     2701 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     2761 actcttcctt tttcaatgtt atgaagcatt tatcagggtt attgtctcat gagcggatac
     2821 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     2881 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     2941 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     3001 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     3061 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     3121 cagattgtac tgagagtgca ccattcgacg ctctccctta tgcgactcct gcattaggaa
     3181 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     3241 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     3301 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     3361 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     3421 gatctggcta gcgatgaccc tgctgattgg ttcgctgacc atttccgggt gcgggacggc
     3481 gttaccagaa actcagaagg ttcgtccaac caaaccgact ctggcggcag tttacgagag
     3541 agatgatagg gtctgcttca gtaagccaga tgctacacaa ttaggcttgt acatactgtc
     3601 gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac gtaatacgac
     3661 tcactatagg gtacacggaa ttcgcgtttt tatttttaat tttctttcaa atacttctag
     3721 ctagagtatt tttacaacaa ttaccaacaa caacaacaaa caacaacaac attacatttt
     3781 acattctaca actacagata
//
