LOCUS       sequence-222093-        1260 bp    DNA     linear   UNA 14-APR-2025
DEFINITION  Express recombinant proteins with GFP-His fusion in Mycobacterium 
            smegmatis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1260)
  AUTHORS   Radhakrishnan A, Furze CM, Ahangar MS, Fullam E
  TITLE     A GFP-strategy for efficient recombinant protein overexpression and 
            purification in Mycobacterium smegmatis.
  JOURNAL   RSC Adv. 2018 Sep 27;8(58):33087-33095. doi: 10.1039/c8ra06237d. 
            Epub 2018 Sep 25.
  PUBMED    30319771
REFERENCE   2  (bases 1 to 1260)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1039/c8ra06237d"; journalName: "RSC Adv"; date: "2018-09-27- 
            27"; volume: "8"; issue: "58"; pages: "33087-33095"
COMMENT     Sequence Label: pYUB1062-GFP
FEATURES             Location/Qualifiers
     source          1..1260
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     RBS             15..37
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             48..65
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             75..92
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             99..143
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             159..173
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             222..242
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             249..959
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(276..304)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     899..919
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             981..998
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 ccctctagaa taattttgtt taactttaag aaggagatat acatatgcac catcatcatc
       61 atcattcttc tggtctggtg ccacgcggtt ctggtatgaa agaaaccgct gctgctaaat
      121 tcgaacgcca gcacatggac agcccagatc tgggtaccga cgacgacgac aaggccatgg
      181 ctgatatcgg atccgaattc gagctccgtc gacaagcttc cgaaaacctg tacttccagg
      241 gtcaattcag caaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag
      301 atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgctacat
      361 acggaaaact cacccttaaa tttatttgca ctactggaaa actacctgtt ccatggccaa
      421 cacttgtcac tactctgacc tatggtgttc aatgcttttc ccgttatccg gatcacatga
      481 aacggcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa cgcactatat
      541 ctttcaaaga tgacgggaac tacaagacgc gtgctgaagt caagtttgaa ggtgataccc
      601 ttgttaatcg tatcgagtta aagggtattg attttaaaga agatggaaac attctcggac
      661 acaaactaga gtacaactat aactcacaca atgtatacat cacggcagac aaacaaaaga
      721 atggaatcaa agctaacttc aaaattcgcc acaacattga agatggttcc gttcaactag
      781 cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc
      841 attacctgtc gacacaatct gccctttcga aagatcccaa cgaaaagcgt gaccacatgg
      901 tccttcttga gtttgtaact gctgctggga ttacacatgg catggatgag ctctacaaaa
      961 agggtgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     1021 cccgaaagga agctgattng gctgctgcca ccgctgaaca ataactagca taaccccttg
     1081 ggggcctcta aacggggtct tgaaggggtt ttttggctga aaggagnaan ctatatccgg
     1141 gattggccaa aggggacgcc cccctggaag cggcccattt aagccccggc ggggtgtggg
     1201 gggttaccnn cnaacgtgga ccggtaaact tggcccgggc ccttaggggc ccggtccttt
//
