LOCUS       sequence-362497-        5598 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The plasmid contains LIC cassette upstream of folding reporter GFP 
            and TEV-cleavable 10x histidine purification tag. Compatible with T7
            promoter-based expression system in mycobacteria..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5598)
  AUTHORS   Grave K, Bennett MD, Hogbom M
  TITLE     High-throughput strategy for identification of Mycobacterium 
            tuberculosis membrane protein expression conditions using folding 
            reporter GFP.
  JOURNAL   Protein Expr Purif. 2022 Oct;198:106132. doi: 
            10.1016/j.pep.2022.106132. Epub 2022 Jun 21.
  PUBMED    35750296
REFERENCE   2  (bases 1 to 5598)
  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: "Protein 
            Expr Purif."; date: "2022-10"; volume: "198:106132. doi"; pages: " 
            10.1016/j.pep.2022.106132. Epub 2022 Jun 21"
COMMENT     Sequence Label: pYUBcLIC-GFP
FEATURES             Location/Qualifiers
     source          1..5598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..80
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        61..79
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    80..104
                     /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             119..141
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             188..898
                     /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(215..243)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     838..858
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             905..934
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             979..996
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1049..1067)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1063..1110
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1147..1575
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1234..1253)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1444..1465
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      complement(3678..4266)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3758..3777)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
       61 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
      121 tgtttaactt taagaaggag atataccatg ggtggtggat ttaaatttat acttccaagg
      181 tcaattcagc aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga
      241 tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgctacata
      301 cggaaaactc acccttaaat ttatttgcac tactggaaaa ctacctgttc cttggccaac
      361 acttgtcact actctgacct atggtgttca atgcttttcc cgttatccgg atcacatgaa
      421 acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaac gcactatatc
      481 tttcaaagat gacgggaact acaagacgcg tgctgaagtc aagtttgaag gtgataccct
      541 tgttaatcgt atcgagttaa agggtattga ttttaaagaa gatggaaaca ttctcggaca
      601 caaactagag tacaactata actcacacaa tgtatacatc acggcagaca aacaaaagaa
      661 tggaatcaaa gctaacttca aaattcgcca caacattgaa gatggttccg ttcaactagc
      721 agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca
      781 ttacctgtcg acacaatctg ccctttcgaa agatcccaac gaaaagcgtg accacatggt
      841 ccttcttgag tttgtaactg ctgctgggat tacacatggc atggatgagc tctacaaaac
      901 tagtcatcat catcaccatc atcatcacca tcattaagtc gaccatctag agagctcaag
      961 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     1021 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     1081 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     1141 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1201 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1261 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1321 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1381 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1441 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1501 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1561 aatttaacgc gaattttaac aaaatatcta gatttaaaga tctggtaccg cggccgcgcc
     1621 acggatgcca ccacaggcac tacaacggag ttcgccacgt acatcaccac aaccaccgat
     1681 tctggcggtg agctccccga tattcagcgg aaatggcttg gtatcgacca agattcgtag
     1741 aaccccgtct cgtctggctg gtattcaaaa cgggcgcaac gaaacacgca acgagacagg
     1801 catggcccaa accagaaaac tagcgtctac caggactttt acctgtccga cccgttgcaa
     1861 cggaaccccc cacggaaccc ccgcgacacc cgctccccaa ttgcgttaga acagcggtgg
