LOCUS       sequence-83003-e         976 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses SpyTag-SpyCatcher mediated cyclized version of TEM-1 
            beta-lactamase (BLA). Cyclization of BLA led to an increase in 
            thermal resilience of >60 degrees celsius..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 976)
  AUTHORS   Schoene C, Fierer JO, Bennett SP, Howarth M
  TITLE     SpyTag/SpyCatcher cyclization confers resilience to boiling on a 
            mesophilic enzyme.
  JOURNAL   Angew Chem Int Ed Engl. 2014 Jun 10;53(24):6101-4. doi: 
            10.1002/anie.201402519. Epub 2014 May 9.
  PUBMED    24817566
REFERENCE   2  (bases 1 to 976)
  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"; doi: 
            "10.1002/anie.201402519"; journalName: "Angew Chem Int Ed Engl"; 
            date: "2014-06-10- 10"; volume: "53"; issue: "24"; pages: "6101-4"
COMMENT     Sequence Label: SpyTag-beta-Lactamase-SpyCatcher
FEATURES             Location/Qualifiers
     source          1..976
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     RBS             16..38
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             57..74
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             84..101
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             111..149
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     CDS             171..956
                     /codon_start=1
                     /gene="bla(M)"
                     /product="beta-lactamase lacking the signal sequence"
                     /label=bla(M)
                     /note="allows cytosolic expression of beta-lactamase"
                     /translation="PETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMST
                     FKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMS
                     DNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATT
                     LRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAA
                     LGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     primer_bind     complement(317..336)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc
       61 atcatcatca tcacagcagc ggcctggtgc cgcgcggcag ccatatggga gcccacatcg
      121 tgatggtgga cgcctacaag ccgacgaagg gttcaggggg ttccggtcac ccagaaacgc
      181 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
      241 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
      301 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
      361 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
      421 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
      481 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
      541 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
      601 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
      661 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
      721 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
      781 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
      841 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
      901 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggggga
      961 gtggtggcag cggagg
//
