LOCUS       sequence-62732-e         927 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 927)
  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 927)
  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: pCoofy34
FEATURES             Location/Qualifiers
     source          1..927
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     43..62
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        43..61
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    62..86
                     /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)."
     CDS             115..159
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             join(160..165,166..189,190..225,226..249)
                     /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             259..282
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             398..406
                     /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             408..716
                     /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     595..614
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             736..753
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(806..824)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      820..867
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
       61 gggaattgtg agcggataac aattcccctc tagattagaa ggagatatac catgaaagaa
      121 accgctgctg ctaaattcga acgccagcac atggacagca gcgcttggag ccacccgcag
      181 ttcgaaaaag gtggaggttc tggcggtgga tcgggaggtt cagcgtggag ccacccgcag
      241 ttcgagaaaa ccgcgggtct ggaagttctg ttccaggggc ccatggacta atgaggtacc
      301 ggatccgaat tcgagctccg tcgacaagct tgatatcgaa ttcctgcagc ccaaactgca
      361 gttgacaaca taaaaacttt gtgttatact tgtaacgtaa ggaggtaatg attcagttta
      421 aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata
      481 ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag
      541 ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga
      601 tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca
      661 gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataagctt
      721 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
      781 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
      841 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
      901 gggacgcgcc ctgtagcggc gcattaa
//
