LOCUS       sequence-338514-        3938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses DogCatcher protein in bacterial cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3938)
  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 3938)
  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-DogCatcher
FEATURES             Location/Qualifiers
     source          1..3938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..67
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    82..221
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(147..166)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     307..329
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(323..514)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1023..1042
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1023..1041
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1073..1095
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1115..1132
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1154..1174
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1181..1492
                     /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(1531..1549)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1545..1592
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1629..2084
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1716..1735)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1926..1947
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2110..2214
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2215..2283,2284..3075)
                     /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(2433..2452)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3246..3834
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3735..3754
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
       61 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
      121 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
      181 tatacactcc gctatcacta cgtgactggg tcatggctgc gccccgacac ccgccaacac
      241 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
      301 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
      361 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
      421 ccgcgtccag ctcgttgagc ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
      481 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
      541 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
      601 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
      661 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
      721 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
      781 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
      841 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
      901 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
      961 gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatctcg atcccgcgaa
     1021 attaatacga ctcactatag ggagaccaca acggtttccc tctagaaata attttgttta
     1081 actttaagaa ggagatatac atatgtcgta ctaccatcac catcaccatc acgattacga
     1141 catcccaacg accgaaaacc tgtattttca gggcgccatg aaactgggcg agattgaatt
     1201 tattaaagtg gacaaaaccg ataaaaagcc gctgcgtggt gccgtgttta gcctgcagaa
     1261 acagcatccc gactatcccg atatctatgg cgcgattgat cagaatggga cctatcaaga
     1321 tgtgcgtacc ggcgaagatg gtaaactgac ctttacgaat ctgagcgatg gcaaatatcg
     1381 cctgattgaa aatagcgaac ccccgggcta taaaccggtg cagaataagc cgattgtgag
     1441 ctttcgtatt gtggatggcg aagtgcgtga tgtgaccagt attgtgccgc agtaacaaag
     1501 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     1561 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     1621 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1681 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1741 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1801 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     1861 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1921 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     1981 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2041 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     2101 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2161 ctcatgagac aataaccctg gtaaatgctt caataatatt gaaaaaggaa gagtatgagt
     2221 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2281 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     2341 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     2401 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     2461 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     2521 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     2581 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     2641 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2701 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     2761 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     2821 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2881 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     2941 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     3001 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     3061 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     3121 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3181 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3241 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3301 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3361 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3421 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3481 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3541 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3601 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3661 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3721 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3781 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3841 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3901 cctgcgttat cccctgattc tgtggataac cgtattac
//
