LOCUS       sequence-393681-        2987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pJL1 plasmid encoding E. coli maltose binding protein (MBP) modified
            at the C-terminus with 30 amino acids containing one optimal DQNAT 
            sequon followed by a 6x-His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2987)
  AUTHORS   Hershewe JM, Warfel KF, Iyer SM, Peruzzi JA, Sullivan CJ, Roth EW, 
            DeLisa MP, Kamat NP, Jewett MC
  TITLE     Improving cell-free glycoprotein synthesis by characterizing and 
            enriching native membrane vesicles.
  JOURNAL   Nat Commun. 2021 Apr 22;12(1):2363. doi: 10.1038/s41467-021-22329-3.
  PUBMED    33888690
REFERENCE   2  (bases 1 to 2987)
  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: "Nat 
            Commun."; date: "2021-04-22"; volume: "12(1):2363. doi"; pages: " 
            10.1038/s41467-021-22329-3"
COMMENT     Sequence Label: pJL1-MBP-DQNAT
FEATURES             Location/Qualifiers
     source          1..2987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     506..525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     633..652
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        633..651
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             683..705
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             713..1813
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGTVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1670..1679
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1787..1810
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1841..1870
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1916..1933
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1988..2006)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2002..2049
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2067..2882)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2790..2809
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2874..2893
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
       61 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      601 ggaaaaacgc cagcaacgcg atcccgcgaa attaatacga ctcactatag ggagaccaca
      661 acggtttccc tctagaaata attttgttta actttaagaa ggagatatac atatgaaaat
      721 cgaagaaggt aaactggtaa tctggattaa cggcgataaa ggctataacg gtctcgctga
      781 agtcggtaag aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa
      841 actggaagag aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg
      901 ggcacacgac cgctttggtg gctacgctca atctggcctg ttggctgaaa tcaccccgga
      961 caaagcgttc caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa
     1021 gctgattgct tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct
     1081 gccgaacccg ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa
     1141 aggtaagagc gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc
     1201 tgctgacggg ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg
     1261 cgtggataac gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa
     1321 acacatgaat gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac
     1381 agcgatgacc atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta
     1441 tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct
     1501 gagcgcaggt attaacgccg ccagtccgaa caaagagctg gcgaaagagt tcctcgaaaa
     1561 ctatctgctg actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtaccgt
     1621 agcgctgaag tcttacgagg aagagttggc gaaagatcca cgtattgccg ccaccatgga
     1681 aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc
     1741 cgtgcgtact gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa
     1801 agacgcgcag actcgtatca ccaagctagg tgcggccgca gaacaaaaac tcatctcaga
     1861 agaggatctg aatggggccg cactcgaggg tggcgatcag aacgcgaccg gcggtcatca
     1921 ccatcatcac cattaagtcg accggctgct aacaaagccc gaaaggaagc tgagttggct
     1981 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     2041 ggttttttgc tgaaagccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc
     2101 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     2161 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     2221 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     2281 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag cttatgcatt
     2341 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     2401 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     2461 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     2521 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     2581 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     2641 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     2701 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     2761 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     2821 gcatccatgt tggaatttaa tcgcggcttc gagcaagacg tttcccgttg aatatggctc
     2881 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata
     2941 tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtg
//
