LOCUS       sequence-99547-e        2045 bp    DNA     linear   UNA 15-APR-2025
DEFINITION  Protein expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 2045)
  AUTHORS   Goulas T, Cuppari A, Garcia-Castellanos R, Snipas S, Glockshuber R, 
            Arolas JL, Gomis-Ruth FX
  TITLE     The pCri System: A Vector Collection for Recombinant Protein 
            Expression and Purification.
  JOURNAL   PLoS One. 2014 Nov 11;9(11):e112643. doi: 
            10.1371/journal.pone.0112643. eCollection 2014.
  PUBMED    25386923
REFERENCE   2  (bases 1 to 2045)
  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: "PLoS One.";
            date: "2014-11-11"; volume: "9(11):e112643. doi"; pages: " 
            10.1371/journal.pone.0112643. eCollection 2014"
COMMENT     Sequence Label: pCri-1a
FEATURES             Location/Qualifiers
     source          1..2045
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     RBS             15..37
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             50..67
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             71..1171
                     /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
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1145..1168
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1196..1216
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1229..1231,1232..1945)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1271..1292)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1532..1551)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1879..1900
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1998..2015
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 ccctctagaa taattttgtt taactttaag aaggagatat accatgaaac atcaccatca
       61 ccatcacccc atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg
      121 ctataacggt ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac
      181 cgttgagcat ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg
      241 ccctgacatt atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt
      301 ggctgaaatc accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc
      361 cgtacgttac aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat
      421 ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga
      481 taaagaactg aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt
      541 cacctggccg ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta
      601 cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt
      661 tgacctgatt aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc
      721 ctttaataaa ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga
      781 caccagcaaa gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa
      841 accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc
      901 aaaagagttc ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga
      961 caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg
     1021 tattgccgcc actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat
     1081 gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac
     1141 tgtcgatgaa gccctgaaag acgcgcagac taattcggga tctggcagtg gttctgagaa
     1201 tctttatttt cagggcgcca tggccaaagt gagcaagggc gaggagctgt tcaccggggt
     1261 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     1321 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     1381 caagctgccc gtgccctggc ccaccctcgt gaccaccttc ggctacggcc tgcagtgctt
     1441 cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     1501 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     1561 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1621 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     1681 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     1741 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1801 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1861 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     1921 cggcatggac gagctgtaca agtaaggtac cggatccgaa ttcgagctcc gtcgacaagc
     1981 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     2041 gaaag
//
