LOCUS       sequence-62769-e         948 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 948)
  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 948)
  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: pCoofy36
FEATURES             Location/Qualifiers
     source          1..948
                     /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)."
     CDS             125..169
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             join(170..175,176..199,200..235,236..259)
                     /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             269..292
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             408..416
                     /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             418..726
                     /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     605..624
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             746..775
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(828..846)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      842..889
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
       61 ctcactatag gggaattgtg agcggataac aattcccctc tagattagaa ggagatatac
      121 catgaaagaa accgctgctg ctaaattcga acgccagcac atggacagca gcgcttggag
      181 ccacccgcag ttcgaaaaag gtggaggttc tggcggtgga tcgggaggtt cagcgtggag
      241 ccacccgcag ttcgagaaaa ccgcgggtct ggaagttctg ttccaggggc ccatggacta
      301 atgaggtacc ggatccgaat tcgagctccg tcgacaagct tgatatcgaa ttcctgcagc
      361 ccaaactgca gttgacaaca taaaaacttt gtgttatact tgtaacgtaa ggaggtaatg
      421 attcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta
      481 cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt
      541 ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc
      601 tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg
      661 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga
      721 atataagctt gcggccgcac tcgagcatca tcatcatcac caccaccacc accactgaga
      781 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
      841 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
      901 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcatta
//
