LOCUS       sequence-227376-        5708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express recombinant proteins with GFP-His fusion in Mycobacterium 
            smegmatis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5708)
  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 5708)
  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"; 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..5708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             119..136
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             143..187
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             203..217
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             266..286
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             293..1003
                     /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(320..348)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     943..963
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             1025..1042
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1095..1113)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1109..1156
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1193..1621
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1280..1299)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1490..1511
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      complement(3724..4312)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3804..3823)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtct
      121 ggtgccacgc ggttctggta tgaaagaaac cgctgctgct aaattcgaac gccagcacat
      181 ggacagccca gatctgggta ccgacgacga cgacaaggcc atggctgata tcggatccga
      241 attcgagctc cgtcgacaag cttccgaaaa cctgtacttc cagggtcaat tcagcaaagg
      301 agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg
      361 gcacaaattt tctgtcagtg gagagggtga aggtgatgct acatacggaa aactcaccct
      421 taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactactct
      481 gacctatggt gttcaatgct tttcccgtta tccggatcac atgaaacggc atgacttttt
      541 caagagtgcc atgcccgaag gttatgtaca ggaacgcact atatctttca aagatgacgg
      601 gaactacaag acgcgtgctg aagtcaagtt tgaaggtgat acccttgtta atcgtatcga
      661 gttaaagggt attgatttta aagaagatgg aaacattctc ggacacaaac tagagtacaa
      721 ctataactca cacaatgtat acatcacggc agacaaacaa aagaatggaa tcaaagctaa
      781 cttcaaaatt cgccacaaca ttgaagatgg ttccgttcaa ctagcagacc attatcaaca
      841 aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc tgtcgacaca
      901 atctgccctt tcgaaagatc ccaacgaaaa gcgtgaccac atggtccttc ttgagtttgt
      961 aactgctgct gggattacac atggcatgga tgagctctac aaaaagggtg cggccgcact
     1021 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     1081 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1141 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     1201 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1261 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1321 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     1381 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     1441 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     1501 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     1561 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     1621 tttaacaaaa tatctagatt taaagatctg gtaccgcggc cgcgccacgg atgccaccac
     1681 aggcactaca acggagttcg ccacgtacat caccacaacc accgattctg gcggtgagct
     1741 ccccgatatt cagcggaaat ggcttggtat cgaccaagat tcgtagaacc ccgtctcgtc
     1801 tggctggtat tcaaaacggg cgcaacgaaa cacgcaacga gacaggcatg gcccaaacca
     1861 gaaaactagc gtctaccagg acttttacct gtccgacccg ttgcaacgga accccccacg
     1921 gaacccccgc gacacccgct ccccaattgc gttagaacag cggtggattg tcggcttcgt
     1981 tgtgggcctt ttgagccgct tcctgttctg ccgcacgctc tttcctcgcc cgatagccga
     2041 gtcgcttaac ggtgtccaga tgcagcccga aatgtttggc cgtttgcggc caagagtggc
     2101 cctcgtcgtc gtgataggcg cggatgcgtt cgcggcgtgc agcctgctcg gcgagccact
     2161 cgctgcgttc ctgcgccacg agccggacga cgtggcgttc ggatagtccg gtgattcgag
     2221 cgccttcggc ggcggtcacg cgccgctttt tgcggacagt cggctgccgg ttgtagccgt
