LOCUS       sequence-62734-e         939 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  natural linear DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 939)
  AUTHORS   Scholz J, Besir H, Strasser C, Suppmann S
  TITLE     A new method to customize protein expression vectors for fast, 
            efficient and background free parallel cloning.
  JOURNAL   BMC Biotechnol. 2013 Feb 14;13(1):12.
  PUBMED    23410102
REFERENCE   2  (bases 1 to 939)
  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: "BMC 
            Biotechnol."; date: "2013-02-14"; volume: "13(1)"; pages: "12"
COMMENT     Sequence Label: pCoofy21
FEATURES             Location/Qualifiers
     source          1..939
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     53..72
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        53..71
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    72..96
                     /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             111..133
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             146..175
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             join(179..184,185..208,209..244,245..268)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             278..301
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             417..425
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             427..735
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MIQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSD
                     KVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     614..633
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             755..772
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(825..843)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      839..886
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
       61 ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac
      121 tttaagaagg agatatacca tggaacatca tcatcatcat caccatcacc atcacgggag
      181 cgcttggagc cacccgcagt tcgaaaaagg tggaggttct ggcggtggat cgggaggttc
      241 agcgtggagc cacccgcagt tcgagaaaac cgcgggtctg gaagttctgt tccaggggcc
      301 catggactaa tgaggtaccg gatccgaatt cgagctccgt cgacaagctt gatatcgaat
      361 tcctgcagcc caaactgcag ttgacaacat aaaaactttg tgttatactt gtaacgtaag
      421 gaggtaatga ttcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt
      481 gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc
      541 agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg
      601 gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg
      661 gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg
      721 ttctggggaa tataagcttg cggccgcact cgagcaccac caccaccacc actgagatcc
      781 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
      841 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
      901 tatatccgga ttggcgaatg ggacgcgccc tgtagcggc
//
