LOCUS       sequence-338570-        6807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AviTag-DogCatcher-MBP in bacterial cytoplasm. The AviTag 
            enables site-specific biotinylation by BirA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6807)
  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 6807)
  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: pET28-AviTag-DogCatcher-MBP
FEATURES             Location/Qualifiers
     source          1..6807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     971..990
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1010..1087)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1293..1312
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1396..1415
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1396..1414
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1415..1439
                     /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             1454..1476
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1495..1512
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1522..1539
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1546..1590
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1603..1914
                     /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
                     "
     CDS             1933..3030
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     3004..3027
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3067..3084
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3137..3155)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3151..3198
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3235..3690
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3322..3341)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3532..3553
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3782..4597)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3782..4597)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4505..4524
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4589..4608
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4719..5307
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5208..5227
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5461..5478
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5493..5632
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5558..5577)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5718..5740
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5734..5925)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6734..6807,1..1009))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
       61 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
      121 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
      181 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
      241 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
      301 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
      361 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
      421 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
      481 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
      541 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
      601 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
      661 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
      721 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
      781 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
      841 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
      901 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
      961 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     1021 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     1081 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     1141 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     1201 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     1261 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     1321 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     1381 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     1441 ctatagaaat aattttgttt aactttaaga aggagatata ccatgggcag cagccatcat
     1501 catcatcatc acagcagcgg cctggtgccg cgcggcagcc atatgggtct gaatgatatt
     1561 ttcgaagcgc agaaaattga atggcatgaa ggtagcggaa tgaaactggg cgagattgaa
     1621 tttattaaag tggacaaaac cgataaaaag ccgctgcgtg gtgccgtgtt tagcctgcag
     1681 aaacagcatc ccgactatcc cgatatctat ggcgcgattg atcagaatgg gacctatcaa
     1741 gatgtgcgta ccggcgaaga tggtaaactg acctttacga atctgagcga tggcaaatat
     1801 cgcctgattg aaaatagcga acccccgggc tataaaccgg tgcagaataa gccgattgtg
     1861 agctttcgta ttgtggatgg cgaagtgcgt gatgtgacca gtattgtgcc gcagggtagt
     1921 ggtgaaagtg gtaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
     1981 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
     2041 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
     2101 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
     2161 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
     2221 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt
     2281 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
     2341 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
     2401 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
     2461 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt
     2521 gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc
     2581 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
     2641 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
     2701 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
     2761 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
     2821 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt
     2881 attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     2941 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     3001 gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgtaaaagct tgcggccgca
     3061 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     3121 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     3181 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     3241 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3301 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3361 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3421 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3481 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3541 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3601 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3661 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     3721 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     3781 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     3841 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     3901 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     3961 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     4021 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     4081 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     4141 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     4201 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     4261 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     4321 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     4381 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     4441 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     4501 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     4561 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     4621 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4681 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4741 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4801 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4861 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4921 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4981 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5041 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5101 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5161 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5221 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5281 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5341 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5401 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5461 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     5521 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     5581 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     5641 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5701 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5761 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     5821 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     5881 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     5941 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     6001 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     6061 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     6121 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     6181 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     6241 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     6301 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     6361 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     6421 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     6481 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     6541 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     6601 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     6661 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     6721 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6781 atgaatcggc caacgcgcgg ggagagg
//