     2281 cgctgtagcc gtcgctcata gcaatgcctc catgcatggc tgacgcggac tttgcgcgcc
     2341 gcgcaactgt gctcgccgcc gtgcgcgctg ctgcgccctt ccgcgagatg gccgactggc
     2401 gcgcactgag tgtggcctcg tagaccacga tcccgtccgc ccaaatgcgc gacttggttg
     2461 tgatccaacg ccaaatgctg ttggcgatgg cgcggacctc gctgtccggt agcggtccgg
     2521 gacacacgtc gttgcacgga aattcggcgt ttcgcgcgtg gcactcggca tagatcgcgc
     2581 ggccgagtcc gtccacgttc cgggtcggca ggtagatccg catgagggcg ggacgatagg
     2641 cccacaacct gacggaatcg aacagtgcgc aattccgccc tagcggcgtc ggagccgctt
     2701 tgtacgtggt ctgctgacgc cagcgcggcg gtggcatgtt cgcgccgagc tcggcctcga
     2761 tgtggctgag tgtgtagaga tctgagtgga gccattccgt ttcccaggcg atgtggccgg
     2821 ggtttttggt catgaggcct gagtaactgc ggtcgccatc gacggcgcgc cgaaggcctt
     2881 cggcgcacgc cgccatgtat gcgagcggct tacgccgcgc gtattcggtg cgtggaacag
     2941 gggcgttgag tgcccacact gcgtgtgcgt ggccgttggc gcgattgccc acgatcgcgt
     3001 tgggcagcgg atgggacccc cgggcgctga gcgctcggag cgctgcgtct ggatggtcta
     3061 cgtccacgac cagcaggttt gccagcgctg ttgggttcgc ctcgatgtac cggcggccta
     3121 gggccgacgc gcggctttgg cggtagatcc cctcgagcag atcgtcgctt gccagcggcc
     3181 agtacggcag ccagagctgc tcaaattcgt cggcgacgtg gctcacgctt ggtagtagac
     3241 cacgattaat caccggtgta tggtccgaca cgagctccaa gtcagatatt tcgctgaggg
     3301 gccaccccac aactgcacac tcccccgctc tcccgtcgag ccctggtggt ggaacaccag
     3361 cgacagccga gcacccccaa ccacctgtac caaccaggag gaacacatgc gtcgtttcga
     3421 ggacgtttcc gggccgctga gagccgctgt ggcggccgta cacgccgcct tagacccgtt
     3481 agaccccctg ccgcctgaat gcgcgggtac gagccacaca gcgcccgaac ttacggagct
     3541 ggtgggctca cgcgtgcggc cgcacgcgtt gcgctcggtc gttcggctgc ggcgagcggt
     3601 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3661 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgttggc
     3721 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3781 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3841 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccctcggg
     3901 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3961 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4021 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4081 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4141 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     4201 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4261 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4321 tttgatcttt tctacggggt ctgacgctca tggaactaga gcatgagatt atcaaaaagg
     4381 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4441 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4501 tgtctatttc gttcatccat agttgcctga ctccccgacg tggccgacca gcccgtcatc
     4561 gtcaacgcct gatccgcggt gcggacaggc cgtgtcgcga ccggccgtgc ggaattaagc
     4621 cggcccgtac cctgtgaata gaggtccgct gtgacacaag aatccctgtt acttctcgac
     4681 cgtattgatt cggatgattc ctacgcgagc ctgcggaacg accaggaatt ctgggagccg
     4741 ctggcccgcc gagccctgga ggagctcggg ctgccggtgc cgccggtgct gcgggtgccc
     4801 ggcgagagca ccaaccccgt actggtcggc gagcccggcc cggtgatcaa gctgttcggc
     4861 gagcactggt gcggtccgga gagcctcgcg tcggagtcgg aggcgtacgc ggtcctggcg
     4921 gacgccccgg tgccggtgcc ccgcctcctc ggccgcggcg agctgcggcc cggcaccgga
     4981 gcctggccgt ggccctacct ggtgatgagc cggatgaccg gcaccacctg gcggtccgcg
     5041 atggacggca cgaccgaccg gaacgcgctg ctcgccctgg cccgcgaact cggccgggtg
     5101 ctcggccggc tgcacagggt gccgctgacc gggaacaccg tgctcacccc ccattccgag
     5161 gtcttcccgg aactgctgcg ggaacgccgc gcggcgaccg tcgaggacca ccgcgggtgg
     5221 ggctacctct cgccccggct gctggaccgc ctggaggact ggctgccgga cgtggacacg
     5281 ctgctggccg gccgcgaacc ccggttcgtc cacggcgacc tgcacgggac caacatcttc
     5341 gtggacctgg ccgcgaccga ggtcaccggg atcgtcgact tcaccgacgt ctatgcggga
     5401 gactcccgct acagcctggt gcaactgcat ctcaacgcct tccggggcga ccgcgagatc
     5461 ctggccgcgc tgctcgacgg ggcgcagtgg aagcggaccg aggacttcgc ccgcgaactg
     5521 ctcgccttca ccttcctgca cgacttcgag gtgttcgagg agaccccgct ggatctctcc
     5581 ggcttcaccg atccggagga actggcgcag ttcctctggg ggccgccgga caccgccccc
     5641 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc
     5701 cgcgaaat
//
