LOCUS       sequence-300413-        4688 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Used for producing recombinant proteins by wheat germ-based 
            cell-free translation, contains T7 promoter, Halo-tag and NotI 
            restriction site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4688)
  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 4688)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-HaloLICNot
FEATURES             Location/Qualifiers
     source          1..4688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..897
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             910..930
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(1621..1637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1621..1637)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1634..1656)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1645..1661
                     /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(1669..1675,1676..1693,1694..1699))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1714..1735
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1852..1869)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2023..2611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2103..2122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2782..3573,3574..3642))
                     /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     3405..3424
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3643..3746)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3814..3832
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3870..3892)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3992..4011
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4225..4244
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4534..4553
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4534..4552
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 actagtatgg cagaaatcgg tactggcttt ccattcgacc cccattatgt ggaagtcctg
       61 ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg
      121 cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc
      181 catcgctgca ttgctccaga cctgatcggt atgggcaaat ccgacaaacc agacctgggt
      241 tatttcttcg acgaccacgt ccgcttcatg gatgccttca tcgaagccct gggtctggaa
      301 gaggtcgtcc tggtcattca cgactggggc tccgctctgg gtttccactg ggccaagcgc
      361 aatccagagc gcgtcaaagg tattgcattt atggagttca tccgccctat cccgacctgg
      421 gacgaatggc cagaatttgc ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc
      481 cgcaagctga tcatcgatca gaacgttttt atcgagggta cgctgccgat gggtgtcgtc
      541 cgcccgctga ctgaagtcga gatggaccat taccgcgagc cgttcctgaa tcctgttgac
      601 cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc
      661 gtcgcgctgg tcgaagaata catggactgg ctgcaccagt cccctgtccc gaagctgctg
      721 ttctggggca ccccaggcgt tctgatccca ccggccgaag ccgctcgcct ggccaaaagc
      781 ctgcctaact gcaaggctgt ggacatcggc ccgggtctga atctgctgca agaagacaac
      841 ccggacctga tcggcagcga gatcgcgcgc tggctgtcga cgctcgagat ttccggcgag
      901 ccaaccactg aggatctgta cttccaatcc aatattggaa gtggataacg gatccatata
      961 tagggcccgg gttataatta cctcaggtcg acgtcccatg gttttgtata gaatttacgg
     1021 ctagcgccgg atgcgacgcc ggtcgcgtct tatccggcct tcctatatca ggctgtgttt
     1081 aagacgccgc cgcttcgccc aaatccttat gccggttcga cgactggaca aaatactgtt
     1141 tatcttccca gcgcaggcag gttaatgtac caccccagca gcagccggta tccagcgcgt
     1201 atataccttc cggcgtacct ttgccctcca gcgatgccca gtgaccaaag gcgatgctgt
     1261 attcttcagc gacagggcca ggaatcgcaa accacggttt cagtggggca ggggcctctt
     1321 ccggcgattc ttactagcta gtatgcatag gtgctgaaat ataaagtttg tgtttctaaa
     1381 acacacgtgg tacgtacgat aacgtacagt gtttttccct ccacttaaat cgaagggtag
     1441 tgtcttggag cgcgcggagt aaacatatat ggttcatata tgtccgtagg cacgtaaaaa
     1501 aagcgaggga ttcgaattcc cccggaaccc ccggttgggg cccacgcctc gatcgagcaa
     1561 aaaaaaaaag aaaaaaaaaa aaaaaaaaag ctttcccgcg gccagcttgg cgtaatcatg
     1621 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     1681 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     1741 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1801 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     1861 tgactcgctg cgctcggtcg ctcggctgcg gcgagcggta tcagctcact caaaggcggt
     1921 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1981 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2041 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2101 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2161 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2221 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2281 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2341 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2401 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2461 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2521 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2581 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2641 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2701 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2761 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2821 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2881 ggagggctta ccacctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2941 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3001 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     3061 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     3121 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3181 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     3241 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     3301 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     3361 gtgtatgcgg ccgccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     3421 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3481 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3541 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3601 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaatg
     3661 ttatgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     3721 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     3781 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     3841 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     3901 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     3961 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     4021 caccattcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     4081 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     4141 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     4201 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     4261 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatctggc tagcgatgac
     4321 cctgctgatt ggttcgctga ccatttccgg gtgcgggacg gcgttaccag aaactcagaa
     4381 ggttcgtcca accaaaccga ctctggcggc agtttacgag agagatgata gggtctgctt
     4441 cagtaagcca gatgctacac aattaggctt gtacatactg tcgttagaac gcggctacaa
     4501 ttaatacata accttatgta tcatacacat acgtaatacg actcactata gggtacacgg
     4561 aattcgcgtt tttattttta attttctttc aaatacttct agctagagta tttttacaac
     4621 aattaccaac aacaacaaca aacaacaaca acattacatt ttacattcta caactacaga
     4681 tactagta
//
