LOCUS       sequence-300400-        3860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for producing recombinant proteins by wheat germ-based 
            cell-free translation, contains T7 promoter, 12xHis-tag and NotI 
            restriction site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3860)
  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 3860)
  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-HxHLICNot
FEATURES             Location/Qualifiers
     source          1..3860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             10..27
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             70..87
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             88..108
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(800..816)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(800..816)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(813..835)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    824..840
                     /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(848..854,855..872,873..878))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    893..914
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1031..1048)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1202..1790)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1282..1301)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1961..2752,2753..2821))
                     /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     2584..2603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2822..2925)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2993..3011
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3049..3071)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3171..3190
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3404..3423
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3713..3732
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3713..3731
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 actagtatgc atcatcatca tcatcattct cgtgcttggc gtcacccgca gttcggtggt
       61 actagtatgc atcatcatca tcatcatgag aacctgtact tccaatccaa tattggaagt
      121 ggataacgga tccatatata gggcccgggt tataattacc tcaggtcgac gtcccatggt
      181 tttgtataga atttacggct agcgccggat gcgacgccgg tcgcgtctta tccggccttc
      241 ctatatcagg ctgtgtttaa gacgccgccg cttcgcccaa atccttatgc cggttcgacg
      301 actggacaaa atactgttta tcttcccagc gcaggcaggt taatgtacca ccccagcagc
      361 agccggtatc cagcgcgtat ataccttccg gcgtaccttt gccctccagc gatgcccagt
      421 gaccaaaggc gatgctgtat tcttcagcga cagggccagg aatcgcaaac cacggtttca
      481 gtggggcagg ggcctcttcc ggcgattctt actagctagt atgcataggt gctgaaatat
      541 aaagtttgtg tttctaaaac acacgtggta cgtacgataa cgtacagtgt ttttccctcc
      601 acttaaatcg aagggtagtg tcttggagcg cgcggagtaa acatatatgg ttcatatatg
      661 tccgtaggca cgtaaaaaaa gcgagggatt cgaattcccc cggaaccccc ggttggggcc
      721 cacgcctcga tcgagcaaaa aaaaaaaaga aaaaaaaaaa aaaaaaaagc tttcccgcgg
      781 ccagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
      841 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
      901 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
      961 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     1021 cttccgcttc ctcgctcact gactcgctgc gctcggtcgc tcggctgcgg cgagcggtat
     1081 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     1141 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     1201 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     1261 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     1321 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     1381 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     1441 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     1501 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     1561 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     1621 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     1681 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     1741 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     1801 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     1861 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     1921 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     1981 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     2041 tgtagataac tacgatacgg gagggcttac cacctggccc cagtgctgca atgataccgc
     2101 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     2161 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     2221 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     2281 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     2341 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     2401 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     2461 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     2521 ccaagtcatt ctgagaatag tgtatgcggc cgccgagttg ctcttgcccg gcgtcaatac
     2581 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     2641 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     2701 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     2761 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     2821 tactcttcct ttttcaatgt tatgaagcat ttatcagggt tattgtctca tgagcggata
     2881 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     2941 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     3001 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     3061 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     3121 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     3181 gcagattgta ctgagagtgc accattcgac gctctccctt atgcgactcc tgcattagga
     3241 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     3301 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     3361 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     3421 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     3481 ggatctggct agcgatgacc ctgctgattg gttcgctgac catttccggg tgcgggacgg
     3541 cgttaccaga aactcagaag gttcgtccaa ccaaaccgac tctggcggca gtttacgaga
     3601 gagatgatag ggtctgcttc agtaagccag atgctacaca attaggcttg tacatactgt
     3661 cgttagaacg cggctacaat taatacataa ccttatgtat catacacata cgtaatacga
     3721 ctcactatag ggtacacgga attcgcgttt ttatttttaa ttttctttca aatacttcta
     3781 gctagagtat ttttacaaca attaccaaca acaacaacaa acaacaacaa cattacattt
     3841 tacattctac aactacagat
//