     1921 attgtcggct tcgttgtggg ccttttgagc cgcttcctgt tctgccgcac gctctttcct
     1981 cgcccgatag ccgagtcgct taacggtgtc cagatgcagc ccgaaatgtt tggccgtttg
     2041 cggccaagag tggccctcgt cgtcgtgata ggcgcggatg cgttcgcggc gtgcagcctg
     2101 ctcggcgagc cactcgctgc gttcctgcgc cacgagccgg acgacgtggc gttcggatag
     2161 tccggtgatt cgagcgcctt cggcggcggt cacgcgccgc tttttgcgga cagtcggctg
     2221 ccggttgtag ccgtcgctgt agccgtcgct catagcaatg cctccatgca tggctgacgc
     2281 ggactttgcg cgccgcgcaa ctgtgctcgc cgccgtgcgc gctgctgcgc ccttccgcga
     2341 gatggccgac tggcgcgcac tgagtgtggc ctcgtagacc acgatcccgt ccgcccaaat
     2401 gcgcgacttg gttgtgatcc aacgccaaat gctgttggcg atggcgcgga cctcgctgtc
     2461 cggtagcggt ccgggacaca cgtcgttgca cggaaattcg gcgtttcgcg cgtggcactc
     2521 ggcatagatc gcgcggccga gtccgtccac gttccgggtc ggcaggtaga tccgcatgag
     2581 ggcgggacga taggcccaca acctgacgga atcgaacagt gcgcaattcc gccctagcgg
     2641 cgtcggagcc gctttgtacg tggtctgctg acgccagcgc ggcggtggca tgttcgcgcc
     2701 gagctcggcc tcgatgtggc tgagtgtgta gagatctgag tggagccatt ccgtttccca
     2761 ggcgatgtgg ccggggtttt tggtcatgag gcctgagtaa ctgcggtcgc catcgacggc
     2821 gcgccgaagg ccttcggcgc acgccgccat gtatgcgagc ggcttacgcc gcgcgtattc
     2881 ggtgcgtgga acaggggcgt tgagtgccca cactgcgtgt gcgtggccgt tggcgcgatt
     2941 gcccacgatc gcgttgggca gcggatggga cccccgggcg ctgagcgctc ggagcgctgc
     3001 gtctggatgg tctacgtcca cgaccagcag gtttgccagc gctgttgggt tcgcctcgat
     3061 gtaccggcgg cctagggccg acgcgcggct ttggcggtag atcccctcga gcagatcgtc
     3121 gcttgccagc ggccagtacg gcagccagag ctgctcaaat tcgtcggcga cgtggctcac
     3181 gcttggtagt agaccacgat taatcaccgg tgtatggtcc gacacgagct ccaagtcaga
     3241 tatttcgctg aggggccacc ccacaactgc acactccccc gctctcccgt cgagccctgg
     3301 tggtggaaca ccagcgacag ccgagcaccc ccaaccacct gtaccaacca ggaggaacac
     3361 atgcgtcgtt tcgaggacgt ttccgggccg ctgagagccg ctgtggcggc cgtacacgcc
     3421 gccttagacc cgttagaccc cctgccgcct gaatgcgcgg gtacgagcca cacagcgccc
     3481 gaacttacgg agctggtggg ctcacgcgtg cggccgcacg cgttgcgctc ggtcgttcgg
     3541 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3601 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3661 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3721 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3781 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3841 tttctcccct cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3901 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3961 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4021 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4081 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     4141 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4201 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4261 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcatggaac tagagcatga
     4321 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4381 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4441 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gacgtggccg
     4501 accagcccgt catcgtcaac gcctgatccg cggtgcggac aggccgtgtc gcgaccggcc
     4561 gtgcggaatt aagccggccc gtaccctgtg aatagaggtc cgctgtgaca caagaatccc
     4621 tgttacttct cgaccgtatt gattcggatg attcctacgc gagcctgcgg aacgaccagg
     4681 aattctggga gccgctggcc cgccgagccc tggaggagct cgggctgccg gtgccgccgg
     4741 tgctgcgggt gcccggcgag agcaccaacc ccgtactggt cggcgagccc ggcccggtga
     4801 tcaagctgtt cggcgagcac tggtgcggtc cggagagcct cgcgtcggag tcggaggcgt
     4861 acgcggtcct ggcggacgcc ccggtgccgg tgccccgcct cctcggccgc ggcgagctgc
     4921 ggcccggcac cggagcctgg ccgtggccct acctggtgat gagccggatg accggcacca
     4981 cctggcggtc cgcgatggac ggcacgaccg accggaacgc gctgctcgcc ctggcccgcg
     5041 aactcggccg ggtgctcggc cggctgcaca gggtgccgct gaccgggaac accgtgctca
     5101 ccccccattc cgaggtcttc ccggaactgc tgcgggaacg ccgcgcggcg accgtcgagg
     5161 accaccgcgg gtggggctac ctctcgcccc ggctgctgga ccgcctggag gactggctgc
     5221 cggacgtgga cacgctgctg gccggccgcg aaccccggtt cgtccacggc gacctgcacg
     5281 ggaccaacat cttcgtggac ctggccgcga ccgaggtcac cgggatcgtc gacttcaccg
     5341 acgtctatgc gggagactcc cgctacagcc tggtgcaact gcatctcaac gccttccggg
     5401 gcgaccgcga gatcctggcc gcgctgctcg acggggcgca gtggaagcgg accgaggact
     5461 tcgcccgcga actgctcgcc ttcaccttcc tgcacgactt cgaggtgttc gaggagaccc
     5521 cgctggatct ctccggcttc accgatccgg aggaactggc gcagttcctc tgggggccgc
     5581 cggacaccgc ccccggcg
//
