LOCUS       sequence-104783-        1121 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  bacterial expression of N-terminal fusion protein of CNA35 with 
            EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1121)
  AUTHORS   Aper SJ, van Spreeuwel AC, van Turnhout MC, van der Linden AJ, 
            Pieters PA, van der Zon NL, de la Rambelje SL, Bouten CV, Merkx M
  TITLE     Colorful Protein-Based Fluorescent Probes for Collagen Imaging.
  JOURNAL   PLoS One. 2014 Dec 9;9(12):e114983. doi: 
            10.1371/journal.pone.0114983. eCollection 2014.
  PUBMED    25490719
REFERENCE   2  (bases 1 to 1121)
  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: "PLoS One.";
            date: "2014-12-9"; volume: "9(12):e114983. doi"; pages: " 
            10.1371/journal.pone.0114983. eCollection 2014"
COMMENT     Sequence Label: pET28a-EGFP-CNA35
FEATURES             Location/Qualifiers
     source          1..1121
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     65..84
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        65..83
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    84..108
                     /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             123..145
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             164..181
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             191..208
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(221..223,224..226,227..937)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(221..223,224..226,227..937)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(266..287)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(527..546)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     874..895
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
       61 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
      121 attttgttta actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca
      181 cagcagcggc ctggtgccgc gcggcagcca tatggctagc atggtgagca agggcgagga
      241 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
      301 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
      361 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
      421 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
      481 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
      541 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
      601 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
      661 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
      721 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
      781 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
      841 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
      901 cgccgggatc actctcggca tggacgagct gtacaagtcc ggagaattcc acggatccgc
      961 acgagatatt tcatcaacga atgttacaga tttaactgta tcaccgtcta agatagaaga
     1021 tggtggtaaa acgacagtaa aaatgacgtt cgacgataaa aatggaaaaa tacaaaatgg
     1081 tgacatgatt aaagtggcat ggccgacaag cggtacagta a
//
