LOCUS       sequence-62771-e         920 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 920)
  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 920)
  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: pCoofy24
FEATURES             Location/Qualifiers
     source          1..920
                     /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             143..220
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     CDS             230..253
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             369..377
                     /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             379..687
                     /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     566..585
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             707..736
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(789..807)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      803..850
                     /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 agatatacaa tgaagcgacg atggaaaaag aatttcatag ccgtctcagc
      181 agccaaccgc tttaagaaaa tctcatcctc cggggcactt ttaccgcggc tggaagttct
      241 gttccagggg cccatggact aatgaggtac cggatccgaa ttcgagctcc gtcgacaagc
      301 ttgatatcga attcctgcag cccaaactgc agttgacaac ataaaaactt tgtgttatac
      361 ttgtaacgta aggaggtaat gattcagttt aaggtttaca cctataaaag agagagccgt
      421 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg
      481 atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg
      541 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc
      601 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc
      661 attaacctga tgttctgggg aatataagct tgcggccgca ctcgagcatc atcatcatca
      721 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
      781 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
      841 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
      901 ggcgcattaa gcgcggcggg
//
