LOCUS       sequence-338611-        3944 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cys-DogCatcher protein in bacterial cytoplasm. The cysteine 
            enables convenient coupling to maleimide-dye or thiol-reactive 
            resin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3944)
  AUTHORS   Keeble AH, Yadav VK, Ferla MP, Bauer CC, Chuntharpursat-Bon E, Huang
            J, Bon RS, Howarth M
  TITLE     DogCatcher allows loop-friendly protein-protein ligation.
  JOURNAL   Cell Chem Biol. 2021 Jul 28. pii: S2451-9456(21)00315-9. doi: 
            10.1016/j.chembiol.2021.07.005.
  PUBMED    34324879
REFERENCE   2  (bases 1 to 3944)
  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: "Cell Chem 
            Biol. 2021 Jul 28. pii: S2451-9456(21)00315-9. doi: 
            10.1016/j.chembiol.2021.07.005."
COMMENT     Sequence Label: pDEST14-Cys-DogCatcher
FEATURES             Location/Qualifiers
     source          1..3944
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     98..115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    130..269
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(195..214)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     355..377
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(371..562)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1071..1090
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1071..1089
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1121..1143
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1163..1180
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1202..1222
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1235..1546
                     /codon_start=1
                     /product="engineered fragment of the RrgA adhesin of 
                     Streptococcus pneumoniae"
                     /label=DogCatcher
                     /note="forms a covalent bond with an intra-domain protein 
                     loop containing the DogTag peptide (Keeble et al., 2022)"
                     /translation="KLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTY
                     QDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQ
                     "
     primer_bind     complement(1585..1603)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1599..1646
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1683..2138
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1770..1789)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1980..2001
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2164..2268
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2269..2337,2338..3129)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2487..2506)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3300..3888
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3789..3808
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
       61 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      121 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
      181 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
      241 tatcactacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
      301 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
      361 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
      421 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
      481 cgttgagctt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
      541 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
      601 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
      661 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
      721 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
      781 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
      841 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
      901 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
      961 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     1021 gcacccgtgg ccaggaccca acgctgcccg agatctcgat cccgcgaaat taatacgact
     1081 cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac tttaagaagg
     1141 agatatacat atgtcgtact accatcacca tcaccatcac gattacgaca tcccaacgac
     1201 cgaaaacctg tattttcagg gcggttgcgg tatgaaactg ggcgagattg aatttattaa
     1261 agtggacaaa accgataaaa agccgctgcg tggtgccgtg tttagcctgc agaaacagca
     1321 tcccgactat cccgatatct atggcgcgat tgatcagaat gggacctatc aagatgtgcg
     1381 taccggcgaa gatggtaaac tgacctttac gaatctgagc gatggcaaat atcgcctgat
     1441 tgaaaatagc gaacccccgg gctataaacc ggtgcagaat aagccgattg tgagctttcg
     1501 tattgtggat ggcgaagtgc gtgatgtgac cagtattgtg ccgcagtaac aaagcccgaa
     1561 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1621 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     1681 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1741 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1801 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1861 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1921 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1981 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2041 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2101 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     2161 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2221 agacaataac cctggtaaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2281 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2341 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2401 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2461 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     2521 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     2581 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2641 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     2701 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     2761 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg
     2821 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     2881 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     2941 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     3001 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3061 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3121 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3181 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3241 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3301 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3361 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3421 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3481 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3541 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3601 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3661 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3721 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3781 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3841 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3901 gcggcctttt tacggttcct ggccttttgc tggccttttg ctca
//
